[go: up one dir, main page]

WO2025042428A1 - Molécules bispécifiques de liaison à l'antigène et leurs utilisations - Google Patents

Molécules bispécifiques de liaison à l'antigène et leurs utilisations Download PDF

Info

Publication number
WO2025042428A1
WO2025042428A1 PCT/US2023/084330 US2023084330W WO2025042428A1 WO 2025042428 A1 WO2025042428 A1 WO 2025042428A1 US 2023084330 W US2023084330 W US 2023084330W WO 2025042428 A1 WO2025042428 A1 WO 2025042428A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
antigen
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2023/084330
Other languages
English (en)
Other versions
WO2025042428A9 (fr
Inventor
Christos Kyratsous
Andrew J. Murphy
Sven MOLLER-TANK
Yang Shen
Tri Nguyen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Regeneron Pharmaceuticals Inc
Original Assignee
Regeneron Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Regeneron Pharmaceuticals Inc filed Critical Regeneron Pharmaceuticals Inc
Priority to EP23848162.6A priority Critical patent/EP4630457A1/fr
Publication of WO2025042428A1 publication Critical patent/WO2025042428A1/fr
Publication of WO2025042428A9 publication Critical patent/WO2025042428A9/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • 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.
  • TfR transferrin receptor
  • CACNG1 calcium voltage-gated channel auxiliary subunit gamma 1
  • the antibodies described herein can bridge the virus to target cells that express TfR or CACNG1 , thereby redirecting virus delivery and transduction.
  • 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
  • the scFv is linked to the first heavy chain region via a linker.
  • 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
  • the scFv is linked to the first heavy chain region via a linker.
  • 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
  • the scFv is linked to the Fc domain via a linker.
  • 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
  • Fab2 is linked to the Fc domain via a linker.
  • 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
  • the first scFv and/or second scFv is linked to the Fc domain via a linker.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the first scFv is linked to Fab1 and/or the second scFv is linked to Fab2 via a linker.
  • 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
  • the first scFv and/or second scFv is linked to the Fc domain via a linker.
  • 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.
  • 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.
  • 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.
  • 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.
  • the linker is or comprises a 3xG4S (SEQ ID NO: 1115).
  • the bispecific antibody or bispecific antigen-binding fragment comprises an Fc heterodimer.
  • the Fc domains in the Fc heterodimer comprise knob-in-hole mutations as compared to a wild type Fc domain.
  • 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
  • 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.
  • At least one Fc domain in the Fc heterodimer comprises a star mutation as compared to a wild type Fc domain.
  • 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.
  • the capsid comprises one or more wild-type AAV capsid polypeptides.
  • the capsid comprises one or more non-wild-type AAV capsid polypeptides.
  • a pharmaceutical composition comprising the bispecific antibody or bispecific antigen-binding fragment and a pharmaceutically acceptable carrier or excipient.
  • a molecular complex comprising an AAV particle bound to one or more bispecific antibodies and/or bispecific antigen-binding fragments.
  • the AAV particle comprises one or more mutations in one or more AAV capsid proteins inhibiting the natural tropism of the AAV particle.
  • composition comprising the molecular complex described herein and a pharmaceutically acceptable carrier or excipient.
  • 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.
  • 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.
  • TfR transferrin receptor
  • 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.
  • CACNG1 calcium voltage-gated channel auxiliary subunit gamma 1
  • 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.
  • TfR transferrin receptor
  • 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.
  • CACNG1 calcium voltage-gated channel auxiliary subunit gamma 1
  • the cell is in a subject and the molecular complex is administered to the subject.
  • the AAV particle does not target the cell in the absence of the one or more bispecific antibodies and/or bispecific antigen-binding fragments.
  • 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.
  • FIG. 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.
  • FIG. 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.
  • 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).
  • FIG. 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.
  • FIG. 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).
  • 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.
  • MyHC Myosin Heavy Chain
  • 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.
  • 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.
  • 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., ⁇ ).
  • Figure 11 shows an example experimental setup used to test alternative format antibodies binding to AAV9W503A virus by ELISA.
  • Figure 12 depicts ELISA data for AAV x mTfR alternative format antibodies binding to AAV9W503A.
  • 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.
  • Figure 14 shows a line graph of data generated in experiments testing AAV9W503A retargeting using AAVxmTfR alternative format antibodies on mTfR293T cells.
  • Figures 15A-15C illustrate a retargeting assay using AAV9 scCBH.eGFP.
  • Figures 16A-16B show in vitro test infection results for in vivo injection samples.
  • 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).
  • GFP green fluorescent protein
  • Figure 18 shows relative RNA expression of GFP in liver samples as determined by RT-qPCR.
  • Figure 19 shows RNA expression of GFP in brain samples as determined by RT- qPCR.
  • 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.
  • AAV9 Figures 20A-20B
  • AAV9W503A Figures 20C and 20D
  • 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).
  • 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 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).
  • Figure 24 depicts an example of a study design for testing Hu37 complexed with anti-AAV x CACNG1 2x2 antibodies in D2.MDX mice.
  • Figure 25 shows in vitro transduction in HEK293-hCACNG1 of complexes prepared for in vivo study.
  • Figures 26A-26C illustrate improved Hu37 transduction in muscle tissues when complexed with an 2x2 anti-CACNG1xAAV alternative format antibody.
  • Figures 27A-27B illustrate bispecific antibody enhancement of Hu37 transduction into skeletal muscle.
  • 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).
  • 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.
  • agent e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portions thereof, or combinations thereof
  • 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.
  • 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.
  • 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.
  • MHC major histocompatibility complex
  • an antigen-binding molecule refers in its broadest sense to a molecule that specifically binds to an antigen.
  • an antigen-binding molecule is an antibody or an antigen-binding fragment of an antibody, including, e.g., bispecific antibodies or fragments thereof.
  • 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.
  • bispecific antigen-binding molecules e.g., antibodies
  • 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).
  • 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.
  • antibody 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.
  • CDR complementarity determining region
  • 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).
  • 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).
  • CDRs complementarity determining regions
  • 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.
  • 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.
  • antibody also includes antigen-binding fragments of full antibody molecules.
  • 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.
  • 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.
  • 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.
  • CDR complementarity determining region
  • 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.
  • the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain H- H, H- L or L- L dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric H or VL domain.
  • an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • 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.
  • 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)).
  • 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.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region
  • LCDR1, LCDR2, LCDR3 three CDRs in each light chain variable region
  • 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.
  • 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.
  • 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.
  • VH variable heavy
  • L variable light
  • C2 second constant domain
  • the VH is N- terminal to the first constant domain (CH1) of the heavy chain
  • 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.
  • 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.”
  • Fc domain can refer to a portion of the heavy chain that pairs with the corresponding portion of another heavy chain.
  • 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.
  • 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.
  • 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
  • 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' 2 M, less than 10' 3 M, less than 5x1 O' 4 M, less than 10' 4 M, less than 5x10' 5 M, less than 10' 5 M, less than 5x10' 6 M, less than 10' 6 M, less than 5x10' 7 M, less than 10' 7 M, less than 5x10' 8 M, less than 10' 8 M, less than 5x10' 9 M, less than 10' 9 M, or less than 10' 1 ° M).
  • an antibody or an antibody fragment e.g., an antigen-binding molecule or antigen-binding domain
  • a target antigen e.g., an antigen-binding molecule or antigen-binding domain
  • FACS fluorescent-activated cell sorting
  • operably linked 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.
  • 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.
  • 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.
  • 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).
  • R group side chain
  • a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • 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.
  • 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.
  • Sequence similarity for polypeptides 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.
  • 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
  • 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.
  • subject or "patient” as used herein includes all members of the animal kingdom including non-human primates and humans.
  • antibodies and antigen-binding fragments thereof that bind a capsid of an AAV particle also known as “anti-AAV antibodies” herein.
  • the capsid comprises any of various wild-type and/or non- wild-type AAV capsid protein(s) described herein.
  • 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.
  • 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.
  • ITR inverted terminal repeats
  • ORFs open reading frames
  • 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.
  • VP1 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.
  • 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.
  • PHA2 phospholipase A2 domain
  • 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.
  • ITR inverted terminal repeat
  • 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.
  • 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.
  • 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.
  • the rep encoding sequence encodes a Rep protein or Rep protein equivalent that is capable of binding an ITR comprised on the transfer plasmid.
  • 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.
  • 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.
  • a chimeric 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.
  • 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.
  • 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).
  • SPR surface plasmon resonance
  • FACS fluorescent- activated cell sorting
  • 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,
  • 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).
  • a non-specific antigen e.g., BSA
  • 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.
  • KD 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.
  • KD 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.
  • binding affinity There is an inverse relationship between KD and binding affinity, therefore the smaller the KD value, the higher, i.e. stronger, the affinity.
  • the terms “higher affinity” or “stronger affinity” relate to a higher ability to form an interaction and therefore a smaller KD value
  • the terms “lower affinity” or “weaker affinity” relate to a lower ability to form an interaction and therefore a larger KD value.
  • 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)
  • 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.
  • 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 O ff value.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • nucleic acid molecules encoding anti-AAV antibodies or antigen-binding fragments thereof.
  • 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.
  • 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.
  • the scFv can comprise VL and VH sequences from any suitable species, such as murine, human or humanized VH and VL sequences.
  • 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).
  • a linker e.g., encoding any of the linkers described herein
  • 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.
  • the Fab domains are recombinantly expressed as part of a larger molecule.
  • the Fab domains can comprise constant domain and variable region sequences from any suitable species, and thus can be murine, chimeric, human or humanized.
  • Fab domains typically comprise a CH1 domain attached to a VH domain which pairs with a CL domain attached to a VL domain.
  • VH domain is paired with the VL domain to constitute the Fv region
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an ABD e.g., a H and a VL of an scFv
  • ABDs e.g., an scFv and a Fab, or a Fab and a Fab
  • a non-ABD component e.g., an Fc region
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Charged (e.g., charged hydrophilic linkers) and/or flexible linkers are particularly preferred.
  • 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.
  • bispecific antigen-binding molecules of the disclosure comprise constant regions (e.g., CH1 , hinge, CH2, CH3, CL) derived from any suitable class of antibody.
  • the constant regions are derived from a human antibody. Hinge Regions
  • 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.
  • 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.
  • 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.
  • the hinge region can be a chimeric hinge region.
  • the bispecific antigen-binding molecules of the disclosure can include an Fc region derived from any suitable species.
  • the Fc region is derived from a human Fc domain.
  • 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).
  • mouse constant regions e.g., a mouse IgG 1 Fc or a mouse lgG2 Fc (a or b isotype
  • human constant regions e.g., a human IgG 1 Fc or a human lgG4 Fc
  • 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.
  • 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.
  • the two Fc domains within the Fc region can be the same or different from one another.
  • 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.
  • 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.
  • 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.
  • the Fc region comprises CH2 and CH3 domains derived from lgG1.
  • the Fc region comprises CH2 and CH3 domains derived from lgG2.
  • the Fc region comprises CH2 and CH3 domains derived from lgG3.
  • the Fc region comprises CH2 and CH3 domains derived from lgG4.
  • the Fc region comprises a CH4 domain from IgM.
  • the IgM CH4 domain is typically located at the C-terminus of the CH3 domain.
  • the Fc region comprises CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.
  • 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.
  • 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.
  • the variant constant domains are at least 60% identical or similar to a wild-type constant domain.
  • the variant constant domains are at least 70% identical or similar.
  • the variant constant domains are at least 80% identical or similar.
  • the variant constant domains are at least 90% identical or similar.
  • the variant constant domains are at least 95% identical or similar.
  • 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.
  • 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.
  • the same one or more amino acid substitution is present in each of the two Fc domains of an Fc region.
  • the Fc domain is an lgG1 Fc domain, particularly a human lgG1 Fc domain.
  • the same one or more amino acid substitution is present in each of the two Fc domains of an Fc region.
  • 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.
  • the present disclosure provides bispecific antigen-binding molecules comprising Fc heterodimers, i.e., Fc regions comprising heterologous, non-identical Fc domains.
  • 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.
  • 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.
  • IgG lgG1 , lgG2, lgG3 and lgG4
  • 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.
  • 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.
  • 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).
  • an Fc domain can be modified to allow a purification strategy that enables selections of Fc heterodimers.
  • 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.
  • 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.
  • 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.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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").
  • 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
  • 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.
  • AAV adeno-associated virus
  • the bispecific antigenbinding molecule is a bispecific antibody.
  • 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.
  • the capsid comprises a wild-type AAV capsid protein(s).
  • the capsid comprises a non-wild-type AAV capsid protein(s).
  • 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.
  • 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).
  • 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.
  • REGN13880 comprises of anti-AAV#70 Fab.
  • REGN 13880 was purified from the starting material using affinity capture chromatography followed by size exclusion chromatography.
  • 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.
  • HCVR heavy chain variable region
  • 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
  • nucleic acid molecules encoding the HCVR and LCVR sequences of the anti-AAV/anti-TfR bispecific antigen-binding molecules disclosed herein.
  • 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.
  • HCDR1 GGATTTACTTTCTCAAATTATGGAATGCAT (SEQ ID NO: 1120)
  • HCDR3 CCAACTAGTCATTACGTCGTCGATGTT (SEQ ID NO: 1124)
  • HCDR1 GFTFSNYGMH (SEQ ID NO: 1121)
  • HCDR2 MIYYDSSKMNYADTVKG (SEQ ID NO: 1123)
  • HCDR3 PTSHYVVDV (SEQ ID NO: 1125)
  • LCDR1 CAAGCATCTCAAGATATCGGCAACTGGCTCGCA (SEQ I D NO: 1128)
  • LCDR2 GGCGCAACATCCCTCGCAGAC (SEQ ID NO: 1130)
  • LCDR3 CAAGCATATAACACACCATGGACAT (SEQ I D NO: 1132)
  • LCDR2 GATSLAD (SEQ ID NO: 1131)
  • LCDR3 QAYNTPWT (SEQ I D NO: 1133)
  • 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:
  • 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.
  • CACNG calcium voltage-gated channel auxiliary subunit gamma 1
  • 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.
  • IWHDGSDK (SEQ ID NO: 799)
  • ARRGIRGTVFDH (SEQ ID NO: 801)
  • AAGGCGTCT (SEQ ID NO: 806)
  • CTCTGGGTAAATGA SEQ ID NO: 810
  • VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1173)
  • AAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAG (SEQ ID NO: 812)
  • 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.
  • TfR transferrin receptor
  • CACNG1 calcium voltage-gated channel auxiliary subunit gamma 1
  • the antibodies described herein can bridge the virus to target cells that express TfR or CACNG1 , thereby redirecting virus delivery and transduction.
  • 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
  • the scFv is linked to the first heavy chain region via a linker.
  • 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
  • the scFv is linked to the first heavy chain region via a linker.
  • 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
  • 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
  • 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
  • the first scFv and/or second scFv is linked to the Fc domain via a linker.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the first scFv is linked to Fab1 and/or the second scFv is linked to Fab2 via a linker.
  • 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
  • the first scFv and/or second scFv is linked to the Fc domain via a linker.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the capsid comprises one or more non-wild-type AAV capsid polypeptides.
  • composition comprising the molecular complex described herein and a pharmaceutically acceptable carrier or excipient.
  • 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.
  • 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.
  • the fully human sequence is derived from a protein endogenous to a human.
  • 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.
  • the disclosure also provides host cells comprising a nucleic acid of the disclosure.
  • the host cells are genetically engineered to comprise one or more nucleic acids described herein.
  • the host cells are genetically engineered by using an expression cassette.
  • 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.
  • the disclosure also provides host cells comprising the vectors described herein.
  • 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.
  • 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.
  • an antigen-binding molecule e.g., anti-AAV monospecific antibody or anti-AAV x anti-CACNG1 or anti-TfR bispecific antibody
  • viral capsid protein e.g., a capsid protein of an AAV particle
  • pharmaceutical compositions comprising a viral capsid protein, e.g., an AAV particle may be useful as a gene transfer vector.
  • compositions of the disclosure are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.
  • 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.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTM, 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.
  • vesicles such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • 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.
  • 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).
  • 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.
  • viral vector compositions can be administered to a subject at a dose ranging from about 1 xio 5 plaque forming units (pfu) to about 1 xio 15 pfu, depending on mode of administration, the route of administration, the nature of the disease and condition of the subject.
  • the viral vector compositions can be administered at a dose ranging from about 1 xio 8 pfu to about 1 x 1 o 15 pfu, or from about 1 x 1 o 10 pfu to about 1 x 1 o 15 pfu, or from about 1 x 1 o 8 pfu to about 1 X 10 12 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.
  • a juvenile human subject can receive from about 1 xio 8 pfu to about 1 X 10 1 ° pfu, while an adult human subject can receive a dose from about 1 xio 10 pfu to about 1 X 10 12 pfu.
  • 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.
  • 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.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • Administration can be systemic or local.
  • a pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe.
  • 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.
  • 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.
  • 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 AUTOPENTM (Owen Mumford, Inc., Woodstock, UK), DISETRONICTM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25TM pen, HUMALOGTM pen, HUMALIN 70/30TM pen (Eli Lilly and Co., Indianapolis, IN), NOVOPENTM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIORTM (Novo Nordisk, Copenhagen, Denmark), BDTM pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENTM, OPTIPEN PROTM, OPTIPEN STARLETTM, and OPTICLIKTM (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 SOLOSTARTM pen (sanofi-aventis), the FLEXPENTM (Novo Nordisk), and the KWIKPENTM (Eli Lilly), the SURECLICKTM Autoinjector (Amgen, Thousand Oaks, CA), the PENLETTM (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRATM Pen (Abbott Labs, Abbott Park IL), to name only a few.
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).
  • polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • 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.
  • 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.
  • the present disclosure includes methods of using the any of antigen-binding molecules as disclosed herein or a pharmaceutically acceptable carrier or diluent.
  • 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.
  • 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.
  • 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.
  • the cell is in a subject and the antibody or antigen-binding fragment is administered to said subject.
  • 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.
  • 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.
  • 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.
  • an antigenbinding molecule and an AAV particle may be administered as a molecular complex described herein.
  • the antigen-binding molecule 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.
  • an antigen-binding molecule and an AAV particle may be administered separately over a defined time course.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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.
  • 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.
  • 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").
  • 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, 8 1 / 2 , 9, 9 1 / 2 , 10, 10 1 / 2 , 11 , 1134, 12, 12 1 / 2 , 13, 13 1 / 2 , 14, 14 1 / 2 , 15, 15 1 / 2 , 16, 16 1 / 2 , 17, 17 1 / 2 , 18, 1814, 19, 1914, 20, 2014, 21 , 21 1 / 2 , 22, 22 1 / 2 , 23, 23 1 / 2 , 24, 24 1 / 2 , 25, 25 1 / 2 , 26, 26 1 / 2 , or more) weeks after the immediately preceding dose.
  • 1 to 26 e.g., 1 , 114, 2, 214, 3, 314, 4, 414, 5, 514, 6, 614, 7, 714, 8, 8 1 / 2 , 9, 9 1 / 2
  • the immediately preceding dose 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.
  • 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.
  • 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
  • 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 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.
  • 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).
  • Example 1 Transduction assay to assess the efficacy of AAV x CACNG1 bispecific antibodies
  • Adeno-associated viruses 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.
  • AAVR adeno-associated virus receptor
  • 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.
  • 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.
  • GFP green fluorescent protein
  • DMEM supplied with 10% FBS, MEM NEAA, Pen Strep
  • 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.
  • 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.
  • 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 TrypLETM Select Enzyme (1X), no phenol red, and incubated for 4-5 minutes in 37°C.
  • BSA BD PharmingenTM Stain Buffer
  • 100 pL of BD PharmingenTM Stain Buffer 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.
  • 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.
  • 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.
  • MF-20 DSHB
  • MyHC Myosin Heavy Chain
  • 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).
  • ELISA enzyme-linked immunoassay
  • Figure 11 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).
  • RT room temperature
  • BSA bovine serum albumin
  • alternative format (AF) antibodies primary antibodies
  • ADB assay dilution buffer
  • 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.
  • 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).
  • AAV9W503A was diluted in 1xPBS+0.001% Pluronic.
  • MOI multiplicity of infection
  • VGs total vector genomes
  • 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.
  • 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.
  • FLuc Firefly luciferase
  • 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.
  • 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
  • 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.
  • AAV9 was diluted in 1xPBS+0.001% Pluronic.
  • MOI multiplicity of infection
  • 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.
  • 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
  • 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).
  • CBh hybrid chicken b-actin
  • 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.
  • brain left hemisphere
  • liver left lobe
  • NBF neutral buffered formalin
  • 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).
  • 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 anti-green fluorescent protein
  • anti-GFP IgG 2 mg/ml
  • Invitrogen Cat# A11122
  • a goat anti-rabbit biotinylated antibody Vector Labs, Cat# BA-1000
  • AAV9 scCBH.eGFP AAV9 1/8 a-TfRC scCBH.eGFP
  • FIG. 17B 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.
  • Example 6 In vivo retargeting of AAV9 versus AAV9W503A using AAV x mTfR alternative format antibodies
  • 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 1x10 11 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.
  • mice 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.
  • qPCR quantitative PCR
  • brain left hemisphere
  • liver left lobe
  • heart was 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.
  • NBF neutral buffered formalin
  • 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).
  • 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.
  • 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
  • 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).
  • 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.
  • HEK293 cells overexpressing hCACNGI were transduced with Hu37 alone (group 2).
  • 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.
  • HEK 293 cells overexpressing human CACNG1 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).
  • 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)).
  • Figure 27A live and heart
  • Figure 27B skeletal muscles
  • GFP mRNA level of each condition was compared in relative to Hu37 1 :0 condition.
  • 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.
  • the GFP mRNA level with control viruses in AAV9 background 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.
  • transduction in skeletal muscles was significantly improved as revealed by IHC and taqman results.
  • the total transduction level of complexed Hu37 was lower compared to complexed AAV9-W503A or spytagged-AAV controls.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des anticorps bispécifiques qui se lient à la fois à une capside d'une particule d'AAV et à un récepteur de transferrine (TfR) ou à une sous-unité auxiliaire de canal calcique voltage-dépendant gamma 1 (CACNG1), des complexes moléculaires associés, des compositions pharmaceutiques, et des procédés d'utilisation associés.
PCT/US2023/084330 2023-08-18 2023-12-15 Molécules bispécifiques de liaison à l'antigène et leurs utilisations Pending WO2025042428A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP23848162.6A EP4630457A1 (fr) 2023-08-18 2023-12-15 Molécules bispécifiques de liaison à l'antigène et leurs utilisations

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202363520485P 2023-08-18 2023-08-18
US63/520,485 2023-08-18
US202363578899P 2023-08-25 2023-08-25
US63/578,899 2023-08-25

Publications (2)

Publication Number Publication Date
WO2025042428A1 true WO2025042428A1 (fr) 2025-02-27
WO2025042428A9 WO2025042428A9 (fr) 2025-11-13

Family

ID=89772040

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/084330 Pending WO2025042428A1 (fr) 2023-08-18 2023-12-15 Molécules bispécifiques de liaison à l'antigène et leurs utilisations

Country Status (2)

Country Link
EP (1) EP4630457A1 (fr)
WO (1) WO2025042428A1 (fr)

Citations (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5582996A (en) 1990-12-04 1996-12-10 The Wistar Institute Of Anatomy & Biology Bifunctional antibodies and method of preparing same
US5677425A (en) 1987-09-04 1997-10-14 Celltech Therapeutics Limited Recombinant antibody
WO1998011244A2 (fr) 1996-09-11 1998-03-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Vecteur de vaa4 et ses utilisations
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
WO1998025971A1 (fr) 1996-12-10 1998-06-18 Celltech Therapeutics Limited Fragments d'anticorps monovalents
WO1999015549A2 (fr) 1997-09-19 1999-04-01 Celltech Therapeutics Limited Peptides
US5910573A (en) 1992-01-23 1999-06-08 Merck Patent Gesellschaft Mit Beschrankter Haftung Monomeric and dimeric antibody-fragment fusion proteins
US5932448A (en) 1991-11-29 1999-08-03 Protein Design Labs., Inc. Bispecific antibody heterodimers
WO1999061601A2 (fr) 1998-05-28 1999-12-02 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Vecteurs d'aav5 et leurs utilisation
WO2000028061A2 (fr) 1998-11-05 2000-05-18 The Trustees Of The University Of Pennsylvania Sequences d'acide nucleique du serotype i du virus associe aux adenovirus, vecteurs et cellules hotes contenant ces derniers
US6156303A (en) 1997-06-11 2000-12-05 University Of Washington Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom
US6833441B2 (en) 2001-08-01 2004-12-21 Abmaxis, Inc. Compositions and methods for generating chimeric heteromultimers
WO2005003171A2 (fr) 2003-07-01 2005-01-13 Celltech R & D Limited Fragments d'anticorps modifies
WO2005003170A2 (fr) 2003-07-01 2005-01-13 Celltech R & D Limited Fragments d'anticorps modifies
WO2005003169A2 (fr) 2003-07-01 2005-01-13 Celltech R & D Limited Fragments d'anticorps fab modifies
US20060204493A1 (en) 2004-09-02 2006-09-14 Genentech, Inc. Heteromultimeric molecules
US7183076B2 (en) 1997-05-02 2007-02-27 Genentech, Inc. Method for making multispecific antibodies having heteromultimeric and common components
EP1870459A1 (fr) 2005-03-31 2007-12-26 Chugai Seiyaku Kabushiki Kaisha Procede pour la production de polypeptide au moyen de la regulation d'un ensemble
WO2009089004A1 (fr) 2008-01-07 2009-07-16 Amgen Inc. Méthode de fabrication de molécules hétérodimères fc d'anticorps utilisant les effets de conduite électrostatique
US20110195454A1 (en) 2010-02-08 2011-08-11 Regeneron Pharmaceuticals, Inc. Common Light Chain Mouse
US20170130245A1 (en) 2014-06-09 2017-05-11 Voyager Therapeutics, Inc. Chimeric capsids
US20200140491A1 (en) * 2017-06-27 2020-05-07 Regeneron Pharmaceuticals, Inc. Tropism-Modified Recombinant Viral Vectors and Uses Thereof for the Targeted Introduction of Genetic Material into Human Cells
WO2021263060A2 (fr) * 2020-06-24 2021-12-30 Lycia Therapeutics, Inc. Compositions de pontage bifonctionnelles pour la transduction virale

Patent Citations (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5677425A (en) 1987-09-04 1997-10-14 Celltech Therapeutics Limited Recombinant antibody
US5582996A (en) 1990-12-04 1996-12-10 The Wistar Institute Of Anatomy & Biology Bifunctional antibodies and method of preparing same
US5932448A (en) 1991-11-29 1999-08-03 Protein Design Labs., Inc. Bispecific antibody heterodimers
US5910573A (en) 1992-01-23 1999-06-08 Merck Patent Gesellschaft Mit Beschrankter Haftung Monomeric and dimeric antibody-fragment fusion proteins
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US7695936B2 (en) 1995-03-01 2010-04-13 Genentech, Inc. Knobs and holes heteromeric polypeptides
WO1998011244A2 (fr) 1996-09-11 1998-03-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Vecteur de vaa4 et ses utilisations
WO1998025971A1 (fr) 1996-12-10 1998-06-18 Celltech Therapeutics Limited Fragments d'anticorps monovalents
US7183076B2 (en) 1997-05-02 2007-02-27 Genentech, Inc. Method for making multispecific antibodies having heteromultimeric and common components
US6156303A (en) 1997-06-11 2000-12-05 University Of Washington Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom
WO1999015549A2 (fr) 1997-09-19 1999-04-01 Celltech Therapeutics Limited Peptides
WO1999061601A2 (fr) 1998-05-28 1999-12-02 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Vecteurs d'aav5 et leurs utilisation
WO2000028061A2 (fr) 1998-11-05 2000-05-18 The Trustees Of The University Of Pennsylvania Sequences d'acide nucleique du serotype i du virus associe aux adenovirus, vecteurs et cellules hotes contenant ces derniers
US6833441B2 (en) 2001-08-01 2004-12-21 Abmaxis, Inc. Compositions and methods for generating chimeric heteromultimers
WO2005003171A2 (fr) 2003-07-01 2005-01-13 Celltech R & D Limited Fragments d'anticorps modifies
WO2005003169A2 (fr) 2003-07-01 2005-01-13 Celltech R & D Limited Fragments d'anticorps fab modifies
WO2005003170A2 (fr) 2003-07-01 2005-01-13 Celltech R & D Limited Fragments d'anticorps modifies
US20060204493A1 (en) 2004-09-02 2006-09-14 Genentech, Inc. Heteromultimeric molecules
EP1870459A1 (fr) 2005-03-31 2007-12-26 Chugai Seiyaku Kabushiki Kaisha Procede pour la production de polypeptide au moyen de la regulation d'un ensemble
WO2009089004A1 (fr) 2008-01-07 2009-07-16 Amgen Inc. Méthode de fabrication de molécules hétérodimères fc d'anticorps utilisant les effets de conduite électrostatique
US20110195454A1 (en) 2010-02-08 2011-08-11 Regeneron Pharmaceuticals, Inc. Common Light Chain Mouse
US20170130245A1 (en) 2014-06-09 2017-05-11 Voyager Therapeutics, Inc. Chimeric capsids
US20200140491A1 (en) * 2017-06-27 2020-05-07 Regeneron Pharmaceuticals, Inc. Tropism-Modified Recombinant Viral Vectors and Uses Thereof for the Targeted Introduction of Genetic Material into Human Cells
WO2021263060A2 (fr) * 2020-06-24 2021-12-30 Lycia Therapeutics, Inc. Compositions de pontage bifonctionnelles pour la transduction virale

Non-Patent Citations (41)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. NC_006261
AL-LAZIKANI ET AL., J. MOL. BIOL., vol. 273, 1997, pages 927 - 948
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 402
ANGAL ET AL., MOLECULAR IMMUNOLOGY, vol. 30, 1993, pages 105
BANTEL-SCHAAL ET AL., J. VIROLOGY, vol. 73, 1999, pages 3994
BARTLETT J ET AL: "Targeted adeno-associated virus vector transduction of nonpermissive cells mediated by a bispecific F(ab'gamma)2 antibody", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 17, 1 February 1999 (1999-02-01), pages 181 - 186, XP002135910, ISSN: 1087-0156, DOI: 10.1038/6185 *
BENEDICT, CA, J IMMUNOL METHODS., vol. 201, no. 2, 1997, pages 223 - 31
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
CARTER, IMMUNOL METH, vol. 248, 2001, pages 7 - 15
CHEN, ADV DRUG DELIV REV., vol. 65, no. 10, 2013, pages 1357 - 1369
CHIORINI ET AL., J. VIROLOGY, vol. 71, 1998, pages 6823
EHRING, ANALYTICAL BIOCHEMISTRY, vol. 267, no. 2, 1999, pages 252 - 259
ENGENSMITH, ANAL. CHEM., vol. 73, 2001, pages 256A - 265A
GAO ET AL., PROC. NAT. ACAD. SCI. USA, vol. 99, 2002, pages 11854
GEUIJEN, CA ET AL., J IMMUNOL METHODS., vol. 302, no. 1-2, 2005, pages 68 - 77
GONNET ET AL., SCIENCE, vol. 256, 1992, pages 1443 - 1445
GOODSON, MEDICAL APPLICATIONS OF CONTROLLED RELEASE, vol. 2, 1984, pages 115 - 138
HUSTON, PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883
JUNGHANS ET AL., CANCER RES., vol. 50, 1990, pages 1495 - 1502
KABAT: "Sequences of Proteins of Immunological Interest", 1991, NATIONAL INSTITUTES OF HEALTH
KASPER BENDIX JOHNSEN: "Targeting the transferrin receptor for brain drug delivery", PROGRESS IN NEUROBIOLOGY, vol. 181, 1 October 2019 (2019-10-01), AMSTERDAM, NL, pages 101665, XP093159431, ISSN: 0301-0082, Retrieved from the Internet <URL:https://pdf.sciencedirectassets.com/271063/1-s2.0-S0301008219X00106/1-s2.0-S0301008219301340/main.pdf?X-Amz-Security-Token=IQoJb3JpZ2luX2VjEGEaCXVzLWVhc3QtMSJGMEQCIDlvAlwVkur2kWZvbaG+0s98iCem0aS3KOHZHiLt9VfYAiAVU0gytRsbfnk6K3RGLGe0Nyj5Dx5OMt3BFLk0GV1BuCq8BQi5//////////8BEAUaDDA1OTAwMzU0Njg2NSIME2oNT> DOI: 10.1016/j.pneurobio.2019.101665 *
KLEIN, PROTEIN ENGINEERING, DESIGN & SELECTION, vol. 27, no. 10, 2014, pages 325 - 330
LANGER, SCIENCE, vol. 249, 1990, pages 1527 - 1533
LEFRANC, DEV. COMP. IMMUNOL., vol. 27, 2003, pages 55 - 77
MARTIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 9268 - 9272
MCCAFFERTY, NATURE, vol. 348, 1990, pages 552 - 554
MORIS ET AL., VIROLOGY, vol. 33, 2004, pages 375 - 383
MURAMATSU ET AL., VIROLOGY, vol. 221, 1996, pages 208
PADRON ET AL., J. VIROLOGY, vol. 79, 2005, pages 5047 - 58
PEARSON, METHODS MOL. BIOL., vol. 24, 1994, pages 307 - 331
POWELL ET AL.: "Compendium of excipients for parenteral formulations", J PHDOMAIN SCI TECHNOL, vol. 52, 1998, pages 238 - 311, XP009119027
REINEKE, METHODS MOL BIOL, vol. 248, 2004, pages 443 - 463
RIDGWAY ET AL., PROT ENG, vol. 9, 1996, pages 617 - 621
SHADE ET AL., J. VIROL., vol. 58, 1986, pages 921
SRIVISTAVA ET AL., J. VIROLOGY, vol. 45, 1983, pages 555
TAYLOR ET AL., NUCL. ACIDS RES., vol. 20, 1992, pages 6287 - 6295
TOMER, PROTEIN SCIENCE, vol. 9, 2000, pages 487 - 496
ULRICH BRINKMANN ET AL: "The making of bispecific antibodies", MABS, vol. 9, no. 2, 10 January 2017 (2017-01-10), US, pages 182 - 212, XP055374463, ISSN: 1942-0862, DOI: 10.1080/19420862.2016.1268307 *
WU ET AL., J. BIOL. CHEM., vol. 262, 1987, pages 4429 - 4432
XINTAO ZHANG: "Blood-brain barrier shuttle peptides enhance AAV transduction in the brain after systemic administration", BIOMATERIALS, vol. 176, 1 September 2018 (2018-09-01), AMSTERDAM, NL, pages 71 - 83, XP093157024, ISSN: 0142-9612, Retrieved from the Internet <URL:https://pdf.sciencedirectassets.com/271870/1-s2.0-S0142961218X00180/1-s2.0-S0142961218303922/main.pdf?X-Amz-Security-Token=IQoJb3JpZ2luX2VjELv//////////wEaCXVzLWVhc3QtMSJGMEQCIG89EdU6sz/YHCS6v99sBMphmIYOb+Qfs0iTMj8Eve5GAiAc99nUxfLPiXVFaljF6DLXkQhWP+zk+3XMaEyW1Zx8MiqzBQgUEAUaDDA1OTAwMzU0Njg2NSIMpDLhe> DOI: 10.1016/j.biomaterials.2018.05.041 *

Also Published As

Publication number Publication date
WO2025042428A9 (fr) 2025-11-13
EP4630457A1 (fr) 2025-10-15

Similar Documents

Publication Publication Date Title
JP7410522B2 (ja) 抗ｆａｍ１９ａ５抗体のアデノ関連ウイルス（ａａｖ）伝達
CN118922449A (zh) 特异性结合psma和cd28的抗原结合分子及其医药用途
US20250011412A1 (en) Antigen-binding molecules and uses thereof
TW202246333A (zh) 特異性結合ｂｃｍａ和ｃｄ３的抗原結合分子及其醫藥用途
EP4630457A1 (fr) Molécules bispécifiques de liaison à l&#39;antigène et leurs utilisations
AU2024220647A1 (en) Treatment of muscle related disorders with anti-human cacng1 antibodies
CN115772544B (zh) 抗vegf-a和ang-2的aav载体
KR20240139065A (ko) Psma 및 cd3에 특이적으로 결합하는 항원 결합 분자 및 이들의 의약적 용도
WO2024130175A2 (fr) Molécules de liaison à l&#39;antigène qui se lient à des particules d&#39;aav et utilisations
WO2025255452A2 (fr) Molécules de liaison à l&#39;antigène qui se lient à des particules d&#39;aav et leurs utilisations
WO2020243659A1 (fr) Thérapie basée sur un vecteur pour une maladie thyroïdienne
TW202507010A (zh) 工程化之aav衣殼蛋白
WO2024229105A1 (fr) Anticorps anti-m-cadhérine humaine (cdh15), conjugués et leurs utilisations pour l&#39;administration de charges utiles génétiques à des cellules musculaires
CN120082564A (zh) 可用腺相关病毒表达的Her2抗体
WO2025166281A1 (fr) Plateforme d&#39;analyse par spectrométrie de masse à détection de charge d&#39;aavs
EA047634B1 (ru) Доставка антител к fam19a5 с помощью аденоассоциированного вируса (aav)

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23848162

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023848162

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2023848162

Country of ref document: EP

Effective date: 20250710

WWP Wipo information: published in national office

Ref document number: 2023848162

Country of ref document: EP