EP4630457A1 - Bispecific antigen-binding molecules and uses thereof - Google Patents

Abstract

Provided herein are bispecific antibodies that bind to both a capsid of an AAV particle and to transferrin receptor (TfR) or calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1), related molecular complexes, and pharmaceutical compositions, and methods of use thereof.

Description

BISPECIFIC ANTIGEN-BINDING MOLECULES AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/520,485, filed August 18, 2023, and U.S. Provisional Application No. 63/578,899, filed August 25, 2023, the disclosure of both of which is herein incorporated by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 12, 2023, is named 250298_000567_SL.xml and is 404,527 bytes in size.

FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to bispecific antigen-binding molecules such as bispecific antibodies, or antigen-binding fragments thereof, that bind to a capsid of an AAV and to transferrin receptor (TfR) or calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1), as well as related molecular complexes and pharmaceutical compositions. Methods for using the bispecific antibodies disclosed herein, molecular complexes and/or pharmaceutical composition are also provided.

BACKGROUND

[0004] Viral particles have emerged as vectors for gene therapy and the treatment of disease. Viral vectors, such as those based on the genome of adeno-associated virus (AAV), offer promising platforms for gene delivery. Despite advances providing the ability to direct AAV infection, retargeted AAV as a gene delivery vehicle remains less than ideal, at least in part, due to limited success from the efforts to redirect vector tropism. The efficiency of AAV vector-mediated gene delivery to different cell types also varies greatly. One possible mechanism for inefficient AAV transduction of certain cells may be a lack of cellular receptor(s) to mediate virus binding and entry. There remains a need for selective and restrictive targeting of AAVs with enhanced cellular binding and transduction capabilities.

SUMMARY OF THE DISCLOSURE

[0005] In general, the present disclosure provides bispecific antibodies, which can bind both to the capsid of an AAV particle and to transferrin receptor (TfR) or calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1), and methods of use thereof. By binding to both a capsid of an AAV and transferrin receptor (TfR) or calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1), the antibodies described herein can bridge the virus to target cells that express TfR or CACNG1 , thereby redirecting virus delivery and transduction.

[0006] In one aspect, provided herein is a bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) an scFv comprising a first antigen-binding domain (“ABD1”) operably linked to (ii) a first heavy chain region of a first Fab (“Fab1”), operably linked to (iii) an Fc domain; b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a second antigen-binding domain (“ABD2”); and d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a third antigen-binding domain (“ABD3”); wherein ABD2 and ABD3 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD2 and ABD3 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD1 binds to transferrin receptor (TfR) and comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

[0007] In one embodiment, the scFv is linked to the first heavy chain region via a linker.

[0008] In another aspect, provided herein is a bispecific antibody, or a bispecific antigenbinding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an scFv comprising a first antigen-binding domain (“ABD1”), operably linked to (iii) an Fc domain; b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a second antigen-binding domain (“ABD2”); and d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a third antigen-binding domain (“ABD3”); wherein ABD2 and ABD3 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD2 and ABD3 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD1 binds to transferrin receptor (TfR) and comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

[0009] In one embodiment, the scFv is linked to the first heavy chain region via a linker.

[0010] In a further aspect, provided herein is a bispecific antibody, or a bispecific antigenbinding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an Fc domain, operably linked to (iii) an scFv comprising a first antigen-binding domain (“ABD1”); b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a second antigen-binding domain (“ABD2”); and d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a third antigen-binding domain (“ABD3”); wherein ABD2 and ABD3 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD2 and ABD3 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD1 binds to transferrin receptor (TfR) and comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

[0011] In one embodiment, the scFv is linked to the Fc domain via a linker.

[0012] In a further aspect, provided herein is a bispecific antibody, or a bispecific antigenbinding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an Fc domain, operably linked to (iii) a second heavy chain region of a second Fab (“Fab2”); b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) an scFv comprising a first antigen-binding domain (“ABD1”) operably linked to (ii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a second antigen-binding domain (“ABD2”); d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a third antigen-binding domain (“ABD3”); wherein ABD2 and ABD3 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD2 and ABD3 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD1 binds to transferrin receptor (TfR) and comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

[0013] In one embodiment, Fab2 is linked to the Fc domain via a linker.

[0014] In another aspect, provided herein is a bispecific antibody, or a bispecific antigenbinding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an Fc domain, operably linked to (iii) a first scFv comprising a first antigen-binding domain (“ABD1”); b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain, operably linked to (iii) a second scFv comprising a second antigen-binding domain (“ABD2”); c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a third antigen-binding domain (“ABD3”); d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a fourth antigen-binding domain (“ABD4”); wherein ABD3 and ABD4 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD3 and ABD4 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein the ABD1 and ABD2 bind to transferrin receptor (TfR), and each of ABD1 and ABD2 comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

[0015] In one embodiment, the first scFv and/or second scFv is linked to the Fc domain via a linker.

[0016] In a further embodiment, the ABD that binds to a capsid of an AAV particle comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 58; and/or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 16.

[0017] In some embodiments, the ABD that binds to a capsid of an AAV particle comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157.

[0018] In a further embodiment, the ABD that binds to TfR comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1121 , a HCDR2 comprising the amino acid sequence of SEQ ID NO: 1123, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 1125; and/or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 1129, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 1131 , and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 1133.

[0019] In some embodiments, the ABD that binds to TfR comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

[0020] In a further aspect, provided herein is a bispecific antibody, or a bispecific antigenbinding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first scFv comprising a first antigen-binding domain (“ABD1”) operably linked to (ii) a first heavy chain region of a first Fab (“Fab1”), operably linked to (iii) an Fc domain; b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second scFv comprising a second antigen-binding domain (“ABD2”) operably linked to (ii) a second heavy chain region of a second Fab (“Fab2”), operably linked to (iii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a third antigen-binding domain (“ABD3”); d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a fourth antigen-binding domain (“ABD4”); wherein ABD1 and ABD2 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD1 and ABD2 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD3 and ABD4 bind to calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1), and each of ABD3 and ABD4 comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 795, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 803.

[0021] In one embodiment, the first scFv is linked to Fab1 and/or the second scFv is linked to Fab2 via a linker.

[0022] In another aspect, provided herein is a bispecific antibody, or a bispecific antigenbinding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an Fc domain, operably linked to (iii) a first scFv comprising a first antigen-binding domain (“ABD1”); b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain, operably linked to (iii) a second scFv comprising a second antigen-binding domain (“ABD2”); c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a third antigen-binding domain (“ABD3”); d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a fourth antigen-binding domain (“ABD4”); wherein ABD1 and ABD2 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD1 and ABD2 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD3 and ABD4 bind to calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1), and each of ABD3 and ABD4 comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 795, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 803.

[0023] In one embodiment, the first scFv and/or second scFv is linked to the Fc domain via a linker.

[0024] In a further embodiment, each of ABD1 and ABD2 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 58; and/or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 16.

[0025] In some embodiments, each of ABD1 and ABD2 comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a LCVR comprising the amino acid sequence of SEQ I D NO: 10 or 1157.

[0026] In some embodiments, each of ABD3 and ABD4 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 797, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 799, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 801 ; and/or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 805, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 807, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 809.

[0027] In a further embodiment, each of ABD3 and ABD4 comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 795, and/or a LCVR comprising the amino acid sequence of SEQ ID NO: 803.

[0028] In one embodiment, the linker is or comprises a 3xG4S (SEQ ID NO: 1115).

[0029] In some embodiments, the bispecific antibody or bispecific antigen-binding fragment comprises an Fc heterodimer.

[0030] In one embodiment, the Fc domains in the Fc heterodimer comprise knob-in-hole mutations as compared to a wild type Fc domain.

[0031] In some embodiments, one Fc domain in the Fc heterodimer comprises amino acid substitutions S354C and T366W (according to Ell numbering) as compared to a wild type Fc domain, and the other Fc domain in the Fc heterodimer comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (according to Ell numbering) as compared to a wild type Fc domain.

[0032] In a further embodiment, at least one Fc domain in the Fc heterodimer comprises a star mutation as compared to a wild type Fc domain.

[0033] In some embodiments, one Fc domain in the Fc heterodimer comprises amino acid mutations H435R and/or Y436F (according to Ell numbering) as compared to a wild type Fc domain.

[0034] In one embodiment, the capsid comprises one or more wild-type AAV capsid polypeptides.

[0035] In some embodiments, the capsid comprises one or more non-wild-type AAV capsid polypeptides.

[0036] In one aspect, provided herein is a pharmaceutical composition comprising the bispecific antibody or bispecific antigen-binding fragment and a pharmaceutically acceptable carrier or excipient.

[0037] In a further aspect, provided herein is a molecular complex comprising an AAV particle bound to one or more bispecific antibodies and/or bispecific antigen-binding fragments.

[0038] In one embodiment, the AAV particle comprises one or more mutations in one or more AAV capsid proteins inhibiting the natural tropism of the AAV particle.

[0039] In a further aspect, provided herein is a pharmaceutical composition comprising the molecular complex described herein and a pharmaceutically acceptable carrier or excipient.

[0040] In another aspect, provided herein is a method of preparing the molecular complex comprising incubating the AAV particle in the presence of one or more bispecific antibodies and/or bispecific antigen-binding fragments under conditions allowing specific binding of the one or more bispecific antibodies and/or bispecific antigen-binding fragments to the AAV particle capsid.

[0041] In a further aspect, provided herein is a method for targeting an AAV particle to a cell expressing transferrin receptor (TfR) on the cell surface comprising contacting the cell with the molecular complex or the pharmaceutical composition described herein, wherein the molecular complex comprises one or more bispecific antibodies and/or bispecific antigenbinding fragments which bind to TfR.

[0042] In another aspect, provided herein is a method for targeting an AAV particle to a cell expressing calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1) on the cell surface comprising contacting the cell with the molecular complex or the pharmaceutical composition described herein, wherein the molecular complex comprises one or more bispecific antibodies and/or bispecific antigen-binding fragments which bind to CACNG1.

[0043] In another aspect, provided herein is a method for delivering a polynucleotide to a cell expressing transferrin receptor (TfR) on the cell surface comprising contacting the cell with the molecular complex or the pharmaceutical composition described herein, wherein the molecular complex comprises the AAV particle comprising the polynucleotide and bound to one or more bispecific antibodies and/or bispecific antigen-binding fragments which bind to TfR.

[0044] In another aspect, provided herein is a method for delivering a polynucleotide to a cell expressing calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1) on the cell surface comprising contacting the cell with the molecular complex or the pharmaceutical composition described herein, wherein the molecular complex comprises the AAV particle comprising the polynucleotide and bound to one or more bispecific antibodies and/or bispecific antigen-binding fragments which bind to CACNG1.

[0045] In one embodiment, the cell is in a subject and the molecular complex is administered to the subject.

[0046] In a further embodiment, the AAV particle does not target the cell in the absence of the one or more bispecific antibodies and/or bispecific antigen-binding fragments.

[0047] In various embodiments, any of the features or components of embodiments discussed above or herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value discussed above or herein may be combined with another related value discussed above or herein to recite a range with the values representing the upper and lower ends of the range, and such ranges are encompassed within the scope of the present disclosure.

[0048] Other embodiments will become apparent from a review of the ensuing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 shows a schematic representation of AAV x CACNG1 alternative format (AF) antibodies AF70 and AF71. AF70 comprises anti-AAV#70 scFv fused to the N-terminus of anti-CACNG1 REGN10717 hlgG1 N297G antibody. AF71 comprises anti-AAV#70 scFv fused to the C-terminus of anti-CACNG1 REGN10717 hlgG1 N297G antibody. Disulfide bonds are indicated by S-S.

[0050] Figure 2 shows flow cytometry data illustrating co-incubation of AAV9 W503A with AF70 (top two rows) and AF71 (bottom two rows) resulted in improved transduction efficiency over control AAV without antibody on HEK 293 cells overexpressing mouse CACNG1 (mCACNGI). Transduction efficiency was optimal between molar ratios of 1 AAV to 1 antibody (AAV:Ab ratio 1:1) and 1 AAV to 9 antibodies (AAV:Ab ratio 1 :9) as assessed by percent (%) GFP positive cells.

[0051] Figure 3 depicts line graphs showing co-incubation of AAV9 W503A with AF70 (top) and AF71 (bottom) resulted in improved transduction efficiency over control AAV without antibody on HEK 293 cells overexpressing mouse CACNG1 (mCACNGI).

Transduction efficiency was optimal between molar ratios of 1 AAV to 1 antibody (AAV:Ab ratio 1:1) and 1 AAV to 9 antibodies (AAV:Ab ratio 1:9) as assessed by mean fluorescence intensity (MFI).

[0052] Figure 4 shows flow cytometry data illustrating co-incubation of AAV9 W503A with AF70 (top two rows) and AF71 (bottom two rows) resulted in improved AAV transduction efficiency over control AAV without antibody on HEK 293 cells overexpressing human CACNG1 (hCACNGI). Transduction efficiency was optimal between molar ratios of 1 AAV to 1 antibody (AAV:Ab ratio 1:1) and 1 AAV to 9 antibodies (AAV:Ab ratio 1 :9) as assessed by percent (%) GFP positive cells.

[0053] Figure 5 depicts line graphs showing co-incubation of AAV9 W503A with AF70 (top) and AF71 (bottom) resulted in improved AAV transduction efficiency over control AAV without antibody on HEK 293 cells overexpressing human CACNG1 (hCACNGI). Transduction efficiency was optimal between molar ratios of 1 AAV to 1 antibody (AAV:Ab ratio 1:1) and 1 AAV to 9 antibodies (AAV:Ab ratio 1:9) as assessed by mean fluorescence intensity (MFI).

[0054] Figure 6 shows immunohistochemical staining of differentiated C2C12 myotubes for Myosin Heavy Chain (MyHC) and demonstration that incubation of AAV with AF70 and AF71 enhanced transduction into the myotubes as determined by GFP fluorescence.

[0055] Figure 7 shows immunohistochemical staining of differentiated human myotubes for Myosin Heavy Chain (MyHC) and demonstration that incubation of AAV with AF70 and AF71 enhanced transduction into the myotubes as determined by GFP fluorescence. Human myotubes were most efficiently transduced with AAV complexed with AF71 at molar ratio 1:3 and 1 :9.

[0056] Figures 8A-8D demonstrate incubation of AAV with AF70 and AF71 enhanced transduction into differentiated C2C12 myotubes as determined by quantification of GFP positive cells.

[0057] Figures 9A-9D demonstrate incubation of AAV with AF70 and AF71 enhanced transduction into differentiated human myotubes as determined by quantification of GFP positive cells.

[0058] Figures 10A-10B show non-limiting examples of AAV x mTfR alternative format (AF) antibodies described herein. Star mutations are depicted with an asterisk (*) and knob- in-hole (KiH) mutations are depicted with a triangle (e.g., ◄).

[0059] Figure 11 shows an example experimental setup used to test alternative format antibodies binding to AAV9W503A virus by ELISA.

[0060] Figure 12 depicts ELISA data for AAV x mTfR alternative format antibodies binding to AAV9W503A.

[0061] Figure 13 shows a schematic diagram of a FLuc assay protocol used to test retargeting of AAV9W503A using mTfR alternative format antibodies on 293T cells expressing mTfR receptor.

[0062] Figure 14 shows a line graph of data generated in experiments testing AAV9W503A retargeting using AAVxmTfR alternative format antibodies on mTfR293T cells. [0063] Figures 15A-15C illustrate a retargeting assay using AAV9 scCBH.eGFP.

[0064] Figures 16A-16B show in vitro test infection results for in vivo injection samples.

[0065] Figures 17A-17F depict AAV x mTfR liver and brain (hippocampus, cortex, and cerebellum) green fluorescent protein (GFP) staining for control groups (Figure 17A) and alternative format designs, AAV x mTfR AF1 (Figure 17B), AAV x mTfR AF3 (Figure 17C), AAV x mTfR AF5 (Figure 17D), AAV x mTfR AF7 (Figure 17E), and AAV x mTfR AF9 (Figure 17F).

[0066] Figure 18 shows relative RNA expression of GFP in liver samples as determined by RT-qPCR.

[0067] Figure 19 shows RNA expression of GFP in brain samples as determined by RT- qPCR.

[0068] Figures 20A-20D depict GFP staining in brain, heart and liver tissues from mice receiving AAV9 (Figures 20A-20B) or AAV9W503A (Figures 20C and 20D) complexed with AAV x mTfR alternative format AF7 at AAV vector genome (VG) to antibody (Ab) ratios (VG:Ab ratios) of 1 :9 and 1:3.

[0069] Figures 21 A-211 depict AAV x mTfR alternative format designs AF3 and AF7. Figure 21 A provides a description of AAV x mTfR alternative format designs AF3 and AF7. Star mutations are depicted with an asterisk (*) and knob-in-hole (KiH) mutations are depicted with a triangle (e.g., ◄). Figures 21B-21E show examples of AAV x mTfR AF3 amino acid sequences (Figure 21 B) and corresponding nucleotide sequences (Figures 21C-21E). Figures 21F-21I show examples of AAV x mTfR AF7 amino acid sequences (Figure 21 F) and corresponding nucleotide sequences (Figures 21G-21I). Figure discloses “3xG4S” as SEQ ID NO: 1115.

[0070] Figures 22A-22G depict AAV x CACNG1 alternative format designs AF70 and AF71. Figure 22A provides a description of AAV x CACNG1 alternative format designs AF70 and AF71. Figures 22B-22G show examples of AAV x mTfR AF70 amino acid sequences (Figure 22B) and corresponding nucleotide sequences (Figures 22C-22D). Figures 22E-22G show examples of AAV x mTfR AF71 amino acid sequences (Figure 22E) and corresponding nucleotide sequences (Figures 22F-22G). Figure discloses “3xG4S” as SEQ I D NO: 1115 and “4xG4S” as SEQ I D NO: 1161.

[0071] Figure 23 illustrates AAV x CACNG1 bispecific antibody enhancement of transduction into CACNG1 overexpressing 293 cells. GFP expression was assessed by flow cytometry and is shown as % GFP positive cells (top panels) or MFI of GFP expressing cells (bottom panels).

[0072] Figure 24 depicts an example of a study design for testing Hu37 complexed with anti-AAV x CACNG1 2x2 antibodies in D2.MDX mice.

[0073] Figure 25 shows in vitro transduction in HEK293-hCACNG1 of complexes prepared for in vivo study.

[0074] Figures 26A-26C illustrate improved Hu37 transduction in muscle tissues when complexed with an 2x2 anti-CACNG1xAAV alternative format antibody.

[0075] Figures 27A-27B illustrate bispecific antibody enhancement of Hu37 transduction into skeletal muscle.

[0076] Figures 28A-28I depict AAV x mTfR alternative format designs AF1 , AF5, and AF9. Figures 28A-28C show examples of AAV x mTfR AF1 amino acid sequences (Figure 28A) and corresponding nucleotide sequences (Figures 28B-28C). Figures 28D-28F show examples of AAV x mTfR AF5 amino acid sequences (Figure 28D) and corresponding nucleotide sequences (Figures 28E-28F). Figures 28G-28I show examples of AAV x mTfR AF9 amino acid sequences (Figure 28G) and corresponding nucleotide sequences (Figures 28H-28I). Figure discloses “3xG4S” as SEQ ID NO: 1115 and “4xG4S” as SEQ ID NO: 1161.

DETAILED DESCRIPTION

[0077] Before the present disclosure is described, it is to be understood that this disclosure is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0078] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entireties.

Definitions

[0079] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0080] The term “antigen” encompasses any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portions thereof, or combinations thereof) that, when introduced into an immunocompetent host is recognized by the immune system of the host and is capable of eliciting an immune response by the host.

[0081] The term "epitope" can refer to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen. Epitopes may also be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and are defined as those residues that directly contribute to the affinity of the interaction between a major histocompatibility complex (MHC) molecule and the antigen.

[0082] The term "antigen-binding molecule" refers in its broadest sense to a molecule that specifically binds to an antigen. In certain embodiments, an antigen-binding molecule is an antibody or an antigen-binding fragment of an antibody, including, e.g., bispecific antibodies or fragments thereof.

[0083] According to certain exemplary embodiments, the present disclosure includes bispecific antigen-binding molecules (e.g., antibodies) that specifically bind a capsid of an AAV particle and either CACNG1 or TfR. Such antigen-binding molecules may be referred to herein as, e.g., “anti-AAV x anti-CACNG1”, “anti-AAV x anti-TfR”, or other similar terminology (e.g., anti-AAV/anti-CACNG1 or anti-AAV/anti-TfR).

[0084] The term “antigen-binding domain” or “ABD” as used herein can refer to the portion of an antigen-binding molecule that is capable of specific binding to an antigen.

[0085] The term "antibody", as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). The term “antibody” also includes immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or H) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1 , CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (Ci_1 ). The H and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the disclosure, the FRs of the anti-AAV antibody or anti-CACNG1 or anti-TfR antibody (or antigen-binding portion thereof) may be identical to the human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0086] The term "antibody", as used herein, also includes antigen-binding fragments of full antibody molecules. The terms "antigen-binding fragment" of an antibody, "antigen-binding portion" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0087] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide.

[0088] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain H- H, H- L or L- L dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric H or VL domain.

[0089] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. In any configuration of variable and constant domains, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non- covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)). [0090] As with full antibody molecules, antigen-binding fragments may be monospecific or bispecific. A bispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any bispecific antibody format, including the non-limiting example formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.

[0091] The terms “complementarity determining region” or “CDR,” as used herein, can refer to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. In general, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3).

[0092] Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, the ABS definition and the IMGT definition. See, e.g., Kabat, 1991, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-948 (Chothia numbering scheme); Martin et al., 1989, Proc. Natl. Acad. Sci. USA 86:9268-9272 (ABS numbering scheme); and Lefranc et ai, 2003, Dev. Comp. Immunol. 27:55-77 (IMGT numbering scheme). Public databases are also available for identifying CDR sequences within an antibody.

[0093] The term “single chain Fv” or “scFv” as used herein can refer to a polypeptide chain comprising the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain.

[0094] The term “Fab” can refer to a pair of polypeptide chains, the first polypeptide chain comprising a variable heavy (VH) domain of an antibody N- terminal to a first constant domain (referred to herein as C1), and the second polypeptide chain comprising a variable light ( L) domain of an antibody N-terminal to a second constant domain (referred to herein as C2) capable of pairing with the first constant domain. In a native antibody, the VH is N- terminal to the first constant domain (CH1) of the heavy chain and the VL is N- terminal to the constant domain of the light chain (CL). The Fabs of the disclosure can be arranged according to the native orientation or include domain substitutions or swaps on that facilitate correct VH and VL pairings, particularly where the antigen-binding molecules of the disclosure comprise non-identical Fabs.

[0095] The term “universal light chain” as used herein in the context of an antigen-binding molecule described herein can refer to a light chain polypeptide capable of pairing with the heavy chain region of a first Fab to form the first Fab and capable of pairing with the heavy chain region of a second Fab to form the second Fab. Universal light chains are also known as “common light chains.”

[0096] The term “Fc domain” can refer to a portion of the heavy chain that pairs with the corresponding portion of another heavy chain. The term “Fc region” can refer to the region of antibody-based binding molecules formed by association of two heavy chain Fc domains. The two Fc domains within the Fc region may be the same or different from one another. In a native antibody the Fc domains are typically identical, but for the purpose of producing the antigen-binding molecules of the disclosure, one or both Fc domains might advantageously be modified to allow for heterodimerization.

[0097] As used herein, the term “derived from” indicates a relationship between a first and a second molecule. It generally can refer to structural similarity between the first molecule and a second molecule and does not connote or include a process or source limitation on a first molecule that is derived from a second molecule

[0098] The term “specifically binds” as used herein means that an antigen-binding molecule forms a complex with a target antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by a KD of about 5x1 O'² M or less (e.g., less than 5x1 O'² M, less than 10'² M, less than 5x10'²M, less than 10'³ M, less than 5x1 O'⁴ M, less than 10'⁴M, less than 5x10'⁵M, less than 10'⁵M, less than 5x10'⁶M, less than 10'⁶ M, less than 5x10'⁷M, less than 10'⁷M, less than 5x10'⁸M, less than 10'⁸M, less than 5x10'⁹M, less than 10'⁹M, or less than 10'¹° M). Methods for determining the binding affinity of an antibody or an antibody fragment, e.g., an antigen-binding molecule or antigen-binding domain, to a target antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., Biacore assays), fluorescent-activated cell sorting (FACS) binding assays and the like. A antigen-binding molecule that specifically binds to a target antigen from one species can, however, have cross- reactivity to the target antigen from one or more other species.

[0099] The term “operably linked” as used herein can refer to a functional relationship between two or more regions of a polypeptide chain in which the two or more regions are linked so as to produce a functional polypeptide.

[00100] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well- known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule. [00101] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalaninetyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[00102] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GOG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GOG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GOG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each herein incorporated by reference.

[00103] The term "subject" or "patient" as used herein includes all members of the animal kingdom including non-human primates and humans.

Anti-AAV Antibodies and Antigen-Binding Fragments Thereof

[00104] In a one aspect provided herein are antibodies and antigen-binding fragments thereof that bind a capsid of an AAV particle (also known as “anti-AAV antibodies” herein). In some embodiments, the capsid comprises any of various wild-type and/or non- wild-type AAV capsid protein(s) described herein. In some embodiments, the antibodies and/or antigen-binding fragments thereof bind an epitope on a capsid described herein. The anti-AAV antibodies provided herein, or antigen-binding portions thereof, may be included as part of a bispecific antigen-binding molecule, e.g., a bispecific antibody or bispecific antigenbinding fragment thereof, described herein.

[00105] "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. AAVs are members of the Parvovirus family of small, non-enveloped, single-stranded DNA viruses. Generally, a wildtype AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITR) and two open reading frames (ORFs), rep and cap. The wildtype rep reading frame encodes four proteins of molecular weight 78 kD (“Rep78”), 68 kD (“Rep68”), 52 kD (“Rep52”) and 40 kD (“Rep 40”). Rep78 and Rep68 are transcribed from the p5 promoter, and Rep52 and Rep40 are transcribed from the p19 promoter. These proteins function mainly in regulating the transcription and replication of the AAV genome. The wildtype cap reading frame encodes three structural (capsid) viral proteins (VPs) having molecular weights of 83-85 kD (VP1), 72-73 kD (VP2) and 61-62 kD (VP3). More than 80% of total proteins in an AAV virion (capsid) comprise VP3; in mature virions VP1 , VP2 and VP3 are found at relative abundance of approximately 1:1:10, although ratios of 1:1:8 have been reported. Padron et al. (2005) J. Virology 79:5047-58.

[00106] The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_002077 (AAV1), AF063497 (AAV1), NC001401 (AAV-2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8); the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71 :6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al. ,(1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33:375-383; US Patent Publication 20170130245; international patent publications WO 00/28061 , WO 99/61601, WO 98/11244; and U.S. Pat. No. 6,156,303, each of which is incorporated by reference in its entirety by reference.

[00107] The term "capsid protein,” “Cap protein” and the like, includes a protein that is part of the capsid of the virus. For adeno-associated viruses, the capsid proteins are generally referred to as VP1, VP2 and/or VP3, and may be encoded by the single cap gene. The three AAV capsid proteins can be produced in nature an overlapping fashion from the cap ORF alternative translational start codon usage, although all three proteins use a common stop codon. The ORF of a wildtype cap gene encodes from 5’ to 3’ three alternative start codons: “the VP1 start codon,” “the VP2 start codon,” and “the VP3 start codon”; and one “common stop codon”. The largest viral protein, VP1, is generally encoded from the VP1 start codon to the “common stop codon.” VP2 is generally encoded from the VP2 start codon to the common stop codon. VP3 is generally encoded from the VP3 start codon to the common stop codon. Accordingly, VP1 comprises at its N-terminus sequence that it does not share with the VP2 or VP3, referred to as the VP1-unique region (VP1-u).

The VP1-u region is generally encoded by the sequence of a wildtype cap gene starting from the VP1 start codon to the “VP2 start codon.” VP1-u comprises a phospholipase A2 domain (PLA2), which may be important for infection, as well as nuclear localization signals which may aid the virus in targeting to the nucleus for uncoating and genome release. The VP1, VP2, and VP3 capsid proteins share the same C-terminal sequence that makes up the entirety of VP3, which may also be referred to herein as the VP3 region. The VP3 region is encoded from the VP3 start codon to the common stop codon. VP2 has an additional ~ 60 amino acids that it shares with the VP1. This region is called the VP1/VP2 common region. [00108] The phrase “inverted terminal repeat” or “ITR” includes symmetrical nucleic acid sequences in the genome of adeno-associated viruses required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. The ITRs serve as the origins of replication for viral DNA synthesis and are essential cis components for generating AAV particles, e.g., packaging into AAV particles.

[00109] AAV ITR comprise recognition sites for replication proteins Rep78 or Rep68. A "D" region of the ITR comprises the DNA nick site where DNA replication initiates and provides directionality to the nucleic acid replication step. An AAV replicating in a mammalian cell typically comprises two ITR sequences.

[00110] A single ITR may be engineered with Rep binding sites on both strands of the “A” regions and two symmetrical D regions on each side of the ITR palindrome. Such an engineered construct on a double-stranded circular DNA template allows Rep78 or Rep68 initiated nucleic acid replication that proceeds in both directions. A single ITR is sufficient for AAV replication of a circular particle. In methods of producing an AAV viral particle of the disclosure, the rep encoding sequence encodes a Rep protein or Rep protein equivalent that is capable of binding an ITR comprised on the transfer plasmid.

[00111] The Cap proteins of the disclosure, when expressed with appropriate Rep proteins by a packaging cell, may encapsidate a transfer plasmid comprising a nucleotide of interest and an even number of two or more ITR sequences.

[00112] A “chimeric AAV capsid protein” includes an AAV capsid protein that comprises amino acid sequences, e.g., portions, from two or more different AAV and that is capable of forming and/or forms an AAV viral capsid/viral particle. A chimeric AAV capsid protein is encoded by a chimeric AAV capsid gene, e.g., a chimeric nucleotide comprising a plurality, e.g., at least two, nucleic acid sequences, each of which plurality is identical to a portion of a capsid gene encoding a capsid protein of distinct AAV, and which plurality together encodes a functional chimeric AAV capsid protein. Association of a chimeric capsid protein to a specific AAV indicates that the capsid protein comprises one or more portions from a capsid protein of that AAV and one or more portions from a capsid protein of a different AAV. For example, a chimeric AAV2 capsid protein includes a capsid protein comprising one or more portions of a VP1 , VP2, and/or VP3 capsid protein of AAV2 and one or more portions of a VP1 , VP2, and/or VP3 capsid protein of a different AAV.

[00113] The anti-AAV antibodies or antigen-binding fragments thereof described herein may bind to a capsid of an AAV particle with a KD value of about 5x1 O'⁶ M or less, such as about 10^-7 M or less, about 10'⁸ M or less, such as about 10'⁹ M or less when determined by, for instance, surface plasmon resonance (SPR) technology in a BIAcore instrument using the antigen as the ligand and the antibody, Ig, antibody-binding fragment, or Fc-containing protein as the analyte (or antiligand). Cell-based binding strategies, such as fluorescent- activated cell sorting (FACS) binding assays, are also routinely used, and FACS data correlates well with other methods such as radioligand competition binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201 (2):223-31 ; Geuijen, CA, et al. J Immunol Methods. 2005, 302(1-2):68-77). In some embodiments, an anti-AAV antibody or antigenbinding fragment thereof described herein may bind to a capsid of an AAV particle with a KD value of about 5x1 O’⁶ M to 1x1 O’⁶ M (e.g., 1x1 O'⁶ M, 1.5x1 O’⁶ M, 2x1 O’⁶ M, 3x1 O’⁶ M, 4x1 O’⁶ M, 5x1 O’⁶ M), about 1x10’⁶ M to 1x10’⁷ M (e.g., 1x10’⁷ M, 2x10’⁷ M, 3x10’⁷ M, 4x1 O’⁷ M, 5x10’⁷ M, 6x1 O’⁷ M, 7x10'⁷ M, 8x10’⁷ M, 9x10’⁷ M), about 1x10’⁷ M to 1x10’⁸ M (e.g., 1x10’⁸ M, 2x10’ ⁸ M, 3x1 O’⁸ M, 4x1 O’⁸ M, 5x1 O’⁸ M, 6x1 O’⁸ M, 7x1 O’⁸ M, 8x1 O’⁸ M, 9x1 O’⁸ M), about 1x1 O’⁸ M to 1x1 O’⁹ M (e.g., 1x10’⁹ M, 2x10’⁹ M, 3x10’⁹ M, 4x10’⁹ M, 5x10’⁹ M, 6x10’⁹ M, 7x10’⁹ M, 8x10’⁹ M, 9x1 O’⁹ M), or about 1x10’⁹ M to IxlO’¹⁰ M (e.g., IxlO’¹⁰ M, 2x1O-¹⁰ M, 3x1O-¹⁰ M, 4x1O-¹⁰ M, 5x1 O’¹⁰ M, 6x1 O’¹⁰ M, 7x1 O’¹⁰ M, 8x1 O’¹⁰ M, 9x1 O'¹⁰ M).

[00114] The antibody or antigen-binding protein of the disclosure may bind to the predetermined antigen or CACNG1 or TfR (receptor) having an affinity corresponding to a KD value that is at least ten-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA). According to the present disclosure, the affinity of an antibody corresponding to a KD value that is equal to or less than ten-fold lower than a non-specific antigen may be considered non-detectable binding, however such an antibody may be paired with a second antigen-binding domain for the production of a bispecific antibody of the disclosure.

[00115] The term "KD" (M) can refer to the dissociation equilibrium constant of a particular antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. There is an inverse relationship between KD and binding affinity, therefore the smaller the KD value, the higher, i.e. stronger, the affinity. Thus, the terms “higher affinity” or “stronger affinity” relate to a higher ability to form an interaction and therefore a smaller KD value, and conversely the terms “lower affinity” or “weaker affinity” relate to a lower ability to form an interaction and therefore a larger KD value. In some circumstances, a higher binding affinity (or KD) of a particular molecule (e.g. antibody) to its interactive partner molecule (e.g. antigen X) compared to the binding affinity of the molecule (e.g. antibody) to another interactive partner molecule (e.g. antigen Y) may be expressed as a binding ratio determined by dividing the larger KD value (lower, or weaker, affinity) by the smaller KD (higher, or stronger, affinity), for example expressed as 5-fold or 10-fold greater binding affinity, as the case may be.

[00116] The term "kd" (sec -1 or 1/s) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibodybinding fragment. Said value is also referred to as the k_Off value.

[00117] The term "k_a" (M-1 x sec-1 or 1/M/s) can refer to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody-binding fragment.

[00118] The term "KA" (M-1 or 1/M) can refer to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is obtained by dividing the k_a by the kd.

[00119] The term “EC50” or “ECso” can refer to the half maximal effective concentration, which includes the concentration of an antibody which induces a response halfway between the baseline and maximum after a specified exposure time. The ECso essentially represents the concentration of an antibody where 50% of its maximal effect is observed. In certain embodiments, the ECso value equals the concentration of an antibody of the disclosure that gives half-maximal binding to a capsid protein of an AAV particle (e.g., a wild-type or a non- wild-type AAV capsid protein(s) or to cells expressing CACNG1 or TfR, as determined by e.g., a FACS binding assay. Thus, reduced or weaker binding is observed with an increased EC50, or half maximal effective concentration value.

[00120] Also provided herein are nucleic acid molecules encoding anti-AAV antibodies or antigen-binding fragments thereof.

Bispecific Antigen-binding Molecules

[00121] Single chain Fv or “scFv” antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain, are capable of being expressed as a single chain polypeptide and retain the specificity of the intact antibodies from which they are derived. Generally, an scFv polypeptide may further comprise a polypeptide linker between the VH and VL domain that enables the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of an scFV are the linkers are described herein.

[00122] The scFv can comprise VL and VH sequences from any suitable species, such as murine, human or humanized VH and VL sequences.

[00123] To create an scFv-encoding nucleic acid, the VL and Vn-encoding DNA fragments are operably linked to another fragment encoding a linker, e.g., encoding any of the linkers described herein, such that the VL and VH sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see, e.g., Bird et al., 1988, Science 242:423- 426; Huston et ai, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et ai, 1990, Nature 348:552-554).

[00124] The bispecific antigen-binding molecule of the disclosure may comprise at least one Fab domain. Fab domains were traditionally produced from by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the bispecific antigenbinding molecule of the disclosure, the Fab domains are recombinantly expressed as part of a larger molecule.

[00125] The Fab domains can comprise constant domain and variable region sequences from any suitable species, and thus can be murine, chimeric, human or humanized.

[00126] Fab domains typically comprise a CH1 domain attached to a VH domain which pairs with a CL domain attached to a VL domain. In a wild-type immunoglobulin, the VH domain is paired with the VL domain to constitute the Fv region, and the CH1 domain is paired with the CL domain to further stabilize the binding module. A disulfide bond between the two constant domains can further stabilize the Fab domain.

[00127] The anti-AAV monospecific antibodies or anti-AAV x anti-CACNG1 or anti-AAV x anti-TfR bispecific antibodies disclosed herein can comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived.

[00128] Provided herein are antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”), and having weak or no detectable binding to a AAV capsid antigen or CACNG1 or TfR.

[00129] Furthermore, the antigen-binding domains may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antigen-binding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner are encompassed within the present disclosure.

Linkers

[00130] In certain aspects, the present disclosure provides bispecific antigen-binding molecules in which two or more components of an ABD (e.g., a H and a VL of an scFv), two or more ABDs (e.g., an scFv and a Fab, or a Fab and a Fab), or an ABD and a non-ABD component (e.g., an Fc region) are connected to one another by a peptide linker.

[00131] A peptide linker can range from 2 amino acids to 60 or more amino acids, and in certain aspects a peptide linker ranges from 3 amino acids to 50 amino acids, from 4 to 30 amino acids, from 5 to 25 amino acids, from 10 to 25 amino acids, 10 amino acids to 60 amino acids, from 12 amino acids to 20 amino acids, from 20 amino acids to 50 amino acids, or from 25 amino acids to 35 amino acids in length.

[00132] In particular aspects, a peptide linker, e.g., a peptide linker separating an scFv domain and a heavy chain constant region, is at least 5 amino acids, at least 6 amino acids or at least 7 amino acids in length and optionally is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids or up to 60 amino acids in length. [00133] In some embodiments of the foregoing, the linker ranges from 5 amino acids to 50 amino acids in length, e.g., ranges from 5 to 50, from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, or from 5 to 20 amino acids in length. In other embodiments of the foregoing, the linker ranges from 6 amino acids to 50 amino acids in length, e.g., ranges from 6 to 50, from 6 to 45, from 6 to 40, from 6 to 35, from 6 to 30, from 6 to 25, or from 6 to 20 amino acids in length. In yet other embodiments of the foregoing, the linker ranges from 7 amino acids to 50 amino acids in length, e.g., ranges from 7 to 50, from 7 to 45, from 7 to 40, from 7 to 35, from 7 to 30, from 7 to 25, or from 7 to 20 amino acids in length.

[00134] Charged (e.g., charged hydrophilic linkers) and/or flexible linkers are particularly preferred.

[00135] Examples of flexible linkers that can be used in the bispecific antigen-binding molecules of the disclosure include those disclosed by Chen et ai, 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et a/., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible linkers are or comprise repeats of glycines and serines. Polyglycine linkers can suitably be used in the bispecific antigen-binding molecules of the disclosure.

Constant Regions

[00136] In some embodiments, bispecific antigen-binding molecules of the disclosure comprise constant regions (e.g., CH1 , hinge, CH2, CH3, CL) derived from any suitable class of antibody. In some embodiments, the constant regions are derived from a human antibody. Hinge Regions

[00137] The bispecific antigen-binding molecules of the disclosure can also comprise hinge regions, e.g., connecting an ABD module to an Fc region. The hinge region can be a native or a modified hinge region. Hinge regions are typically found at the N-termini of Fc regions.

[00138] A native hinge region is the hinge region that would normally be found between Fab and Fc domains in a naturally occurring antibody. A modified hinge region is any hinge that differs in length and/or composition from the native hinge region. Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama or goat hinge regions. Other modified hinge regions may comprise a complete hinge region derived from an antibody of a different class or subclass from that of the heavy chain Fc region. Alternatively, the modified hinge region may comprise part of a natural hinge or a repeating unit in which each unit in the repeat is derived from a natural hinge region. In a further alternative, the natural hinge region may be altered by converting one or more cysteine or other residues into neutral residues, such as serine or alanine, or by converting suitably placed residues into cysteine residues. By such means the number of cysteine residues in the hinge region may be increased or decreased. Other modified hinge regions may be entirely synthetic and may be designed to possess desired properties such as length, cysteine composition and flexibility.

[00139] A number of modified hinge regions have already been described for example, in U.S. Patent No. 5,677,425, WO9915549, W02005003170, W02005003169, W02005003170, WO9825971 and W02005003171 and these are incorporated herein by reference.

[00140] The hinge region can be a chimeric hinge region. Fc Domains

[00141] The bispecific antigen-binding molecules of the disclosure can include an Fc region derived from any suitable species. In one embodiment the Fc region is derived from a human Fc domain.

[00142] In some embodiments, bispecific antigen-binding molecules disclosed herein can have, e.g., fully human variable regions but can have mouse constant regions (e.g., a mouse IgG 1 Fc or a mouse lgG2 Fc (a or b isotype)) or human constant regions (e.g., a human IgG 1 Fc or a human lgG4 Fc). As will be appreciated by a person of ordinary skill in the art, a bispecific antigen-binding molecules having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse IgG 1 Fc can be converted to an antibody with a human I gG4, etc.), but in any event, the variable domains (including the CDRs) will remain the same, and the binding properties to antigen are expected to be identical or substantially similar regardless of the nature of the constant domain.

[00143] The Fc domain can be derived from any suitable class of antibody, including IgA (including subclasses lgA1 and lgA2), IgD, IgE, IgG (including subclasses lgG1, lgG2, lgG3 and lgG4), and IgM.

[00144] The two Fc domains within the Fc region can be the same or different from one another. In a native antibody the Fc domains are typically identical, but for the purpose of producing bispecific binding molecules of the disclosure, the Fc domains might advantageously be different to allow for heterodimerization.

[00145] In native antibodies, the heavy chain Fc domain of IgA, IgD and IgG is composed of two heavy chain constant domains (CH2 and CH3) and that of IgE and IgM is composed of three heavy chain constant domains (CH2, CH3 and CH4). These dimerize to create an Fc region.

[00146] In bispecific antigen-binding molecules of the present disclosure, the Fc region, and/or the Fc domains within it, can comprise heavy chain constant domains from one or more different classes of antibody, for example one, two or three different classes. [00147] In some embodiments, the Fc region comprises CH2 and CH3 domains derived from lgG1. [00148] In some embodiments, the Fc region comprises CH2 and CH3 domains derived from lgG2.

[00149] In some embodiments, the Fc region comprises CH2 and CH3 domains derived from lgG3.

[00150] In some embodiments, the Fc region comprises CH2 and CH3 domains derived from lgG4.

[00151] In some embodiments, the Fc region comprises a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[00152] In some embodiments, the Fc region comprises CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[00153] It will be appreciated that the heavy chain constant domains for use in producing an Fc region for the bispecific antigen-binding molecules of the present disclosure may include variants of the naturally occurring constant domains described above. Such variants may comprise one or more amino acid variations compared to wild type constant domains. In one example the Fc region of the present disclosure comprises at least one constant domain that varies in sequence from the wild-type constant domain. It will be appreciated that the variant constant domains may be longer or shorter than the wild-type constant domain. Preferably the variant constant domains are at least 60% identical or similar to a wild-type constant domain. In another example the variant constant domains are at least 70% identical or similar. In another example the variant constant domains are at least 80% identical or similar. In another example the variant constant domains are at least 90% identical or similar. In another example the variant constant domains are at least 95% identical or similar.

[00154] IgM and IgA occur naturally in humans as covalent multimers of the common H2L2 antibody unit. IgM occurs as a pentamer when it has incorporated a J-chain, or as a hexamer when it lacks a J-chain. IgA occurs as monomer and dimer forms. The heavy chains of IgM and IgA possess an 18 amino acid extension to the C-terminal constant domain, known as a tailpiece. The tailpiece includes a cysteine residue that forms a disulfide bond between heavy chains in the polymer and is believed to have an important role in polymerization. The tailpiece also contains a glycosylation site.

[00155] The Fc domains can also be altered to include modifications that improve manufacturability of asymmetric bispecific antigen-binding molecules, for example by allowing heterodimerization, which is the preferential pairing of non-identical Fc domains over identical Fc domains. Heterodimerization permits the production of bispecific antigenbinding molecules in which different A BSs are connected to one another by an Fc region containing Fc domains that differ in sequence.

[00156] It will be appreciated that any of the modifications mentioned above can be combined in any suitable manner to achieve the desired functional properties and/or combined with other modifications to alter the properties of the bispecific antigen-binding molecules.

[00157] Typically, the same one or more amino acid substitution is present in each of the two Fc domains of an Fc region.

[00158] In one embodiment, the Fc domain is an lgG1 Fc domain, particularly a human lgG1 Fc domain.

[00159] Typically, the same one or more amino acid substitution is present in each of the two Fc domains of an Fc region.

[00160] Many bispecific molecule formats entail dimerization between two Fc domains that, unlike a native immunoglobulin, are operably linked to non-identical antigen-binding domains (or portions thereof, e.g., a VH or VH-CH1 of a Fab). Inadequate heterodimerization of two Fc regions to form an Fc domain has can be an obstacle for increasing the yield of desired bispecific molecules and represents challenges for purification. A variety of approaches available in the art can be used in for enhancing dimerization of Fc domains that might be present in the bispecific antigen-binding molecules of the disclosure, for example as disclosed in EP 1870459A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WG2009/089004A1.

[00161] The present disclosure provides bispecific antigen-binding molecules comprising Fc heterodimers, i.e., Fc regions comprising heterologous, non-identical Fc domains. Heterodimerization strategies are used to enhance dimerization of Fc regions operably linked to different ABDs (or portions thereof, e.g., a VH or H-CH1 of a Fab) and reduce dimerization of Fc domains operably linked to identical ABDs. Typically, each Fc domain in the Fc heterodimer comprises a CH3 domain of an antibody. The CH3 domains are derived from the constant region of an antibody of any isotype, class or subclass, and preferably of IgG (lgG1 , lgG2, lgG3 and lgG4) class, as described in the preceding section. [00162] Heterodimerization of the two different heavy chains at CH3 domains give rise to the desired bispecific antigen-binding molecule, while homodimerization of identical heavy chains will reduce yield of the desired bispecific antigen-binding molecule.

[00163] In a specific embodiment said modification promoting the formation of Fc heterodimers is a so-called “knob-into-hole” or “knob-in-hole” (KiH) modification, comprising a “knob” modification in one of the Fc domains and a “hole” modification in the other Fc domain. The knob-into-hole technology is described e.g. in U.S. Patent No. 5,731,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. Generally, the method involves introducing a protuberance (“knob”) at the 1 interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).

[00164] As an alternative, or in addition, to the use of Fc domains that are modified to promote heterodimerization, an Fc domain can be modified to allow a purification strategy that enables selections of Fc heterodimers.

[00165] In certain embodiments of the disclosure, the anti-AAV monospecific antibodies or anti-AAV x anti-CACNG1 or anti-TfR bispecific antibodies provided herein are human antibodies. The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and, in particular, CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[00166] The antibodies of the disclosure may, in some embodiments, be recombinant human antibodies. The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created, or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.

[00167] Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via inter- chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.

[00168] The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human lgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using a human lgG1 hinge. The instant disclosure encompasses antibodies having one or more mutations in the hinge, CH2 or CH3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.

[00169] The antibodies of the disclosure may be isolated antibodies. An "isolated antibody," as used herein, means an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody" for purposes of the present disclosure. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.

[00170] The anti-AAV monospecific antibodies or anti-AAV x anti-CACNG1 or anti-AAV x anti-TfR bispecific antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present disclosure includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. Bispecific Antigen-Binding Molecules Comprising Anti-AAV and Anti-CACNG1 or Anti-TfR Antigen-Binding Domains

[00171] The present disclosure provides antigen-binding molecules such as antibodies or fragments thereof, including bispecific antigen-binding molecules such as bispecific antibodies or fragments thereof, that can bind to a capsid of an adeno-associated virus (AAV) particle and/or CACNG1 or TfR. In some embodiments the bispecific antigenbinding molecule is a bispecific antibody.

[00172] The bispecific antibodies provided herein comprise a first antigen-binding domain that binds to a capsid of an AAV particle, and a second antigen-binding domain that binds to CACNG1 or TfR. In some embodiments, the capsid comprises a wild-type AAV capsid protein(s). In some embodiments, the capsid comprises a non-wild-type AAV capsid protein(s).

[00173] In some embodiments, provided herein are anti-AAV/anti-CACNG1 or anti- TfR bispecific antibodies, wherein the second antigen-binding domain binds to CACNG1 or TfR. Such second antigen-binding domains may, therefore, bind to a protein(s) that is expressed on the surface of a cell in vitro or in vivo such that at least a portion of the protein is exposed to the extracellular side of the cell membrane and is accessible to the antigenbinding portion of the antigen-binding domain.

[00174] In some embodiments, a bispecific antibody described herein comprises an antigen-binding domain that binds to AAV, wherein the anti-AAV antigen-binding domain comprises one or more amino acid sequences as follows for REGN 13880 (anti-AAV#70): SEQ ID NO: 52 or 1159 for heavy chain variable region (HCVR); SEQ ID NO: 54 for heavy chain complementarity determining region 1 (HCDR1); SEQ ID NO: 56 for HCDR2; SEQ ID NO: 58 for HCDR3; SEQ ID NO: 10 or 1157 for light chain variable region (LCVR); SEQ ID NO: 12 for light chain complementarity determining region (LCDR1); SEQ ID NO: 14 for LCDR2; SEQ ID NO: 16 for LCDR3; SEQ ID NO: 60 or 1108 for heavy chain (HC); and SEQ ID NO: 20 for light chain (LC). The sequence identifiers of the nucleic acid molecules encoding the HCVRs, LCVRs, HCDR1 , HCDR2, HCDR3, LCDR1, LCDR2 LCDR3, HC and LC of the exemplary anti-AAV antibodies and antigen-binding fragments for REGN 13880 are as follows: SEQ ID NO: 51 or 1109 or 1158 or 1275 for HCVR; SEQ ID NO: 53 for HCDR1 ; SEQ ID NO: 55 for HCDR2; SEQ ID NO: 57 for HCDR3; SEQ ID NO: 9 or 1156 for LCVR; SEQ ID NO: 11 for LCDR1; SEQ ID NO: 13 for LCDR2; SEQ ID NO: 15 for LCDR3; SEQ ID NO: 59 or 1107 for HC; and SEQ ID NO: 19 or 1145 for LC.

REGN 13880 (anti-AAV#70) (NAC67600)

HCVR DNA Sequence CAGGTACACCTGCAGGAGTCGGGCCCAGGACTGGTGATGCCTTCACAGATCCTGTCCC

TCTCCTGCGTTATTTCTGGTGACTCCATCAGAAATGGCGGCTATTATTGGACCTGGACC

CGCCAGCAACCAGGGAAGGGCCTGGAGTGGATCGGTCACATCCACTATAGTGAAAGG

ACCTCCCACAATCCGTCCCTCCAGAGTCGCGTTATTATGTCAATAGACACGTCTGAGAA

TAAGTTCTCCCTGAAACTGACCTCAGTGACTGTCGCGGACACGGCCATATATTATTGTG

CGCGAGGTCGGGACACCATGGTTCGGGGATCCATTACAACTTCCGGCCACTTCATTGA

CTCCTGGGGTCAGGGAGCCCTGGTCACCGTCTCCTCA

(SEQ ID NO: 51), or

CAGGTACACCTCCAGGAGTCGGGCCCAGGACTGGTGATGCCTTCACAGATCCTGTCCC

TCTCCTGCGTTATTTCTGGTGACTCCATCAGAAATGGCGGCTATTATTGGACCTGGACC

CGCCAGCAACCAGGGAAGGGCCTGGAGTGGATCGGTCACATCCACTATAGTGAAAGG

ACCTCCCACAATCCGTCCCTCCAGAGTCGCGTTATTATGTCAATAGACACGTCTGAGAA

TAAGTTCTCCCTGAAACTGACCTCAGTGACTGTCGCGGACACGGCCATATATTATTGTG

CGCGAGGTCGGGACACCATGGTTCGGGGATCCATTACAACTTCCGGCCACTTCATTGA

CTCCTGGGGTCAGGGAGCCCTGGTCACCGTCTCCTCA

(SEQ ID NO: 1109) (silent mutation underlined in sequence above), or

CAGGTACACCTGCAGGAGTCGGGCCCAGGACTGGTGATGCCTTCACAGATCCTGTCCC

TCTCCTGCGTTATTTCTGGTGACTCCATCAGAAATGGCGGCTATTATTGGACCTGGACC

CGCCAGCAACCAGGGAAGTGTCTGGAGTGGATCGGTCACATCCACTATAGTGAAAGGA

CCTCCCACAATCCGTCCCTCCAGAGTCGCGTTATTATGTCAATAGACACGTCTGAGAAT

AAGTTCTCCCTGAAACTGACCTCAGTGACTGTCGCGGACACGGCCATATATTATTGTGC

GCGAGGTCGGGACACCATGGTTCGGGGATCCATTACAACTTCCGGCCACTTCATTGAC

TCCTGGGGTCAGGGAGCCCTGGTCACCGTCTCCTCA

(SEQ ID NO: 1158) (Cys mutation underlined in sequence above), or

CAGGTACACCTCCAGGAGTCGGGCCCAGGACTGGTGATGCCTTCACAGATCCTGTCCC

TCTCCTGCGTTATTTCTGGTGACTCCATCAGAAATGGCGGCTATTATTGGACCTGGACC

CGCCAGCAACCAGGGAAGTGCCTGGAGTGGATCGGTCACATCCACTATAGTGAAAGGA

CCTCCCACAATCCGTCCCTCCAGAGTCGCGTTATTATGTCAATAGACACGTCTGAGAAT

AAGTTCTCCCTGAAACTGACCTCAGTGACTGTCGCGGACACGGCCATATATTATTGTGC

GCGAGGTCGGGACACCATGGTTCGGGGATCCATTACAACTTCCGGCCACTTCATTGAC

TCCTGGGGTCAGGGAGCCCTGGTCACCGTCTCCTCA

(SEQ ID NO: 1275) (2 silent mutations underlined in sequence above)

HCVR Amino Acid Sequence

QVHLQESGPGLVMPSQILSLSCVISGDSIRNGGYYWTWTRQQPGKGLEWIGHIHYSERTSH

NPSLQSRVIMSIDTSENKFSLKLTSVTVADTAIYYCARGRDTMVRGSITTSGHFIDSWGQGA

LVTVSS

(SEQ ID NO: 52), or

QVHLQESGPGLVMPSQILSLSCVISGDSIRNGGYYWTWTRQQPGKCLEWIGHIHYSERTSH

NPSLQSRVIMSIDTSENKFSLKLTSVTVADTAIYYCARGRDTMVRGSITTSGHFIDSWGQGA

LVTVSS

(SEQ ID NO: 1159) (Cys mutation underlined in sequence above) HCDR1 DNA Sequence

GGTGACTCCATCAGAAATGGCGGCTATTAT

(SEQ ID NO: 53)

HCDR1 Amino Acid Sequence

GDSIRNGGYY

(SEQ ID NO: 54)

HCDR2 DNA Sequence

ATCCACTATAGTGAAAGGACC

(SEQ ID NO: 55)

HCDR2 Amino Acid Sequence

IHYSERT

(SEQ ID NO: 56)

HCDR3 DNA Sequence

GCGCGAGGTCGGGACACCATGGTTCGGGGATCCATTACAACTTCCGGCCACTTCATTG

ACTCC

(SEQ ID NO: 57)

HCDR3 Amino Acid Sequence

ARGRDTMVRGSITTSGHFIDS

(SEQ ID NO: 58)

LCVR DNA Sequence

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCA

CCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAA

CCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCC

CGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCT

GCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCA

CCTTCGGCCAAGGGACACGACTGGAGATTAAA

(SEQ ID NO: 9), or

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCA

CCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAA

CCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCC

CGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCT

GCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCA

CCTTCGGCTGTGGGACACGACTGGAGATTAAA

(SEQ ID NO: 1156) (Cys mutation underlined in sequence above)

LCVR Amino Acid Sequence

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF

SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

(SEQ ID NO: 10), or DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF

SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGCGTRLEIK

(SEQ ID NO: 1157) (Cys mutation underlined in sequence above

LCDR1 DNA Sequence

CAGAGCATTAGCAGCTAT

(SEQ ID NO: 11)

LCDR1 Amino Acid Sequence

QSISSY

(SEQ ID NO: 12)

LCDR2 DNA Sequence

GCTGCATCC

(SEQ ID NO: 13)

LCDR2 Amino Acid Sequence

AAS

(SEQ ID NO: 14)

LCDR3 DNA Sequence

CAACAGAGTTACAGTACCCCTCCGATCACC

(SEQ ID NO: 15)

LCDR3 Amino Acid Sequence

QQSYSTPPIT

(SEQ ID NO: 16)

HC DNA Sequence

CAGGTACACCTGCAGGAGTCGGGCCCAGGACTGGTGATGCCTTCACAGATCCTGTCCC

TCTCCTGCGTTATTTCTGGTGACTCCATCAGAAATGGCGGCTATTATTGGACCTGGACC

CGCCAGCAACCAGGGAAGGGCCTGGAGTGGATCGGTCACATCCACTATAGTGAAAGG

ACCTCCCACAATCCGTCCCTCCAGAGTCGCGTTATTATGTCAATAGACACGTCTGAGAA

TAAGTTCTCCCTGAAACTGACCTCAGTGACTGTCGCGGACACGGCCATATATTATTGTG

CGCGAGGTCGGGACACCATGGTTCGGGGATCCATTACAACTTCCGGCCACTTCATTGA

CTCCTGGGGTCAGGGAGCCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATC

GGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGG

CTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGC

CCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC

CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGC

AACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATG

GTCCCCCATGA

(SEQ ID NO: 59), or

CAGGTACACCTCCAGGAGTCGGGCCCAGGACTGGTGATGCCTTCACAGATCCTGTCCC

TCTCCTGCGTTATTTCTGGTGACTCCATCAGAAATGGCGGCTATTATTGGACCTGGACC

CGCCAGCAACCAGGGAAGGGCCTGGAGTGGATCGGTCACATCCACTATAGTGAAAGG

ACCTCCCACAATCCGTCCCTCCAGAGTCGCGTTATTATGTCAATAGACACGTCTGAGAA TAAGTTCTCCCTGAAACTGACCTCAGTGACTGTCGCGGACACGGCCATATATTATTGTG

CGCGAGGTCGGGACACCATGGTTCGGGGATCCATTACAACTTCCGGCCACTTCATTGA

CTCCTGGGGTCAGGGAGCCCTGGTCACCGTCTCCTCAGCCTCAACCAAGGGACCGTC

CGTGTTTCCCCTCGCTCCTTCATCAAAATCAACTTCCGGCGGAACCGCAGCGCTGGGC

TGCCTCGTGAAGGATTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCGGGCGCCC

TGACCTCCGGTGTCCACACGTTCCCCGCGGTCCTTCAGTCCTCCGGCTTGTATTCCCT

GTCGTCCGTCGTGACCGTCCCGAGCAGCAGCCTGGGAACTCAGACCTACATCTGCAAC

GTGAACCACAAGCCGTCGAACACCAAGGTCGATAAGAAAGTGGAGCCGAAGTCGTGC

GACAAAACTCATACA

(SEQ ID NO: 1107) (silent mutation underlined in sequence above)

HC Amino Acid Sequence

QVHLQESGPGLVMPSQILSLSCVISGDSIRNGGYYWTWTRQQPGKGLEWIGHIHYSERTSH

NPSLQSRVIMSIDTSENKFSLKLTSVTVADTAIYYCARGRDTMVRGSITTSGHFIDSWGQGA

LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

VLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP*

(SEQ ID NO: 60), or

QVHLQESGPGLVMPSQILSLSCVISGDSIRNGGYYWTWTRQQPGKGLEWIGHIHYSERTSH

NPSLQSRVIMSIDTSENKFSLKLTSVTVADTAIYYCARGRDTMVRGSITTSGHFIDSWGQGA

LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

VLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

(SEQ ID NO: 1108)

LC DNA Sequence

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCA

CCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAA

CCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCC

CGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCT

GCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCA

CCTTCGGCCAAGGGACACGACTGGAGATTAAACGAACTGTGGCTGCACCATCTGTCTT

CATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGC

TGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCA

ATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAG

CCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC

TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA

GAGTGTTAG

(SEQ ID NO: 19), or

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCA

CCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAA

CCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCC

CGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCT

GCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCA

CCTTCGGCCAAGGGACACGACTGGAGATTAAACGAACTGTGGCTGCACCATCTGTCTT

CATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGC

TGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCA

ATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAG CCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGT

(SEQ ID NO: 1145)

LC Amino Acid Sequence

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO: 20)

[00175] REGN13880. REGN 13880 comprises of anti-AAV#70 Fab. REGN 13880 was purified from the starting material using affinity capture chromatography followed by size exclusion chromatography.

[00176] In some embodiments, a bispecific antibody of the present disclosure may comprise an antigen-binding domain that binds to transferrin receptor (TfR), wherein the anti-TfR antigen-binding domain comprises one or more amino acid sequences as follows for 8D3: SEQ ID NO: 1119 for heavy chain variable region (HCVR); SEQ ID NO: 1121 for HCDR1 ; SEQ ID NO: 1123 for HCDR2; SEQ ID NO: 1125 for HCDR3; SEQ ID NO: 1127 for light chain variable region (LCVR); SEQ ID NO: 1129 for LCDR1 ; SEQ ID NO: 1131 for LCDR2; and SEQ ID NO: 1133 for LCDR3.

[00177] In another aspect, provided herein are nucleic acid molecules encoding the HCVR and LCVR sequences of the anti-AAV/anti-TfR bispecific antigen-binding molecules disclosed herein. In some embodiments, the nucleic acid molecules described herein may comprise the nucleic acid sequence identifiers as follows for 8D3: SEQ ID NO: 1118 for HCVR and SEQ ID NO: 1126 for LCVR.

8D3

HCVR (VH) Nucleotide Sequence

GAAGTACAGCTCGTCGAAAGTGGCGGTGGTCTTGTTCAGCCTGGAAACTCCCTCACCC TCTCATGTGTCGCAAGTGGATTTACTTTCTCAAATTATGGAATGCATTGGATTAGGCAAG CACCAAAAAAAGGACTTGAATGGATAGCTATGATATATTACGACTCCTCTAAAATGAATT ACGCAGATACTGTAAAAGGCAGGTTTACAATTTCTCGGGATAACTCTAAAAACACTCTTT ACCTTGAAATGAACTCTCTTAGATCCGAAGATACAGCTATGTACTATTGCGCAGTGCCA ACTAGTCATTACGTCGTCGATGTTTGGGGACAAGGCGTAAGTGTAACAGTCAGCTCA (SEQ ID NO: 1118)

HCDR1 : GGATTTACTTTCTCAAATTATGGAATGCAT (SEQ ID NO: 1120)

HCDR2:

ATGATATATTACGACTCCTCTAAAATGAATTACGCAGATACTGTAAAAGGC (SEQ ID NO: 1122)

HCDR3: CCAACTAGTCATTACGTCGTCGATGTT (SEQ ID NO: 1124)

HCVR (VH) Amino Acid Sequence

EVQLVESGGGLVQPGNSLTLSCVASGFTFSNYGMHWIRQAPKKGLEWIAMIYYDSSKMNY ADTVKGRFTISRDNSKNTLYLEMNSLRSEDTAMYYCAVPTSHYVVDVWGQGVSVTVSS (SEQ ID NO: 1119)

HCDR1 : GFTFSNYGMH (SEQ ID NO: 1121)

HCDR2: MIYYDSSKMNYADTVKG (SEQ ID NO: 1123)

HCDR3: PTSHYVVDV (SEQ ID NO: 1125)

LCVR (VL) Nucleotide Sequence

GACATTCAGATGACACAATCCCCAGCATCCCTCTCAGCATCACTCGAAGAAATCGTAAC AATCACCTGTCAAGCATCTCAAGATATCGGCAACTGGCTCGCATGGTACCAACAGAAAC CCGGCAAATCCCCTCAACTTCTTATTTATGGCGCAACATCCCTCGCAGACGGCGTTCCC TCAAGGTTCTCAGGCTCACGATCAGGCACTCAGTTCTCACTTAAAATCTCCAGAGTTCA AGTCGAAGATATAGGAATTTATTATTGTCTCCAAGCATATAACACACCATGGACATTCGG CGGCGGCACCAAACTCGAACTTAAA (SEQ ID NO: 1126)

LCDR1 : CAAGCATCTCAAGATATCGGCAACTGGCTCGCA (SEQ I D NO: 1128)

LCDR2: GGCGCAACATCCCTCGCAGAC (SEQ ID NO: 1130)

LCDR3: CAAGCATATAACACACCATGGACAT (SEQ I D NO: 1132)

LCVR (VL) Amino Acid Sequence

DIQMTQSPASLSASLEEIVTITCQASQDIGNWLAWYQQKPGKSPQLLIYGATSLADGVPSRF SGSRSGTQFSLKISRVQVEDIGIYYCLQAYNTPWTFGGGTKLELK (SEQ ID NO: 1127) LCDR1 : QASQDIGNWLA (SEQ ID NO: 1129)

LCDR2: GATSLAD (SEQ ID NO: 1131)

LCDR3: QAYNTPWT (SEQ I D NO: 1133)

[00178] In some embodiments, an anti-TfR scFv of the present disclosure, in VL- (Gly4Ser)3 (SEQ ID NO: 1115)-VH format, comprises the amino acid sequence of SEQ ID NO: 1117:

EVQLVESGGGLVQPGNSLTLSCVASGFTFSNYGMHWIRQAPKKGLEWIAMIYYDSSKMNY ADTVKGRFTISRDNSKNTLYLEMNSLRSEDTAMYYCAVPTSHYVVDVWGQGVSVTVSSGG GGSGGGGSGGGGSDIQMTQSPASLSASLEEIVTITCQASQDIGNWLAWYQQKPGKSPQLL IYGATSLADGVPSRFSGSRSGTQFSLKISRVQVEDIGIYYCLQAYNTPWTFGGGTKLELK

[00179] In some embodiments, a bispecific antibody of the present disclosure may comprise an antigen-binding domain that binds to calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG, wherein the anti-CACNG1 antigen-binding domain comprises one or more amino acid sequences as follows for REGN10717: SEQ ID NO: 795 for the heavy chain variable region (HCVR); SEQ ID NO: 797 for HCDR1 ; SEQ ID NO: 799 for HCDR2; SEQ ID NO: 801 for HCDR3; SEQ ID NO: 803 for the light chain variable region (LCVR); SEQ ID NO: 805 for LCDR1; SEQ ID NO: 807 for LCDR2; SEQ ID NO: 809 for LCDR3; SEQ ID NO: 811 or 1173 for HC; and SEQ ID NO: 813 for LC.

[00180] In another aspect, provided herein are nucleic acid molecules encoding the CDR, HCVR, LCVR, HC, or LC sequences of the anti-AAV/anti-CACNG1 bispecific antigenbinding molecules disclosed herein, or variants thereof, including nucleic acid molecules comprising the polynucleotide sequences of nucleic acid sequence identifiers as follows for REGN10717: SEQ ID NO: 794 for HCVR; SEQ ID NO: 796 for HCDR1 ; SEQ ID NO: 798 for HCDR2; SEQ ID NO: 800 for HCDR3; SEQ ID NO: 802 for LCVR; SEQ ID NQ:804 for LCDR1 ; SEQ ID NO: 806 for LCDR2; SEQ ID NO: 808 for LCDR3; SEQ ID NO: 810 or 1172 for HC; and SEQ ID NO: 812 for LC.

REGN10717

HCVR DNA Sequence

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG ACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACATATGGCATGCACTGGGTCCGC CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATTTGGCATGATGGAAGTGAT AAATATTATGTAGACTCCGTGAAGGGCCGATTCTCCATCGCCAGAGACAATTCCAAGAA CACGCTTTATCTGCAAATGAATAGTCTGAGAGTCGAGGACACGGGTATATATTACTGTG CGAGAAGGGGTATACGTGGAACCGTTTTTGACCACTGGGGCCTGGGAACCCTGGTCAC CGTCTCCTCA (SEQ ID NO: 794)

HCVR Amino Acid Sequence

QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWHDGSDK YYVDSVKGRFSIARDNSKNTLYLQMNSLRVEDTGIYYCARRGIRGTVFDHWGLGTLVTVSS (SEQ ID NO: 795)

HCDR1 DNA Sequence

GGATTCACCTTCAGTACATATGGC (SEQ ID NO: 796)

HCDR1 Amino Acid Sequence

GFTFSTYG (SEQ ID NO: 797) HCDR2 DNA Sequence

ATTTGGCATGATGGAAGTGATAAA (SEQ ID NO: 798)

HCDR2 Amino Acid Sequence

IWHDGSDK (SEQ ID NO: 799)

HCDR3 DNA Sequence

GCGAGAAGGGGTATACGTGGAACCGTTTTTGACCAC (SEQ ID NO: 800)

HCDR3 Amino Acid Sequence

ARRGIRGTVFDH (SEQ ID NO: 801)

LCVR DNA Sequence

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCA

CCCTCACTTGTCGGGCCAGTCAGAGTATTAGTAACAAGTTGGCCTGGTATCAGCAGAAA

CCAGGGAAAGCCCCTAACCTCCTGATCTATAAGGCGTCTAATTTAGAAAGTGGGGTCC

CATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCT

GCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCGTGGACGTT

CGGCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO: 802)

LCVR Amino Acid Sequence

DIQMTQSPSTLSASVGDRVTLTCRASQSISNKLAWYQQKPGKAPNLLIYKASNLESGVPSR

FSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIK (SEQ ID NO: 803)

LCDR1 DNA Sequence

CAGAGTATTAGTAACAAG (SEQ ID NO: 804)

LCDR1 Amino Acid Sequence

QSISNK (SEQ ID NO: 805)

LCDR2 DNA Sequence

AAGGCGTCT (SEQ ID NO: 806)

LCDR2 Amino Acid Sequence

KAS (SEQ ID NO: 807)

LCDR3 DNA Sequence CAACAGTATAATAGTTATTCGTGGACG (SEQ ID NO: 808)

LCDR3 Amino Acid Sequence

QQYNSYSWT (SEQ ID NO: 809)

LC Constant Light (CL) Domain DNA Sequence

CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATC

TGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC

AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCA

GGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGA

CTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCC

GTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 1170)

LC Constant Light (CL) Domain Amino Acid Sequence

RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD

SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1147)

HC DNA Sequence

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACATATGGCATGCACTGGGTCCGC

CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATTTGGCATGATGGAAGTGAT

AAATATTATGTAGACTCCGTGAAGGGCCGATTCTCCATCGCCAGAGACAATTCCAAGAA

CACGCTTTATCTGCAAATGAATAGTCTGAGAGTCGAGGACACGGGTATATATTACTGTG

CGAGAAGGGGTATACGTGGAACCGTTTTTGACCACTGGGGCCTGGGAACCCTGGTCAC

CGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAG

GAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGA

ACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC

GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC

AGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCA

AGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACC

TGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTC

ATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGAC

CCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAA

AGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCC

TGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCT

CCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAG

GTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT GCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT CCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTC ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGT

CTCTGGGTAAATGA (SEQ ID NO: 810), or

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACATATGGCATGCACTGGGTCCGC

CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATTTGGCATGATGGAAGTGAT

AAATATTATGTAGACTCCGTGAAGGGCCGATTCTCCATCGCCAGAGACAATTCCAAGAA

CACGCTTTATCTGCAAATGAATAGTCTGAGAGTCGAGGACACGGGTATATATTACTGTG CGAGAAGGGGTATACGTGGAACCGTTTTTGACCACTGGGGCCTGGGAACCCTGGTCAC

CGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG

AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA

CCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCG

GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA

GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAA

GGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGC CCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG

ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA

CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC

AAGACAAAGCCGCGGGAGGAGCAGTACGGCAGCACGTACCGTGTGGTCAGCGTCCTC

ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA

AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA

ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG

CCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG

CAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGG CTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC

GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGTCCCT

CTCCCTGTCTCCGGGTAAA (SEQ ID NO: 1172)

HC Amino Acid Sequence

QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWHDGSDK

YYVDSVKGRFSIARDNSKNTLYLQMNSLRVEDTGIYYCARRGIRGTVFDHWGLGTLVTVSS

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL

FPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF

SCSVMHEALHNHYTQKSLSLSLGK* (SEQ ID NO: 811), or

QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWHDGSDK

YYVDSVKGRFSIARDNSKNTLYLQMNSLRVEDTGIYYCARRGIRGTVFDHWGLGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS

VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTY RWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1173)

LC DNA Sequence

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCA

CCCTCACTTGTCGGGCCAGTCAGAGTATTAGTAACAAGTTGGCCTGGTATCAGCAGAAA

CCAGGGAAAGCCCCTAACCTCCTGATCTATAAGGCGTCTAATTTAGAAAGTGGGGTCC CATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCT GCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCGTGGACGTT

CGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATC

TTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAA

TAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCG

AAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAG (SEQ ID NO: 812)

LC Amino Acid Sequence

DIQMTQSPSTLSASVGDRVTLTCRASQSISNKLAWYQQKPGKAPNLLIYKASNLESGVPSR FSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK

ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 813)

The heavy and light chains of anti-CACNG1 Fabs in anti-AAV/anti-CACNG1 bispecific antibodies of the present disclosure are set forth below.

(1) REGN10717

HC Fab (VH + lgG1 CH1) DNA Sequence

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG ACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACATATGGCATGCACTGGGTCCGC CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATTTGGCATGATGGAAGTGAT AAATATTATGTAGACTCCGTGAAGGGCCGATTCTCCATCGCCAGAGACAATTCCAAGAA

CACGCTTTATCTGCAAATGAATAGTCTGAGAGTCGAGGACACGGGTATATATTACTGTG CGAGAAGGGGTATACGTGGAACCGTTTTTGACCACTGGGGCCTGGGAACCCTGGTCAC CGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG

AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA

CCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCG

GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAA GGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACA (SEQ ID NO: 1174)

(2) REGN10717

HC Fab (VH + IgG 1 CH1) Amino Acid Sequence

QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWHDGSDK

YYVDSVKGRFSIARDNSKNTLYLQMNSLRVEDTGIYYCARRGIRGTVFDHWGLGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 1175)

(3) REGN10717

LC DNA Sequence

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCA

CCCTCACTTGTCGGGCCAGTCAGAGTATTAGTAACAAGTTGGCCTGGTATCAGCAGAAA

CCAGGGAAAGCCCCTAACCTCCTGATCTATAAGGCGTCTAATTTAGAAAGTGGGGTCC

CATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCT GCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCGTGGACGTT CGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATC

TTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAA

TAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG

GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCG AAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT

GTTAG (SEQ ID NO: 812)

(4) REGN10717

LC Fab Amino Acid Sequence

DIQMTQSPSTLSASVGDRVTLTCRASQSISNKLAWYQQKPGKAPNLLIYKASNLESGVPSR FSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK

ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 813) Non-limiting Examples of Alternative Format Antibodies

[00181] In general, the present disclosure provides bispecific antibodies, which can bind both to the capsid of an AAV particle and to transferrin receptor (TfR) or calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1), and methods of use thereof. By binding to both a capsid of an AAV and transferrin receptor (TfR) or calcium voltagegated channel auxiliary subunit gamma 1 (CACNG1), the antibodies described herein can bridge the virus to target cells that express TfR or CACNG1 , thereby redirecting virus delivery and transduction.

[00182] In one aspect, provided herein is a bispecific antibody, or a bispecific antigenbinding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) an scFv comprising a first antigen-binding domain (“ABD1”) operably linked to (ii) a first heavy chain region of a first Fab (“Fab1”), operably linked to (iii) an Fc domain; b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a second antigen-binding domain (“ABD2”); and d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a third antigen-binding domain (“ABD3”); wherein ABD2 and ABD3 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD2 and ABD3 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD1 binds to transferrin receptor (TfR) and comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

[00183] In one embodiment, the scFv is linked to the first heavy chain region via a linker.

[00184] In another aspect, provided herein is a bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an scFv comprising a first antigen-binding domain (“ABD1”), operably linked to (iii) an Fc domain; b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a second antigen-binding domain (“ABD2”); and d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a third antigen-binding domain (“ABD3”); wherein ABD2 and ABD3 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD2 and ABD3 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD1 binds to transferrin receptor (TfR) and comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

[00185] In one embodiment, the scFv is linked to the first heavy chain region via a linker.

[00186] In a further aspect, provided herein is a bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an Fc domain, operably linked to (iii) an scFv comprising a first antigen-binding domain (“ABD1”); b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a second antigen-binding domain (“ABD2”); and d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a third antigen-binding domain (“ABD3”); wherein ABD2 and ABD3 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD2 and ABD3 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD1 binds to transferrin receptor (TfR) and comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127. [00187] In one embodiment, the scFv is linked to the Fc domain via a linker.

[00188] In a further aspect, provided herein is a bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an Fc domain, operably linked to (iii) a second heavy chain region of a second Fab (“Fab2”); b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) an scFv comprising a first antigen-binding domain (“ABD1”) operably linked to (ii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a second antigen-binding domain (“ABD2”); d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a third antigen-binding domain (“ABD3”); wherein ABD2 and ABD3 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD2 and ABD3 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD1 binds to transferrin receptor (TfR) and comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

[00189] In one embodiment, Fab2 is linked to the Fc domain via a linker.

[00190] In another aspect, provided herein is a bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an Fc domain, operably linked to (iii) a first scFv comprising a first antigen-binding domain (“ABD1”); b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain, operably linked to (iii) a second scFv comprising a second antigen-binding domain (“ABD2”); c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a third antigen-binding domain (“ABD3”); d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a fourth antigen-binding domain (“ABD4”); wherein ABD3 and ABD4 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD3 and ABD4 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein the ABD1 and ABD2 bind to transferrin receptor (TfR), and each of ABD1 and ABD2 comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

[00191] In one embodiment, the first scFv and/or second scFv is linked to the Fc domain via a linker.

[00192] In a further embodiment, the ABD that binds to a capsid of an AAV particle comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 58; and/or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 16.

[00193] In some embodiments, the ABD that binds to a capsid of an AAV particle comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157.

[00194] In a further embodiment, the ABD that binds to TfR comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1121 , a HCDR2 comprising the amino acid sequence of SEQ ID NO: 1123, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 1125; and/or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 1129, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 1131 , and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 1133.

[00195] In some embodiments, the ABD that binds to TfR comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

[00196] In a further aspect, provided herein is a bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first scFv comprising a first antigen-binding domain (“ABD1”) operably linked to (ii) a first heavy chain region of a first Fab (“Fab1”), operably linked to (iii) an Fc domain; b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second scFv comprising a second antigen-binding domain (“ABD2”) operably linked to (ii) a second heavy chain region of a second Fab (“Fab2”), operably linked to (iii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a third antigen-binding domain (“ABD3”); d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a fourth antigen-binding domain (“ABD4”); wherein ABD1 and ABD2 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD1 and ABD2 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD3 and ABD4 bind to calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1), and each of ABD3 and ABD4 comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 795, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 803.

[00197] In one embodiment, the first scFv is linked to Fab1 and/or the second scFv is linked to Fab2 via a linker.

[00198] In another aspect, provided herein is a bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an Fc domain, operably linked to (iii) a first scFv comprising a first antigen-binding domain (“ABD1”); b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain, operably linked to (iii) a second scFv comprising a second antigen-binding domain (“ABD2”); c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a third antigen-binding domain (“ABD3”); d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a fourth antigen-binding domain (“ABD4”); wherein ABD1 and ABD2 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD1 and ABD2 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD3 and ABD4 bind to calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1), and each of ABD3 and ABD4 comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 795, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 803.

[00199] In one embodiment, the first scFv and/or second scFv is linked to the Fc domain via a linker.

[00200] In a further embodiment, each of ABD1 and ABD2 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 58; and/or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 16.

[00201] In some embodiments, each of ABD1 and ABD2 comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157.

[00202] In some embodiments, each of ABD3 and ABD4 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 797, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 799, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 801 ; and/or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 805, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 807, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 809.

[00203] In a further embodiment, each of ABD3 and ABD4 comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 795, and/or a LCVR comprising the amino acid sequence of SEQ ID NO: 803.

[00204] In one embodiment, the linker is or comprises a 3xG4S (SEQ ID NO: 1115).

[00205] In some embodiments, the bispecific antibody or bispecific antigen-binding fragment comprises an Fc heterodimer.

[00206] In one embodiment, the Fc domains in the Fc heterodimer comprise knob-in- hole mutations as compared to a wild type Fc domain.

[00207] In some embodiments, one Fc domain in the Fc heterodimer comprises amino acid substitutions S354C and T366W (according to EU numbering) as compared to a wild type Fc domain, and the other Fc domain in the Fc heterodimer comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (according to EU numbering) as compared to a wild type Fc domain.

[00208] In a further embodiment, at least one Fc domain in the Fc heterodimer comprises a star mutation as compared to a wild type Fc domain.

[00209] In some embodiments, one Fc domain in the Fc heterodimer comprises amino acid mutations H435R and/or Y436F (according to EU numbering) as compared to a wild type Fc domain. [00210] In one embodiment, the capsid comprises one or more wild-type AAV capsid polypeptides.

[00211] In some embodiments, the capsid comprises one or more non-wild-type AAV capsid polypeptides.

[00212] In one aspect, provided herein is a pharmaceutical composition comprising the bispecific antibody or bispecific antigen-binding fragment and a pharmaceutically acceptable carrier or excipient.

[00213] In a further aspect, provided herein is a molecular complex comprising an AAV particle bound to one or more bispecific antibodies and/or bispecific antigen-binding fragments. [00214] In one embodiment, the AAV particle comprises one or more mutations in one or more AAV capsid proteins inhibiting the natural tropism of the AAV particle.

[00215] In a further aspect, provided herein is a pharmaceutical composition comprising the molecular complex described herein and a pharmaceutically acceptable carrier or excipient.

[00216] In another aspect, provided herein is a method of preparing the molecular complex comprising incubating the AAV particle in the presence of one or more bispecific antibodies and/or bispecific antigen-binding fragments under conditions allowing specific binding of the one or more bispecific antibodies and/or bispecific antigen-binding fragments to the AAV particle capsid.

[00217] In a further aspect, provided herein is a method for targeting an AAV particle to a cell expressing transferrin receptor (TfR) on the cell surface comprising contacting the cell with the molecular complex or the pharmaceutical composition described herein, wherein the molecular complex comprises one or more bispecific antibodies and/or bispecific antigenbinding fragments which bind to TfR.

[00218] In another aspect, provided herein is a method for targeting an AAV particle to a cell expressing calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1) on the cell surface comprising contacting the cell with the molecular complex or the pharmaceutical composition described herein, wherein the molecular complex comprises one or more bispecific antibodies and/or bispecific antigen-binding fragments which bind to CACNG1.

[00219] In another aspect, provided herein is a method for delivering a polynucleotide to a cell expressing transferrin receptor (TfR) on the cell surface comprising contacting the cell with the molecular complex or the pharmaceutical composition described herein, wherein the molecular complex comprises the AAV particle comprising the polynucleotide and bound to one or more bispecific antibodies and/or bispecific antigen-binding fragments which bind to TfR.

[00220] In another aspect, provided herein is a method for delivering a polynucleotide to a cell expressing calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1) on the cell surface comprising contacting the cell with the molecular complex or the pharmaceutical composition described herein, wherein the molecular complex comprises the AAV particle comprising the polynucleotide and bound to one or more bispecific antibodies and/or bispecific antigen-binding fragments which bind to CACNG1.

[00221] In one embodiment, the cell is in a subject and the molecular complex is administered to the subject.

[00222] In a further embodiment, the AAV particle does not target the cell in the absence of the one or more bispecific antibodies and/or bispecific antigen-binding fragments. [00223] In various embodiments, any of the features or components of embodiments discussed above or herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value discussed above or herein may be combined with another related value discussed above or herein to recite a range with the values representing the upper and lower ends of the range, and such ranges are encompassed within the scope of the present disclosure.

[00224] Other embodiments will become apparent from a review of the ensuing detailed description.

Epitope Mapping and Related Technologies

[00225] The epitope on a capsid of an AAV and/or CACNG1 or TfR to which the antigenbinding molecules of the present disclosure bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of an AAV capsid and/or CACNG1 or TfR protein. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences). [00226] The term "epitope," as used herein, can refer to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[00227] Various techniques known to persons of ordinary skill in the art can be used to determine whether an antigen-binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, e.g., cryogenic electron microscopy (cryo-EM), routine cross-blocking assay such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutational analysis, peptide blots analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding domain of an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except for the residues protected by the antibody (which remain deuterium- labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73.25QA-2Q5A. X-ray crystallography of the antigen/antibody complex may also be used for epitope mapping purposes.

[00228] The provided herein are bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds to a capsid of an AAV, and a second antigenbinding domain that specifically binds to CACNG1 or TfR, wherein the first antigen-binding domain competes for binding to a capsid of an AAV with any of the specific exemplary AAV- specific antigen-binding domains described herein, and/or wherein the second antigenbinding domain competes for binding to CACNG1 or TfR.

[00229] One can easily determine whether a particular antigen-binding molecule (e.g., antibody), or antigen-binding domain thereof, binds to the same epitope as, or competes for binding with, a reference antigen-binding molecule of the present disclosure by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope on an AAV capsid (or CACNG1 or TfR) protein as a reference bispecific antigenbinding molecule of the present disclosure, the reference bispecific molecule is first allowed to bind to the AAV capsid (or CACNG1 or TfR) protein. Next, the ability of a test antibody to bind to the AAV capsid (or CACNG1 or TfR) protein is assessed. If the test antibody is able to bind to the AAV capsid (or CACNG1 or TfR) protein following saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different epitope of the AAV capsid protein (or CACNG1 or TfR) than the reference bispecific antigen-binding molecule. On the other hand, if the test antibody is not able to bind to the AAV capsid protein (or CACNG1 or TfR) following saturation binding with the reference bispecific antigen-binding molecule, then the test antibody may bind to the same epitope of the AAV capsid protein as the epitope bound by the reference bispecific antigen-binding molecule of the disclosure. Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference bispecific antigen-binding molecule or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antibodybinding assay available in the art. In accordance with certain embodiments of the present disclosure, two antigen-binding proteins bind to the same (or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antigen-binding protein inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antigen-binding proteins are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other. Two antigen-binding proteins are deemed to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other.

[00230] To determine if an antibody or antigen-binding domain thereof competes for binding with a reference antigen-binding molecule, the above-described binding methodology is performed in two orientations: As an example, in a first orientation, the reference antigenbinding molecule is allowed to bind to an AAV capsid (or CACNG1 or TfR) protein under saturating conditions followed by assessment of binding of the test antibody to AAV capsid (or CACNG1 or TfR) protein. In a second orientation, the test antibody is allowed to bind to a AAV capsid (or CACNG1 or TfR) protein under saturating conditions followed by assessment of binding of the reference antigen-binding molecule to the AAV capsid (or CACNG1 or TfR) protein. If, in both orientations, only the first (saturating) antigen-binding molecule is capable of binding to the AAV capsid (or CACNG1 or TfR) protein, then it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to the AAV capsid (or CACNG1 or TfR) protein. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antigen-binding molecule may not necessarily bind to the same epitope as the reference antibody but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

Preparation of Antigen-Binding Domains and Construction of Bispecific Molecules [00231] Antigen-binding domains specific for particular antigens can be prepared by any antibody generating technology known in the art. Once obtained, two different antigenbinding domains, specific for two different antigens, can be appropriately arranged relative to one another to produce a bispecific antigen-binding molecule of the present disclosure using routine methods. (A discussion of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules of the present disclosure is provided elsewhere herein). In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the bispecific antigen-binding molecules of the disclosure are derived from chimeric, humanized or fully human antibodies. Methods for making such antibodies are well known in the art. For example, one or more of the heavy and/or light chains of the bispecific antigen-binding molecules of the present disclosure can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generating technology), high affinity chimeric antibodies to a particular antigen are initially isolated having a human variable region and a mouse constant region. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate fully human heavy and/or light chains that can be incorporated into the bispecific antigen-binding molecules of the present disclosure.

[00232] Genetically engineered animals may be used to make human bispecific antigenbinding molecules. For example, a genetically modified mouse can be used which is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa constant gene at the endogenous mouse kappa locus. Such genetically modified mice can be used to produce fully human bispecific antigen-binding molecules comprising two different heavy chains that associate with an identical light chain that comprises a variable domain derived from one of two different human light chain variable region gene segments. (See, e.g., US 2011/0195454). Fully human can refer to an antibody, or antigen-binding fragment or immunoglobulin domain thereof, comprising an amino acid sequence encoded by a DNA derived from a human sequence over the entire length of each polypeptide of the antibody or antigen-binding fragment or immunoglobulin domain thereof. In some instances, the fully human sequence is derived from a protein endogenous to a human. In other instances, the fully human protein or protein sequence comprises a chimeric sequence wherein each component sequence is derived from human sequence. While not being bound by any one theory, chimeric proteins or chimeric sequences are generally designed to minimize the creation of immunogenic epitopes in the junctions of component sequences, e.g. compared to any wild-type human immunoglobulin regions or domains.

Cells

[00233] The disclosure also provides host cells comprising a nucleic acid of the disclosure.

[00234] In one embodiment, the host cells are genetically engineered to comprise one or more nucleic acids described herein.

[00235] In one embodiment, the host cells are genetically engineered by using an expression cassette. The phrase “expression cassette,” refers to nucleotide sequences, which are capable of affecting expression of a gene in hosts compatible with such sequences. Such cassettes may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in effecting expression may also be used, such as, for example, an inducible promoter.

[00236] The disclosure also provides host cells comprising the vectors described herein. [00237] The cell can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

Therapeutic Formulation and Administration

[00238] The present disclosure provides a pharmaceutical composition comprising an antigen-binding molecule (e.g., anti-AAV monospecific antibody or anti-AAV x anti-CACNG1 or anti-TfR bispecific antibody) described herein and/or at least one viral capsid protein (e.g., a capsid protein of an AAV particle) described herein. In some embodiments, pharmaceutical compositions comprising a viral capsid protein, e.g., an AAV particle, may be useful as a gene transfer vector.

[00239] The pharmaceutical compositions of the disclosure are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Phdomain Sci Technol 52:238-311.

[00240] The dose of antigen-binding molecule administered to a patient may vary depending upon the age and the size of the patient, target disease, conditions, route of administration, and the like. The preferred dose is typically calculated according to body weight or body surface area. When a bispecific antigen-binding molecule of the present disclosure is used for therapeutic purposes in an adult patient, it may be advantageous to intravenously administer the bispecific antigen-binding molecule of the present disclosure normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering a bispecific antigenbinding molecule may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991 , Phdomainaceut. Res. 8:1351).

[00241] Dose ranges and frequency of administration of a viral vector described herein can vary depending on the nature of, e.g., the AAV, and the medical condition, as well as parameters of a specific patient and the route of administration used. In some embodiments, viral vector compositions can be administered to a subject at a dose ranging from about 1 xio⁵ plaque forming units (pfu) to about 1 xio¹⁵ pfu, depending on mode of administration, the route of administration, the nature of the disease and condition of the subject. In some cases, the viral vector compositions can be administered at a dose ranging from about 1 xio⁸ pfu to about 1 x 1 o¹⁵ pfu, or from about 1 x 1 o¹⁰ pfu to about 1 x 1 o¹⁵ pfu, or from about 1 x 1 o⁸ pfu to about 1 X 10¹² pfu. A more accurate dose can also depend on the subject in which it is being administered. For example, a lower dose may be required if the subject is juvenile, and a higher dose may be required if the subject is an adult human subject. In certain embodiments, a more accurate dose can depend on the weight of the subject. In certain embodiments, for example, a juvenile human subject can receive from about 1 xio⁸ pfu to about 1 X 10¹° pfu, while an adult human subject can receive a dose from about 1 xio¹⁰ pfu to about 1 X 10¹² pfu.

[00242] Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

[00243] A pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[00244] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present disclosure. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present disclosure include, but are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name only a few.

[00245] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[00246] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[00247] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.

Uses of the Antigen-Binding Molecules

[00248] The present disclosure includes methods of using the any of antigen-binding molecules as disclosed herein or a pharmaceutically acceptable carrier or diluent.

[00249] In one aspect, provided herein is a method for reducing off-target effects of an AAV particle in vivo, comprising administering said AAV particle in a molecular complex comprising said AAV particle bound to one or more antibodies and/or antigen-binding fragments described herein or one or more bispecific antibodies and/or bispecific antigenbinding fragments described herein.

[00250] In another aspect, provided herein is a method for enhancing target specificity of an AAV particle in vivo, comprising administering said AAV particle in a molecular complex comprising said AAV particle bound to one or more antibodies and/or antigen-binding fragments described herein or one or more bispecific antibodies and/or bispecific antigenbinding fragments described herein.

[00251] In another aspect, provided herein is a method for inhibiting an infection or transduction of a cell mediated by an AAV particle comprising contacting said AAV particle with said cell in the presence of the antibody or antigen-binding fragment described herein. In some embodiments, the cell is in a subject and the antibody or antigen-binding fragment is administered to said subject.

[00252] In various aspects, the methods comprise administering to a subject in need thereof a therapeutic composition comprising any of the antibodies or antigen-binding molecules as disclosed herein and a pharmaceutically acceptable carrier or diluent. As used herein, the expression "a subject in need thereof" means a human or non-human animal that exhibits one or more symptoms or indicia of disease, disorder, condition and/or symptom which would benefit from administration of the antibodies or antigen-molecules described herein.

Administration Regimens

[00253] According to certain embodiments of the present disclosure, an antigen-binding molecule described herein may be administered to a subject separately from an AAV particle, or in a pre-complexed form with an AAV particle. In some embodiments, an antigenbinding molecule and an AAV particle may be administered as a molecular complex described herein.

[00254] In some embodiments, when an antigen-binding molecule and an AAV particle are administered separately, the antigen-binding molecule may be administered at the same time as the AAV particle.

[00255] In some embodiments, an antigen-binding molecule and an AAV particle may be administered separately over a defined time course. In certain embodiments, multiple doses of an antigen-binding molecule and/or an AAV particle described herein, may be administered to a subject over a defined time course. The methods according to such aspects of the disclosure may comprise sequentially administering to a subject multiple doses of an antigen-binding molecule and/or an AAV particle of the disclosure. As used herein, "sequentially administering" means that each dose of an antigen-binding molecule and/or AAV is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present disclosure includes methods which comprise sequentially administering to the patient a single initial dose of an antigen-binding molecule and/or AAV, followed by one or more secondary doses of the antigen-binding molecule and/or AAV, and optionally followed by one or more tertiary doses of the antigen-binding molecule and/or AAV.

[00256] The terms "initial dose," "secondary doses," and "tertiary doses," can refer to the temporal sequence of administration of the antigen-binding molecule and/or AAV of the disclosure. Thus, the "initial dose" is the dose which is administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "secondary doses" are the doses which are administered after the initial dose; and the "tertiary doses" are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of the antigen-binding molecule and/or AAV, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of an antigen-binding molecule and/or AAV contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses").

[00257] In one exemplary embodiment of the present disclosure, each secondary and/or tertiary dose is administered 1 to 26 (e.g., 1 , 114, 2, 214, 3, 314, 4, 414, 5, 514, 6, 614, 7, 714, 8, 8¹/₂, 9, 9¹/₂, 10, 10¹/₂, 11 , 1134, 12, 12¹/₂, 13, 13¹/₂, 14, 14¹/₂, 15, 15¹/₂, 16, 16¹/₂, 17, 17¹/₂, 18, 1814, 19, 1914, 20, 2014, 21 , 21¹/₂, 22, 22¹/₂, 23, 23¹/₂, 24, 24¹/₂, 25, 25¹/₂, 26, 26¹/₂, or more) weeks after the immediately preceding dose. The phrase "the immediately preceding dose," as used herein, means, in a sequence of multiple administrations, the dose of antigenbinding molecule and/or AAV which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

[00258] The methods according to this aspect of the disclosure may comprise administering to a patient any number of secondary and/or tertiary doses of an antigen-binding molecule and/or AAV described herein. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[00259] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination. [00260] In some embodiments, when an antigen-binding molecule and an AAV particle described herein are sequentially administered, the AAV particle may be administered as a first component of the dosing regimen and the antigen-binding molecule may be administered as a second component of the dosing regimen (i.e. , the AAV particle may be administered before the antigen-binding molecule). In some embodiments, the AAV particle may be administered as a second component of the dosing regimen and the antigen-binding molecule may be administered as a first component of a dosing regimen (i.e. , the AAV particle may be administered after the antigen-binding molecule). In some embodiments, an AAV particle and/or antigen-binding molecule may be sequentially administered, in either of the above-described orders, with variable time intervals between administration. For example, the time interval between administration of the AAV particle and the antigen binding molecule may be at least about 30 seconds, at least about 35 seconds, at least about 40 seconds, at least about 45 seconds, at least about 50 seconds, at least about 55 seconds, at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 10 to 12 hours, at least about 12 to 14 hours, at least about 14 to 16 hours, at least about 16 to 18 hours, at least about 18 to 20 hours, at least about 20 to 22 hours, at least about 22 to 24 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 10 to 12 days, at least about 12 to 14 days, at least about 14 to 16 days, at least about 16 to 18 days, at least about 18 to 20 days, at least about 20 to 22 days, at least about 22 to 24 days, at least about 24 to 26 days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 31 days, at least about 1 month, or more.

Expression Methods

[00261] Provided herein are recombinant methods for making an antigen-binding molecule of the present disclosure, or an immunoglobulin chain thereof, comprising (i) introducing, into a host cell, one or more polynucleotides encoding light and/or heavy immunoglobulin chains of such an antigen-binding molecule, for example, wherein the polynucleotide is in a vector; and/or integrates into the host cell chromosome and/or is operably linked to a promoter; (ii) culturing the host cell (e.g., mammalian, fungal, Chinese hamster ovary (CHO), Pichia or Pichia pastoris) under conditions favorable to expression of the polynucleotide and, (iii) optionally, isolating the bispecific antigen-binding molecule or immunoglobulin chain from the host cell and/or medium in which the host cell is grown. The product of such a method also forms part of the present disclosure along with a pharmaceutical composition thereof.

[00262] In an embodiment, a method for making an antigen-binding molecule includes a method of purifying the molecule, e.g., by column chromatography, precipitation and/or filtration. The product of such a method also forms part of the present disclosure along with a pharmaceutical composition thereof.

[00263] Host cells comprising an antigen-binding molecule of the present disclosure and/or a polynucleotide encoding immunoglobulin chains of such a molecule (e.g., in a vector) are also part of the present disclosure. Host cells include, for example, mammalian cells such as Chinese hamster ovary (CHO) cells and fungal cells such as Pichia cells (e.g., P. pastoris).

EXAMPLES

[00264] The present disclosure is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the disclosure or of any exemplified term. Likewise, the disclosure is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the disclosure may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the disclosure in spirit or in scope. The disclosure is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.

Example 1. Transduction assay to assess the efficacy of AAV x CACNG1 bispecific antibodies

[00265] Adeno-associated viruses (AAV) are members of the Parvovirus family of nonenveloped single-stranded DNA viruses. The AAV capsid structure comprises three viral proteins, VP1, VP2, and VP3, that are encoded from a single open reading frame. An icosahedral capsid is formed from the assembly of 60 monomers of VP1, VP2, and VP3 in relative amounts of 1:1 :10. The AAV capsid contains residues critical for mediating cell and receptor binding. The capsids of many serotypes bind to the adeno-associated virus receptor (AAVR), which is required for efficient transport of particles to the trans-golgi network, an essential step of the viral transduction pathway. Antibodies that bind to regions of the capsid attributed to either glycan or AAVR binding have been shown to neutralize AAV transduction. It has also been shown that antibodies can be used to bridge AAV capsids to target cells to enhance transduction efficiency. Bispecific antibodies to AAV have been generated and the ability of these to enhance or neutralize transduction was explored.

[00266] AAV x CACNG1 bispecific antibodies of the present disclosure (AF70: anti-AAV#70 scFv fused to N-terminal of anti-CACNG1 REGN10717 hlgG1 N297G antibody; AF71: anti- AAV#70 scFv fused to C-terminal of anti-CACNG1 REGN10717 hlgG1 N297G antibody;

Figure 1, Figures 22A-22G, and Table 3) were assessed for their capacity to enhance internalization of AAV Hu37 particles expressing green fluorescent protein (GFP) genome into mouse CACNG1 -expressing HEK 293 cells and human CACNG1 -expressing HEK 293 cells. The day before transduction, both cell lines were seeded in a clear wall clear bottom 96-well plate at 10,000 cells per well using DMEM (supplemented with 10% FBS, MEM NEAA, Pen Strep) and incubated at 37°C and 5% CO2. The day of transduction, AF70 and AF71 antibodies were first diluted in 1xPBS and then serially diluted (3-fold) in 1xPBS to obtain the appropriate viral genome to antibody molar ratios (1 :1, 1:3, 1:9, 1:27, and 1:81). AAV Hu37 and control virus (1/8 Spytagged AAV9 W503A conjugated with anti-CACNG1 bivalent antibody) were diluted in 1xPBS + 0.001% Pluronic. Equal volumes of AAV Hu37 and antibody dilutions were combined to obtain the above viral genome to antibody molar ratios and incubated for 1 hour at 37°C and 5% CO2. After a 1-hour incubation, the appropriate volumes of the viral genome to antibody complexes were added to the cells to yield an MOI of 2E+04 and 2E+05 VG per cell. 60-hours post transduction, the transduced cells were analyzed by flow cytometry. To prepare samples for flow cytometry, the supplemented media was discarded, and the cells were washed once with 100 pL 1xPBS. Afterwards, the cells were lifted by adding 50 pL of TrypLE™ Select Enzyme (1X), no phenol red, and incubated for 4-5 minutes in 37°C. 100 pL of BD Pharmingen™ Stain Buffer (BSA) was then added and cells resuspended and then transferred to a clear V-bottom 96 well plate. The cells were spun down for 2 minutes at 800xg and the supernatant was discarded. Two extra rounds of washes were performed by adding 100 pL of BSA Stain Buffer and spun down for 2 minutes at 800xg. After the last wash, 100 pL of BSA Stain Buffer was added and cells were resuspended in preparation for Flow Cytometry analysis. GFP expression was measured using a ZE5 Cell Analyzer.

[00267] C2C12 myoblasts were seeded on collagen I coated plates with black walls at 10,000 cells per well; human skeletal muscle derived myoblasts were seeded on collagen I coated plates with black walls at 12,500 cells/well. After 24 hours, human myoblasts were treated with differentiation media (Cook Myosite, MD-5555) for 3 days, and the C2C12 myoblasts were treated with DMEM + 2% horse serum for 2 days. Following confirmation of myotube formation, the cells were treated with the AAVhu37 and antibody complexes (1 E5 viral genomes per cell) as described above. VVT885 (1/8 Spytagged AAV9 W503A conjugated to anti-CACNG1 antibody) diluted in PBS was used as a positive control. Three (3) days post transduction, the cells were fixed with 4% PFA for 15 minutes, washed twice with 1xPBS and blocked with 20% goat serum + 0.3% Triton X-100. The wells were incubated with MF-20 (DSHB) overnight in blocking buffer at 1 :100 to stain for Myosin Heavy Chain (MyHC). The following day, the cells were washed and incubated with anti-mouse IgG Alexa 647, then washed and incubated with DAPI, and imaged on the Axio Observer. Myotube images were analyzed using HALO to determine GFP positive area as a percentage of total MyHC positive area. A separate analysis was also run to determine intensity of GFP signal in MyHC positive regions. [00268] Co-incubation of AAV Hu37 with either AF70 or AF71 resulted in improved transduction efficiency over control AAV without antibody on HEK 293 cells overexpressing mouse CACNG1 (Figure 2 and Figure 3) and human CACNG1 (Figure 4 and Figure 5). Transduction efficiency was optimal between molar ratios of 1 AAV to 1 antibody and 1 AAV to 9 antibodies as assessed by %GFP positive cells (Figure 2 and Figure 4) and mean fluorescence intensity (Figure 3 and Figure 5). Similarly, incubation of AAV with AF70 and AF71 enhanced transduction into differentiated C2C12 myotobes (Figure 6 and Figures 8A- 8D) and differentiated human myotubes (Figure 7 and Figures 9A-9D) as determined by quantification of GFP positive cells (Figures 8A-8D and Figures 9A-9D). C2C12 was most efficiently transduced with AAV complexed with AF71 at molar ratio 1:3 and 1 :9 (Figure 6). Peak transduction efficacy of AAV retargeted with bispecific antibodies was comparable to that of control AAV covalently conjugated to an anti-CACNG1 antibody (VVT885), except for AF70 on human myotubes which showed efficacy but of lesser magnitude. These data show that AAV can be effectively targeted to CACNG1 expressing cells using bispecific antibodies.

Example 2. Testing AAV x mTfR alternative format (AF) antibodies binding to AAV9W503A virus by ELISA

[00269] The present Example was designed to test the binding of AAV x mTfR alternative format (AF) antibodies (Abs) to AAV9W503A virus by enzyme-linked immunoassay (ELISA). A schematic diagram of an example experimental setup used in this Example is shown in Figure 11. Briefly, 96-well plates were coated with AAV9W503A Ubc.Fluc (2e8 VGs/well) and incubated overnight at 4°C. Prior to introducing the primary antibodies, plates were blocked for 1 hour at room temperature (RT) using 3% bovine serum albumin (BSA). In a separate plate, alternative format (AF) antibodies (primary antibodies) were diluted 3-fold in assay dilution buffer (ADB) buffer with a starting concentration of 1 pg/ml. A description the AAV x mTfR alternative format (AF) antibodies tested in the present example (ID 1-13) is shown in Figures 10A-10B (see also, e g., Figures 21A-21I and Table 3). REGN1932 (anti- FelD1), REGN13072 (bivalent with NGS/MS 70), and REGN13221 (bivalent with NGS/MS 64) were also included as control antibodies. Additional control conditions were AAV9W503A only and AAV9W503A + secondary antibody only. Primary antibody dilutions were then transferred to the coated plates and incubated for 1 hour at RT. For the secondary antibody, an a-Human-HRP at 1:10,000 dilution in ADB buffer at RT was used. 1xKPL buffer was used for in-between step washes. The SuperSignal ELISA Pico Chemiluminescent Substrate Kit was used for development. The substrate was added and incubated for ~1 minute at RT and then the readout was performed using the Tecan Infinite m200 Pro instrument. The results of the experiments showed that AAV x mTfR AF antibodies with NGS/MS #70 arm bind well to AAV9W503A Ubc.Fluc at high concentrations. AAV x mTfR AF Abs with anti NGS/MS #64 arm had a lower affinity to AAV9W503A Ubc.Fluc. AAV x mTfR AF11-13 with no anti-AAV arm did not bind (Figure 12).

Example 3. Retargeting AAV9W503A using mTfR alternative format (AF) antibodies on 293T cells Expressing mTfR receptor

[00270] The present Example tested retargeting of AAV9W503A using mouse TfR (mTfR) alternative format antibodies (see, e.g., Figures 10A-10B, Figures 21A-21I and Table 3) on 293T cells expressing the mTfR receptor. A schematic diagram of an example experimental setup used in this Example is shown in Figure 13. Briefly, AAV9W503A was diluted in 1xPBS+0.001% Pluronic. For the virus, the multiplicity of infection (MOI) was 1x10⁵ per well and the total vector genomes (VGs) was 1x10⁹ per well. Alternative format antibodies were diluted in 1XPBS and 4-fold serial dilutions were performed to attain various AAV: Antibody ratios from a starting AAV:Antibody ratio of 1:10,0000. For AF8 (see, e.g., Figures 10A- 10B), the starting AAV:Antibody ratio was 1 :7750 (low stock concentration). The virus and the antibodies were mixed and incubated for 1 hour at 37°C. The Virus:Antibody complex was then added to the 293T cells expressing the mTfR receptor and incubated for 72 hours at 37°C. After the 72-hour incubation period, a Firefly luciferase (FLuc) read-out protocol was performed to measure the luciferase signal.

[00271] For the FLuc assay read-out protocol, the plate was spun at 1500 RPM for 1 minute and then the supernatant was discarded. 50 l lysis buffer was added per well (Gio- Lysis, Promega E2661) for 5 minutes. Then, 100 pl luciferase substrate was added per well and incubated for approximately 1 minute. The plate was analyzed using PerkinElmer2030 (or SpectraMax) with a setting selection of “Luminescence Corning Black Clear Bottom”. The wells to be read and measured were selected and following the reading/measurement, data were exported. The AAV9W503A background value was 0. The results showed that AAV x mTfR alternative format (AF) antibodies with anti-AAV NGS/MS #70 retarget AAV9W503A at low ratios and have higher peak when compared to AAV x mTfR AF Abs w/ anti-AAV NGS/MS #64 (Figure 14). A correlation between the above described ELISA data (see, e.g., Example 2) and the retargeting assay data of the present example revealed consistency between the ELISA and retargeting assay data in terms of binding and retargeting across all AAV x mTfR alternative format antibodies tested (Figures 12, 14). AAV x mTfR alternative format antibodies with NGS/MS#70 bind better and retarget AAV9W503A at lower ratios as compared to AAV x mTfR alternative format antibodies with NGS/MS#64. Example 4. Retargeting assay using AAV9 and an eGFP reporter

[00272] The present Example tested retargeting of AAV9 using mouse TfR (mTfR) alternative format antibodies AF1 and AF7 (see, e.g., Figures 10A-10B and Table 3) on 293T cells expressing the mTfR receptor. Briefly, AAV9 was diluted in 1xPBS+0.001% Pluronic. For the virus, the multiplicity of infection (MOI) was 1x10⁶ per well unless otherwise indicated.. Alternative format antibodies were diluted in 1XPBS and 3-fold serial dilutions were performed to attain various AAV:Antibody ratios from a starting AAV:Antibody ratio of 1 :729. The virus and the antibodies were mixed and incubated for 1 hour at 37°C. The AAV:Antibody complex was then added to the 293T cells expressing the mTfR receptor and incubated for 72 hours at 37°C. After the 72-hour incubation period, a GFP read-out protocol was performed to measure fluorescence.

[00273] To prepare samples for flow cytometry, supplemented media was discarded, and cells were washed once with 100 L 1xPBS. Afterwards, cells were lifted by adding 50 L of TrypLE™ Select Enzyme (1X), no phenol red, and incubated for 4-5 minutes in 37°C. 100 pL of BD Pharmingen™ Stain Buffer (BSA) was then added and cells resuspended and then transferred to a clear V-bottom 96 well plate. Cells were spun down for 2 minutes at 800xg and the supernatant was discarded. Two extra rounds of washes were performed by adding 100 pL of BSA Stain Buffer and spun down for 2 minutes at 800xg. After the last wash, 100 pL of BSA Stain Buffer was added and cells were resuspended in preparation for Flow Cytometry analysis. GFP expression was measured using the ZE5 Cell Analyzer.

[00274] AAV9 was effectively retargeted with AF1 and AF7 at ratios 1 AAV:0.3 antibodies to 1 AAV:27 antibodies and performed similarly to AAV9 or AAV9W503A conjugated to an anti-mTfRc ScFv as assessed by %GFP positive cells (Figure 15A and 15B) or MFI of GFP fluorescence (Figure 15C).

Example 5. In vivo retargeting of AAV9 using AAV x mTfR alternative format antibodies

[00275] The present Example tested in vivo retargeting of AAV9 using various AAV x mTfR alternative format antibodies, specifically AF1, AF3, AF5, AF7 and AF9. The experimental groups consisted of groups 1-3 (AF1), groups 4-6 (AF3), groups 7-9 (AF5), groups 10-12 (AF7), and groups 13-15 (AF9). The AAV x mTfR alternative format antibodies in the experimental groups were complexed with a self-complementary AAV9 encoding eGFP under a hybrid chicken b-actin (CBh) promoter (AAV9 scCBH.eGFP). Each alternative format was tested at three VG to Ab ratios: 1:9, 1 : 3, and 1:1. The control groups consisted of groups 16-18 (AAV9 scCBH.eGFP, AAV9 1/8 a-TfRC scCBH.eGFP, and AAV9W503A 1/8 a-TfRC scFv scCBH.eGFP) and a 1xPBS control group 19. The AAV concentration used in groups 1-18 was 5x1 O'¹¹ vg/mL. Three mice were used in each of groups 1-15,17 and 18, two mice were used in group 16, and one in group 19.

[00276] On Day 0, 200 pL test sample was injected to the mice. 24 hours after the injection, blood samples were collected from the mice for quantitative PCR (qPCR) analysis. In week 5, animals were sacrificed and brain and liver samples were collected for eGFP staining and Taqman qPCR analysis. Figures 16A-16B show in vitro test infection results for in vivo injection samples.

[00277] For tissue staining, brain (left hemisphere) and liver (left lobe) were collected and fixed in 10% neutral buffered formalin (NBF) for approximately 24 hours before being transferred to 70% EtOH for storage until embedding and sectioning.

[00278] Each tissue sample from each individual mouse was embedded into a separate paraffin block, then sectioned and stained using the methods outlined in Table 1.

Table 1. Description of Staining Protocol

[00279] For the liver, the Abeam rabbit green fluorescent protein (GFP) antibody staining protocol comprised a rabbit monoclonal antibody [EPR14104] to GFP (Abeam, Cat# ab183734) as a primary antibody and a goat anti-rabbit biotinylated antibody as a secondary antibody (Vector Labs, Cat# BA-1000). For the brain, the Invitrogen GFP antibody staining protocol comprised an anti-green fluorescent protein (GFP) rabbit IgG Fraction (anti-GFP IgG) (2 mg/ml; Invitrogen, Cat# A11122) as a primary antibody and a goat anti-rabbit biotinylated antibody (Vector Labs, Cat# BA-1000) as a secondary antibody. GFP staining of liver and brain are shown for AAV x mTfR control groups 16-19 (AAV9 scCBH.eGFP, AAV9 1/8 a-TfRC scCBH.eGFP, and AAV9W503A 1/8 a-TfRC scFv scCBH.eGFP) in Figure 17A. Also shown are GFP staining of liver and brain for AAV x mTfR AF1 (Figure 17B), AAV x mTfR AF3 (Figure 17C), AAV x mTfR AF5 (Figure 17D), AAV x mTfR AF7 (Figure 17E), and AAV x mTfR AF9 (Figure 17F). All alternative format antibodies were effectively able to retarget AAV to Cerebellum, Cortex, and Hippocamus with superiority to AAV9 alone and similar efficacy to AAV9 or AAV9W503A covalently conjugated to an anti-TFRc ScFv. [00280] Transduction was also assessed by TaqMan qPCR of GFP mRNA expression in Liver (Figure 18) and Brain (Figure 19). Consistent with immunohistochemistry data, AAV complexed with AFs more effectively transduced the CNS than AAV9 alone. At low antibody ratios (1 :1 and 1:3), comparable liver transduction was seen to AAV9 alone. There was a partial reduction in liver transduction at a ratio of 1:9 for alternative format complexed AAVs.

Example 6. In vivo retargeting of AAV9 versus AAV9W503A using AAV x mTfR alternative format antibodies

[00281] The present Example tested in vivo retargeting of AAV9 versus AAV9W503A using AAV x mTfR alternative format antibodies. A goal of the experiments described herein was to test AAV x mTfR alternative format antibody AF7 on wild-type (WT) and detargeted AAV9 across a wide range of virus vector genome (VG) to antibody (Ab) ratios (VG:Ab). The experimental groups consisted of groups 1-6 (AAV9 scCBH.eGFP), and groups 8-13 (AAV9W503A scCBH.eGFP). Each virus was tested at six VG to Ab ratios: 1:729, 1 : 243, 1 :81 , 1:27, 1:9, and 1:3. The control groups consisted of groups 7, 14, and 15 (AAV9 scCBH.eGFP, AAV9W503A scCBH.eGFP, and AAV9 1/8 a-TfRC scFv scCBH.eGFP) and a 1xPBS control group 16. The total AAV concentration used in groups 1-15 was 1x10¹¹ vg/mouse. Three mice were used in each of groups 1-5, 7, 8-12 and 16 and five in each of groups 6 and 13-15.

[00282] On Day 0, 200 pL test sample was injected to the mice. 24 hours after the injection, blood samples were collected from the mice for quantitative PCR (qPCR) analyses. In weeks 2-4, animals were sacrificed and brain, liver, and heart samples were collected for eGFP staining and Taqman qPCR analysis.

[00283] For tissue staining, brain (left hemisphere), liver (left lobe), and heart were collected and fixed in 10% neutral buffered formalin (NBF) for approximately 24 hours before being transferred to 70% EtOH for storage until embedding and sectioning.

[00284] Each tissue sample from each individual mouse was embedded into a separate paraffin block, then sectioned and stained using the methods outlined in Table 2.

Table 2. Description of Staining Protocol

[00285] For the liver and heart, the Abeam rabbit green fluorescent protein (GFP) antibody staining protocol comprised a rabbit monoclonal antibody [EPR14104] to GFP (Abeam, Cat# ab183734) as a primary antibody and a goat anti-rabbit biotinylated antibody as a secondary antibody (Vector Labs, Cat# BA-1000). For the brain, the Invitrogen GFP antibody staining protocol comprised an anti-green fluorescent protein (GFP) rabbit IgG Fraction (anti-GFP IgG) (2 mg/ml; Invitrogen, Cat# A11122) as a primary antibody and a goat anti-rabbit biotinylated antibody (Vector Labs, Cat# BA- 1000) as a secondary antibody. GFP staining of liver, heart, and brain are shown for AAV9-AAV x mTfR AF7 in Figures 20A-20D.

[00286] When complexed with AF7, both AAV9 (Figures 20A and 20B) and AAV9W503A (Figures 20C and 20D) can effectively transduce the CNS as determined by GFP expression in Hippocampus, Cortex, and Cerebellum (Figures 20A and 20C). AF7 complexed with AAV9 retains liver and heart transduction (Figure 20B), but AAV9 W503A: AF7 complexes are detargeted from both heart and liver (Figure 20D). Optimal CNS transduction efficiency was observed at ratios 1 AAV: 3 antibodies and 1 AAV to 9 antibodies. Together, these data demonstrate AAV can be effectively retargeted to CNS using bispecific antibodies.

Example 7. In vivo retargeting to skeletal muscle with CACNGIxAAV bispecific antibodies

[00287] AAV x CACNG1 bispecific antibodies were validated for retargeting of Hu37 in vitro on HEK 293 cells overexpressing mouse or human CACNG1 (Figure 23). GFP expression was assessed by flow cytometry and is shown as % GFP positive cells (top panels) or MFI of GFP expressing cells (bottom panels). To assess the in vivo retargeting efficacy of AAV x CACNG1 bispecific antibodies, transduction efficiency of AAV and antibody complexes was quantified in a mouse model of Duchenne muscular dystrophy (D2.MDX mice). D2.MDX mice contain a premature stop codon mutation in the dystrophin gene that results in loss of dystrophin expression and development of muscular dystrophy. Hu37 or AAV9 W503A expressing eGFP from CAG promoter and appropriate ratio of antibody (AF71) were mixed, followed by incubation for 1 hour at 37°C. D2.MDX mice were then retro-orbitally injected with 150 pL (1 E11 total VGs) of virus+antibody complexes and Spytagged-AAV9 conjugated with CACNG1 antibody was used as a positive control (Figure 24). Liver, skeletal, and cardiac muscles were harvested at week 3. All the tissues were processed for immunohistochemistry to measure eGFP protein levels and for taqman analysis to measure RNA levels. As a quality control, on mouse injection day, the same virus+antibody complexes and control virus were added to HEK293 cells overexpressing hCACNGI at different MOI; cells were analyzed by flow for eGFP expression three day post-transduction (Figure 25). HEK 293 cells overexpressing human CACNG1 (hCACNGI) were transduced with Hu37 alone (group 2). However, transduction was enhanced when Hu37 was complexed with AF71 at molar ratio 1:1, 1 :3, and 1:9 (groups 3, 4, and 5, respectively) with a minimum transduction efficiency of 76.8% at MOI 2.5E4 VG per cell. A similar pattern was observed when MOI was increased 10-fold to 2.5E5 VG per cell. As a positive control, HEK 293 cells overexpressing human CACNG1 (hCACNGI) were transduced with Spytagged-AAV9 conjugated with CACNG1 spycatcher antibody REGN10717 (group 1) and AAV9 W503A complexed with AF71 (Group 6) (Figure 25). GFP IHC data at week 3 post-AAV+antibody complex injection exhibited GFP staining, indicative of Hu37 transgene expression/retargeting to various skeletal muscles - diaphragm, tongue and tibialis anterior (Figure 26A), gastrocnemius and soleus (Figure 26B), and quadriceps (Figure 26C) of D2.MDX mice with AAVxCACNGI altibody (i.e. , alternative antibody format) AF71 at a variety of virus:antibody ratios (1:1 , 1 :3, and 1:9). Hu37 virus alone transduced the heart and transduction remained when complexing the virus with AF71 (Figure 26B).

Control viruses with AAV9 background (Spytagged-AAV9 conjugated with CACNG1 spycatcher antibody REGN10717 and AAV9 W503A complexed with AF71 at virus:antibody ratio of 1:3) transduced skeletal muscles - diaphragm, tongue and tibialis anterior (Figure 26A), gastrocnemius and soleus (Figure 26B), and quadriceps (Figure 26C). Overall, liver transduction was at a minimum or is not observed by GFP IHC in all conditions tested (Figure 26C) in D2.MDX mice. eGFP mRNA level is shown in various organs (liver and heart (Figure 27A); skeletal muscles such as gastrocnemius, quadriceps, diaphragm, soleus, tibialis anterior, and tongue (Figure 27B)). In each bar graph, GFP mRNA level of each condition was compared in relative to Hu37 1 :0 condition. In the livers (Figure 27A) in all conditions (Hu37 complexed with AF71 and control viruses in AAV9 background), the level of GFP mRNA were lower when compared with Hu37 1:0 condition. In the hearts (Figure 27A), as the ratio increased from 1 :1 to 1:9, the level of GFP mRNA decreased and the level of GFP mRNA were lower than Hu37 1:0 condition. Overall in skeletal muscles (Figure 27B), compared to Hu37 1:0 condition, the GFP mRNA level was 3.82-fold to 142.55-fold higher when Hu37 was complexed with AF71 at ratio virus:antibody 1:1, 1:3, and 1 :9. Also in skeletal muscles, the GFP mRNA level with control viruses in AAV9 background (Spytagged-AAV9 conjugated with CACNG1 spycatcher antibody REGN10717 and AAV9 W503A complexed with AF71 at virus:antibody ratio 1 :3) was at least 29.99-fold higher compared to Hu37 1:0 condition. AF71 with two anti-AAV binding arm and two anti-CACNG1 arm enhanced Hu37 transduction in skeletal muscles in D2.MDX mice. Hu37 alone has a baseline transduction in cardiac and skeletal muscle. When complexed with AF71 , transduction in skeletal muscles was significantly improved as revealed by IHC and taqman results. However, the total transduction level of complexed Hu37 was lower compared to complexed AAV9-W503A or spytagged-AAV controls.

[00288] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Table 3: Listing of Example AAV x mTfR and AAV x CACNG1 Amino Acid and Nucleotide Sequences

Ill

Claims

Claims What is claimed is:

1 . A bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) an scFv comprising a first antigen-binding domain (“ABD1”) operably linked to (ii) a first heavy chain region of a first Fab (“Fab1”), said first heavy chain region operably linked to (iii) an Fc domain; b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a second antigen-binding domain (“ABD2”); and d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a third antigen-binding domain (“ABD3”); wherein ABD2 and ABD3 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD2 and ABD3 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD1 binds to transferrin receptor (TfR) and comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

2. The bispecific antibody or bispecific antigen-binding fragment of claim 1 , wherein the scFv is linked to the first heavy chain region via a linker.

3. A bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an scFv comprising a first antigen-binding domain (“ABD1”), said scFv operably linked to (iii) an Fc domain; b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a second antigen-binding domain (“ABD2”); and d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a third antigen-binding domain (“ABD3”); wherein ABD2 and ABD3 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD2 and ABD3 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD1 binds to transferrin receptor (TfR) and comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

4. The bispecific antibody or bispecific antigen-binding fragment of claim 3, wherein the scFv is linked to the first heavy chain region via a linker.

5. A bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an Fc domain, said Fc domain operably linked to (iii) an scFv comprising a first antigen-binding domain (“ABD1”); b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a second antigen-binding domain (“ABD2”); and d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a third antigen-binding domain (“ABD3”); wherein ABD2 and ABD3 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD2 and ABD3 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD1 binds to transferrin receptor (TfR) and comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

6. The bispecific antibody or bispecific antigen-binding fragment of claim 5, wherein the scFv is linked to the Fc domain via a linker.

7. A bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an Fc domain, said Fc domain operably linked to (iii) a second heavy chain region of a second Fab (“Fab2”); b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) an scFv comprising a first antigen-binding domain (“ABD1”) operably linked to (ii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a second antigen-binding domain (“ABD2”); d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a third antigen-binding domain (“ABD3”); wherein ABD2 and ABD3 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD2 and ABD3 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD1 binds to transferrin receptor (TfR) and comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

8. The bispecific antibody or bispecific antigen-binding fragment of claim 7, wherein Fab2 is linked to the Fc domain via a linker.

9. A bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an Fc domain, said Fc domain operably linked to (iii) a first scFv comprising a first antigen-binding domain (“ABD1”); b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain, said Fc domain operably linked to (iii) a second scFv comprising a second antigen-binding domain (“ABD2”); c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a third antigen-binding domain (“ABD3”); d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a fourth antigen-binding domain (“ABD4”); wherein ABD3 and ABD4 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD3 and ABD4 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein the ABD1 and ABD2 bind to transferrin receptor (TfR), and each of ABD1 and ABD2 comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

10. The bispecific antibody or bispecific antigen-binding fragment of claim 9, wherein the first scFv and/or second scFv is linked to the Fc domain via a linker.

11. The bispecific antibody or bispecific antigen-binding fragment of any one of claims 1- 10, wherein the ABD that binds to a capsid of an AAV particle comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 58; and/or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 16.

12. The bispecific antibody or bispecific antigen-binding fragment of claim 11 , wherein the ABD that binds to a capsid of an AAV particle comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a LCVR comprising the amino acid sequence of SEQ I D NO: 10 or 1157.

13. The bispecific antibody or bispecific antigen-binding fragment of any one of claims 1- 12, wherein the ABD that binds to TfR comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1121 , a HCDR2 comprising the amino acid sequence of SEQ ID NO: 1123, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 1125; and/or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 1129, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 1131 , and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 1133.

14. The bispecific antibody or bispecific antigen-binding fragment thereof of claim 13, wherein the ABD that binds to TfR comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 1119, and/or a LCVR comprising the amino acid sequence of SEQ ID NO: 1127.

15. A bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first scFv comprising a first antigen-binding domain (“ABD1”) operably linked to (ii) a first heavy chain region of a first Fab (“Fab1”), said first heavy chain region of Fab1 operably linked to (iii) an Fc domain; b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second scFv comprising a second antigen-binding domain (“ABD2”) operably linked to (ii) a second heavy chain region of a second Fab (“Fab2”), said second heavy chain region of Fab2 operably linked to (iii) an Fc domain; c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a third antigen-binding domain (“ABD3”); d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a fourth antigen-binding domain (“ABD4”); wherein ABD1 and ABD2 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD1 and ABD2 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD3 and ABD4 bind to calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1), and each of ABD3 and ABD4 comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 795, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 803.

16. The bispecific antibody or bispecific antigen-binding fragment of claim 15, wherein the first scFv is linked to Fab1 and/or the second scFv is linked to Fab2 via a linker.

17. A bispecific antibody, or a bispecific antigen-binding fragment thereof, comprising: a) a first polypeptide chain comprising, in an N- to C-terminal orientation, (i) a first heavy chain region of a first Fab (“Fab1”) operably linked to (ii) an Fc domain, said Fc domain operably linked to (iii) a first scFv comprising a first antigen-binding domain (“ABD1”); b) a second polypeptide chain comprising, in an N- to C-terminal orientation, (i) a second heavy chain region of a second Fab (“Fab2”) operably linked to (ii) an Fc domain, said Fc domain operably linked to (iii) a second scFv comprising a second antigen-binding domain (“ABD2”); c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1 , wherein Fab1 comprises a third antigen-binding domain (“ABD3”); d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, wherein Fab2 comprises a fourth antigen-binding domain (“ABD4”); wherein ABD1 and ABD2 bind to a capsid of an adeno-associated virus (AAV) particle, and each of ABD1 and ABD2 comprises a heavy chain variable region (HCVR) that comprises a HCDR1 , HCDR2, and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a light chain variable region (LCVR) that comprises a LCDR1 , LCDR2, and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157; and wherein ABD3 and ABD4 bind to calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1), and each of ABD3 and ABD4 comprises a HCVR that comprises the HCDR1 , HCDR2 and HCDR3 contained within an HCVR comprising the amino acid sequence of SEQ ID NO: 795, and/or a LCVR that comprises the LCDR1 , LCDR2 and LCDR3 contained within a LCVR comprising the amino acid sequence of SEQ ID NO: 803.

18. The bispecific antibody or bispecific antigen-binding fragment of claim 17, wherein the first scFv and/or second scFv is linked to the Fc domain via a linker.

19. The bispecific antibody or bispecific antigen-binding fragment of any one of claims 15- 18, wherein each of ABD1 and ABD2 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 58; and/or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 16.

20. The bispecific antibody or bispecific antigen-binding fragment of claim 19, wherein each of ABD1 and ABD2 comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 52 or 1159, and/or a LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 1157.

21. The bispecific antibody or bispecific antigen-binding fragment of any one of claims 15- 20, wherein each of ABD3 and ABD4 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 797, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 799, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 801 ; and/or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 805, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 807, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 809.

22. The bispecific antibody or bispecific antigen-binding fragment thereof of claim 21 , wherein each of ABD3 and ABD4 comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 795, and/or a LCVR comprising the amino acid sequence of SEQ ID NO: 803.

23. The bispecific antibody or bispecific antigen-binding fragment of any one of claims 2, 4, 6, 8, 10, 16, and 18, wherein the linker is or comprises a 3xG4S (SEQ ID NO: 1115).

24. The bispecific antibody or bispecific antigen-binding fragment of any one of claims 1- 23, which comprises an Fc heterodimer.

25. The bispecific antibody or bispecific antigen-binding fragment of claim 24, wherein the Fc domains in the Fc heterodimer comprise knob-in-hole mutations as compared to a wild type Fc domain.

26. The bispecific antibody or bispecific antigen-binding fragment of claim 25, wherein one Fc domain in the Fc heterodimer comprises amino acid substitutions S354C and T366W (according to Ell numbering) as compared to a wild type Fc domain, and the other Fc domain in the Fc heterodimer comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (according to Ell numbering) as compared to a wild type Fc domain.

27. The bispecific antibody or bispecific antigen-binding fragment of any one of claims 24- 26, wherein at least one Fc domain in the Fc heterodimer comprises a star mutation as compared to a wild type Fc domain.

28. The bispecific antibody or bispecific antigen-binding fragment of claim 27, wherein one Fc domain in the Fc heterodimer comprises amino acid mutations H435R and/or Y436F (according to Ell numbering) as compared to a wild type Fc domain.

29. The bispecific antibody or bispecific antigen-binding fragment of any one of claims 1- 28, wherein said capsid comprises one or more wild-type AAV capsid polypeptides.

30. The bispecific antibody or bispecific antigen-binding fragment of any one of claims 1- 29, wherein said capsid comprises one or more non-wild-type AAV capsid polypeptides.

31. A pharmaceutical composition comprising the bispecific antibody or bispecific antigenbinding fragment of any one of claims 1-30, and a pharmaceutically acceptable carrier or excipient.

32. A molecular complex comprising an AAV particle bound to one or more bispecific antibodies and/or bispecific antigen-binding fragments of any one of claims 1-30.

33. The molecular complex of claim 32, wherein said AAV particle comprises one or more mutations in one or more AAV capsid proteins inhibiting the natural tropism of said AAV particle.

34. A pharmaceutical composition comprising the molecular complex of claim 32 or 33 and a pharmaceutically acceptable carrier or excipient.

35. A method of preparing the molecular complex of claim 32 or 33, comprising incubating said AAV particle in the presence of said one or more bispecific antibodies and/or bispecific antigen-binding fragments under conditions allowing specific binding of said one or more bispecific antibodies and/or bispecific antigen-binding fragments to said AAV particle capsid.

36. A method for targeting an AAV particle to a cell expressing transferrin receptor (TfR) on the cell surface, comprising contacting the cell with the molecular complex of claim 32 or 33, or the pharmaceutical composition of claim 34, wherein said molecular complex comprises one or more bispecific antibodies and/or bispecific antigen-binding fragments which bind to TfR.

37. A method for targeting an AAV particle to a cell expressing calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1) on the cell surface, comprising contacting the cell with the molecular complex of claim 32 or 33, or the pharmaceutical composition of claim 34, wherein said molecular complex comprises one or more bispecific antibodies and/or bispecific antigen-binding fragments which bind to CACNG1.

38. A method for delivering a polynucleotide to a cell expressing transferrin receptor (TfR) on the cell surface, comprising contacting the cell with the molecular complex of claim 32 or 33 or the pharmaceutical composition of claim 34, wherein said molecular complex comprises the AAV particle comprising said polynucleotide and bound to one or more bispecific antibodies and/or bispecific antigen-binding fragments which bind to TfR.

39. A method for delivering a polynucleotide to a cell expressing calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1) on the cell surface, comprising contacting the cell with the molecular complex of claim 32 or 33 or the pharmaceutical composition of claim 34, wherein said molecular complex comprises the AAV particle comprising said polynucleotide and bound to one or more bispecific antibodies and/or bispecific antigen-binding fragments which bind to CACNG1.

40. The method of any one of claims 36-39, wherein said cell is in a subject and said molecular complex is administered to the subject.

41. The method of any one of claims 36-40 wherein said AAV particle does not target said cell in the absence of said one or more bispecific antibodies and/or bispecific antigenbinding fragments.