Aav-mediated Delivery Of Therapeutic Antibodies To The Inner Ear

Abstract

Provided herein are methods that include introducing into an inner ear of a mammal a therapeutically effective amount of an adeno-associated virus (AAV) vector that includes a nucleotide sequence encoding (a) a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide; (b) a polypeptide including an antigen-binding antibody fragment operably linked to a signal peptide; or (c) a soluble vascular endothelial growth factor receptor operably linked to a signal peptide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 62/607,665, filed Dec. 19, 2017; the entire contents of which are herein incorporated by reference.

TECHNICAL FIELD

[0002] The present disclosure relates generally to the use of nucleic acids to treat hearing loss in a human subject.

BACKGROUND OF THE INVENTION

[0003] Sensorinerual hearing loss is hearing loss that is caused by a malfunction of the cells (e.g., hair cells) in an inner ear of a mammal. Non-limiting causes of sensorineural hearing loss include exposure to loud noise, head trauma, viral infection, autoimmune inner ear disease, genetic hearing loss, aging, malformations in the inner ear, Meniere's disease, otosclerosis, and tumors.

SUMMARY

[0004] The present invention relates to methods that include introducing into an inner ear of a mammal (e.g., a human) a therapeutically effective amount of any adeno-associated virus (AAV) vector that includes a nucleotide sequence encoding: (a) a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide including an antigen-binding antibody fragment operably linked to a signal peptide.

[0005] Provided herein are methods for increasing the level of an antibody or an antigen-binding antibody fragment in an inner ear of a mammal in need thereof that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding (a) a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide including an antigen-binding antibody fragment linked to a signal peptide; wherein the introducing results in an increase in the level of the antibody or the antigen-binding antibody fragment in the inner ear of the mammal.

[0006] In some embodiments, the antibody or the antigen-binding antibody fragment binds specifically to vascular endothelial growth factor (VEGF). In some embodiments, the antibody or antigen-binding antibody fragment decreases VEGF activity. In some embodiments of any of the methods described herein, the AAV vector further includes one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment.

[0007] In some embodiments, the AAV vector includes a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of the methods described herein, the AAV vector further includes a polyadenylation signal sequence.

[0008] In some embodiments of any of the methods described herein, the mammal is a human. In some embodiments of any of the methods described herein, the mammal has been identified as having an inner ear disorder. In some embodiments of any of the methods described herein, the mammal has been diagnosed as having an inner ear disorder.

[0009] In some embodiments of any of the methods described herein, the AAV vector includes a nucleic acid sequence encoding a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide.

[0010] In some embodiments of any of the methods described herein, the AAV vector includes a nucleic acid sequence encoding a polypeptide including an antigen-binding antibody fragment operably linked to a signal.

[0011] Also provided herein are methods for treating an inner ear disorder in a mammal in need thereof that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding: (a) a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide including an antigen-binding antibody fragment linked to a signal peptide; wherein the introducing results in the treatment of the inner ear disorder in the mammal.

[0012] In some embodiments of any of the methods described herein, the AAV vector further includes one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment.

[0013] In some embodiments, the AAV vector includes a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.

[0014] In some embodiments of any of the methods described herein, the AAV vector further includes a polyadenylation signal sequence.

[0015] In some embodiments of any of the methods described herein, the mammal is a human. In some embodiments of any of the methods described herein, the mammal has been identified as having an inner ear disorder. In some embodiments of any of the methods described herein, the mammal has been diagnosed as having an inner ear disorder.

[0016] In some embodiments of any of the methods described herein, the AAV vector includes a nucleic acid sequence encoding a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide.

[0017] In some embodiments of any of the methods described herein, the AAV vector includes a nucleic acid sequence encoding a polypeptide including an antigen-binding antibody fragment operably linked to a signal.

[0018] Also provided herein are methods of reducing VEGF activity in an inner ear of a mammal in need thereof that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding (a) a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide including an antigen-binding antibody fragment linked to a signal peptide; wherein the polypeptide of (a) encodes an antibody that binds specifically to VEGF and reduces VEGF activity, the polypeptide of (b) encodes an antigen-binding antibody fragment that binds specifically to VEGF and reduces VEGF activity; wherein the introducing results in a reduction in VEGF activity in the inner ear of the mammal.

[0019] In some embodiments of any of the methods described herein, the AAV vector further includes one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment.

[0020] In some embodiments, the AAV vector includes a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.

[0021] In some embodiments of any of the methods described herein, the AAV vector further includes a polyadenylation signal sequence. In some embodiments of any of the methods described herein, the mammal is a human.

[0022] In some embodiments of any of the methods described herein, the mammal has been identified or diagnosed as having an acoustic neuroma. In some embodiments, the mammal has been identified or diagnosed as having a vestibular schwannoma. In some embodiments of any of the methods described herein, the mammal has been identified or diagnosed as having a neurofibromatosis type 2.

[0023] In some embodiments of any of the methods described herein, the AAV vector includes a nucleic acid sequence encoding a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide.

[0024] In some embodiments of any of the methods described herein, the AAV vector includes a nucleic acid sequence encoding a polypeptide including an antigen-binding antibody fragment operably linked to a signal peptide.

[0025] Also provided herein are methods of treating acoustic neuroma, vestibular schwannoma, or neurofibromatosis type 2 in an inner ear of a mammal that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding (a) a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide including an antigen-binding antibody fragment linked to a signal peptide; wherein the polypeptide of (a) encodes an antibody that binds specifically to VEGF and reduces VEGF activity, the polypeptide of (b) encodes an antigen-binding antibody fragment that binds specifically to VEGF and reduces VEGF activity; wherein the introducing results in treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II, respectively, in the inner ear of the mammal.

[0026] In some embodiments of any of the methods described herein, the AAV vector further includes one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment.

[0027] In some embodiments of any of the methods described herein, the AAV vector includes a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.

[0028] In some embodiments of any of the methods described herein, the AAV vector further includes a polyadenylation signal sequence.

[0029] In some embodiments of any of the methods described herein, the mammal is a human. In some embodiments of any of the methods described herein, the mammal has been identified or diagnosed as having an acoustic neuroma. In some embodiments of any of the methods described herein, the mammal has been identified or diagnosed as having a vestibular schwannoma. In some embodiments of any of the methods described herein, the mammal has been identified or diagnosed as having neurofibromatosis type 2.

[0030] In some embodiments of any of the methods described herein, the AAV vector includes a nucleic acid sequence encoding a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide.

[0031] In some embodiments of any of the methods described herein, the AAV vector includes a nucleic acid sequence encoding a polypeptide including an antigen-binding antibody fragment operably linked to a signal peptide.

[0032] In some embodiments of any of the methods described herein, the antibody includes a Fc region that includes one or more amino acid substitutions that decreases the half-life of the antibody in a mammal as compared to a control antibody; or the antigen-binding antibody fragment thereof has a decreased in vivo half-life as compared to a control antigen-binding antibody fragment.

[0033] Also provided herein are methods that include introducing into an inner ear of a mammal a therapeutically effective amount of an adeno-associated virus (AAV) vector that includes a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide.

[0034] Also provided herein are methods for increasing the level of a soluble vascular endothelial growth factor (VEGF) receptor in an inner ear of a mammal in need thereof that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide; where the introducing results in an increase in the level of the soluble VEGF receptor in the inner ear of the mammal.

[0035] In some embodiments of any of the methods described herein, the soluble VEGF receptor includes a portion of an extracellular region of VEGF receptor-1 (VEGFR-1). In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-1 includes a contiguous sequence from wildtype human VEGFR-1. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-1 includes one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-1 includes a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-1.

[0036] In some embodiments of any of the methods described herein, the soluble VEGF receptor includes a portion of an extracellular region of VEGF receptor-2 (VEGFR-2). In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-2 includes a contiguous sequence from wildtype human VEGFR-2. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-2 includes one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-2 includes a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-2.

[0037] In some embodiments of any of the methods described herein, the soluble VEGF receptor includes a portion of an extracellular region of VEGFR-1 and a portion of an extracellular region of VEGFR-2. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-1 includes one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1; and the portion of the extracellular region of VEGFR-2 includes one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2. In some embodiments of any of the methods described herein, the soluble VEGF receptor is aflibercept.

[0038] In some embodiments of any of the methods described herein, the soluble VEGF receptor includes a portion of an extracellular region of VEGF receptor-3 (VEGFR-3). In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-3 includes a contiguous sequence from wildtype human VEGFR-3. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-3 includes one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-3. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-3 includes a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-3. In some embodiments of any of the methods described herein, the soluble VEGF receptor comprises a Fc domain. In some embodiments of any of the methods described herein, the Fc domain is an IgG1 Fc domain. In some embodiments of any of the methods described herein, the IgG1 Fc domain is a human wildtype IgG1 Fc domain.

[0039] In some embodiments of any of the methods described herein, the soluble VEGF receptor decreases the ability of a VEGF to bind to one or more of VEGFR-1, VEGFR-2, and VEGFR-3.

[0040] In some embodiments of any of the methods described herein, the AAV vector further includes one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the soluble VEGF receptor. In some embodiments of any of the methods described herein, the AAV vector includes a promoter selected from the group of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of the methods described herein, the AAV vector further includes a polyadenylation signal sequence.

[0041] In some embodiments of any of the methods described herein, the mammal is a human. In some embodiments of any of the methods described herein, the mammal has been identified as having an inner ear disorder. In some embodiments of any of the methods described herein, the mammal has been diagnosed as having an inner ear disorder.

[0042] Also provided herein are methods for treating an inner ear disorder in a mammal in need thereof that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide; where the introducing results in the treatment of the inner ear disorder in the mammal.

[0043] In some embodiments of any of the methods described herein, the AAV vector further includes one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the soluble VEGF receptor. In some embodiments of any of the methods described herein, the AAV vector includes a promoter selected from the group of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of the methods described herein, the AAV vector further includes a polyadenylation signal sequence.

[0044] In some embodiments of any of the methods described herein, the mammal is a human. In some embodiments of any of the methods described herein, the mammal has been identified as having an inner ear disorder. In some embodiments of any of the methods described herein, the mammal has been diagnosed as having an inner ear disorder.

[0045] Also provided herein are methods of reducing a VEGF activity in an inner ear of a mammal in need thereof that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide; where the introducing results in a reduction in the VEGF activity in the inner ear of the mammal.

[0046] In some embodiments of any of the methods described herein, the AAV vector further includes one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the soluble VEGF receptor. In some embodiments of any of the methods described herein, the AAV vector includes a promoter selected from the group of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of the methods described herein, the AAV vector further includes a polyadenylation signal sequence.

[0047] In some embodiments of any of the methods described herein, the mammal is a human. In some embodiments of any of the methods described herein, the mammal has been identified or diagnosed as having an acoustic neuroma. In some embodiments of any of the methods described herein, the mammal has been identified or diagnosed as having a vestibular schwannoma. In some embodiments of any of the methods described herein, the mammal has been identified or diagnosed as having a neurofibromatosis type 2.

[0048] Also provided herein are methods of treating acoustic neuroma, vestibular schwannoma, or neurofibromatosis type 2 in an inner ear of a mammal that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide; where the introducing results in treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II, respectively, in the inner ear of the mammal.

[0049] In some embodiments of any of the methods described herein, the AAV vector further includes one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the soluble VEGF receptor. In some embodiments of any of the methods described herein, the AAV vector includes a promoter selected from the group of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of the methods described herein, the AAV vector further includes a polyadenylation signal sequence.

[0050] In some embodiments of any of the methods described herein, the mammal is a human. In some embodiments of any of the methods described herein, the mammal has been identified or diagnosed as having an acoustic neuroma. In some embodiments of any of the methods described herein, the mammal has been identified or diagnosed as having a vestibular schwannoma. In some embodiments of any of the methods described herein, the mammal has been identified or diagnosed as having neurofibromatosis type 2.

[0051] In some embodiments of any of the methods described herein, the soluble VEGF receptor includes a portion of an extracellular region of VEGF receptor-1 (VEGFR-1). In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-1 includes a contiguous sequence from wildtype human VEGFR-1. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-1 includes one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-1 includes a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-1.

[0052] In some embodiments of any of the methods described herein, the soluble VEGF receptor includes a portion of an extracellular region of VEGF receptor-2 (VEGFR-2). In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-2 includes a contiguous sequence from wildtype human VEGFR-2. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-2 includes one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-2 includes a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-2.

[0053] In some embodiments of any of the methods described herein, the soluble VEGF receptor includes a portion of an extracellular region of VEGFR-1 and a portion of an extracellular region of VEGFR-2. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-1 includes one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1; and the portion of the extracellular region of VEGFR-2 includes one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2. In some embodiments of any of the methods described herein, the soluble VEGF receptor is aflibercept.

[0054] In some embodiments of any of the methods described herein, the soluble VEGF receptor includes a portion of an extracellular region of VEGF receptor-3 (VEGFR-3). In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-3 includes a contiguous sequence from wildtype human VEGFR-3. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-3 includes one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-3. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-3 includes a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-3.

[0055] In some embodiments of any of the methods described herein, the soluble VEGF receptor comprises a Fc domain. In some embodiments of any of the methods described herein, the Fc domain is an IgG1 Fc domain. In some embodiments of any of the methods described herein, the IgG1 Fc domain is a human wildtype IgG1 Fc domain.

[0056] In some embodiments of any of the methods described herein, the soluble VEGF receptor decreases the ability of a VEGF to bind to one or more of VEGFR-1, VEGFR-2, and VEGFR-3. In some embodiments of any of the methods described herein, the AAV vector further includes a secretion sequence.

[0057] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence.

[0058] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0059] The term "transfected," "transformed," or "transduced" refers to a process by which exogenous nucleic acid is transferred or introduced into a cell. A "transfected," "transformed," or "transduced" mammalian cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid.

[0060] The term "expression" refers to the transcription and/or translation of a particular nucleotide sequence encoding a protein.

[0061] The term "transient expression" refers to the expression of a non-integrated coding sequence for a short period of time (e.g., hours or days). The coding sequence that is transiently expressed in a cell (e.g., a mammalian cell) is lost upon multiple rounds of cell division.

[0062] The term "subject" is intended to include any mammal. In some embodiments, the subject is a rodent (e.g., a rat or mouse), a rabbit, a sheep, a goat, a pig, a dog, a cat, a non-human primate, or a human. In some embodiments, the subject has or is at risk of developing non-syndromic deafness. In some embodiments, the subject has been previously identified as having an inner ear disorder. In some embodiments, the subject has previously been diagnosed as having an inner ear disorder. In some embodiments, the subject has been identified as having drug-induced hearing loss. In some embodiments, the subject is an infant (e.g., a human infant).

[0063] A treatment is "therapeutically effective" when it results in a reduction in one or more of the number, severity, and frequency of one or more symptoms of a disease (e.g., non-symptomatic sensorineural hearing loss) in a subject (e.g., a human).

[0064] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some embodiments of any of the nucleic acids described herein, the nucleic acid is DNA. In some embodiments of any of the nucleic acids described herein, the nucleic acid is RNA.

[0065] The term "signal peptide" refers to a sequence present on the N-terminus of a nascent secreted protein but is absent in the naturally-occurring mature protein. A "signal peptide" is cleaved by a protease (e.g., a signal peptidase) after the signal peptide is translated. Signal peptides are known in the art. Non-limiting examples of signal peptides include: MEFFKKTALAALVMGFSGAALA (SEQ ID NO: 9) and MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 10).

[0066] The term "inner ear disorder" refers to a disorder caused by malfunction of the cells (e.g., hair cells, supporting cells, spiral ganglion neurons, macrophages, or schwann cells) in or around the inner ear of a mammal. Non-limiting examples of inner ear disorders include, e.g., sensorineural hearing loss (SNHL), noise-induced hearing loss, drug-induced hearing loss, age-related hearing loss, acoustic neuroma, neurofibromatosis type 2, auditory neuropathy, noise-induced cochlear synaptopathy without hair cell loss, age-related cochlear synaptopathy, acquired sensorineural hearing loss, and vestibular schwannoma. See, e.g., Kujawa et al., Hear Res 330(0 0): 191-199, 2015; and Suzuki et al., Scientific Reports 6: 24907. Non-limiting examples of inner ear disorders are described herein and additional examples of inner ear disorders are known in the art.

[0067] The term "antibody" means a complex of two or more single polypeptide chains that interact to form at least one antigen-binding domain. Non-limiting examples of an antibody include monoclonal antibodies (for example, full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific, trispecific, etc. antibodies so long as they exhibit the desired biological activity). An antibody can be human, humanized, and/or affinity-matured.

[0068] The term "antigen-binding antibody fragment" is a single polypeptide that includes all the amino acids that make up at least one antigen-binding domain (e.g., an scFv).

[0069] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0070] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)).

[0071] An "antigen-binding domain" is one or more protein domain(s) (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides) that is capable of specifically binding to one or more different antigens. In some examples, an antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of naturally-occurring antibodies. In some embodiments, an antigen-binding domain can include an alternative scaffold. Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-binding domains are known in the art. In some examples, an antigen-binding domain can bind to a single antigen.

[0072] "Affinity" refers to the strength of the sum total of non-covalent interactions between an antigen-binding site and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein, "affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of an antigen-binding domain and an antigen or epitope. The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (K.sub.D). Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE.RTM.) or biolayer interferometry (e.g., FORTEBIO.RTM.). Additional methods for determining the affinity between an antigen-binding domain and its corresponding antigen or epitope are known in the art.

[0073] The phrase "half-life" refers to the half-life of an antibody, an antigen-binding antibody fragment thereof, or a soluble VEGF receptor in circulation (e.g., blood) of a mammal (e.g., any of the mammals described herein) and is represented by the time required for 50% of an antibody, an antigen-binding antibody fragment thereof, or soluble VEGF receptor to be cleared from the circulation. In some embodiments, an alteration in half-life (e.g., a decrease in half-life of an antibody, an antigen-binding antibody fragment thereof, or soluble VEGF receptor) is determined by comparing the half-life of an antibody, an antigen-binding antibody fragment, or a soluble VEGF receptor in a subject to the half-life of a control antibody, control antigen-binding antibody fragment, or control soluble VEGF receptor in a similar mammal.

[0074] In some embodiments, the half-life of an antibody, antigen-binding antibody fragment thereof, or soluble VEGF receptor in a mammal is determined by measuring the level of the antibody, antigen-binding antibody fragment thereof, or soluble VEGF receptor in samples obtained from a subject (e.g., a blood sample) at different time points following systemic administration (e.g., intravenous) administration of any of the AAV vectors described herein. In some embodiments, the level of the antibody, antigen-binding antibody fragment thereof, or soluble VEGF receptor present in samples obtained from a mammal is determined using enzyme-linked immunosorbent assay (ELISA) or another assay known to the art, and the determined level of the antibody, antigen-binding antibody fragment thereof, or soluble VEGF receptor present in the samples is plotted as a function of time using a software program (e.g., GraphPad Prism).

[0075] The term "VEGF activity" refers to one or more known activities of a VEGF protein. For example, one activity of a VEGF protein is the ability to bind to one or more VEGF receptors. In another example, one activity of a VEGF protein is the ability of a VEGF to trigger downstream signal transduction pathway(s) in a mammalian cell expressing a VEGF receptor. Methods for detecting one or more activities of VEGF are known in the art.

[0076] The term "soluble VEGF receptor" refers to a polypeptide that includes a portion of an extracellular region of one or more mammalian VEGF receptor(s) (e.g., VEGFR-1, VEGFR-2, and VEGFR-3) operably linked to a signal peptide, where the soluble VEGF receptor is capable of specifically binding to one or more mammalian VEGF proteins (e.g., one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D). In some examples, a soluble VEGF receptor includes a portion of an extracellular region of VEGFR-1 (e.g., a contiguous sequence from wildtype human VEGFR-1 (e.g., one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1) or a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-1). In some examples, a soluble VEGF receptor includes a portion of an extracellular region of VEGFR-2 (e.g., a contiguous sequence from wildtype human VEGFR-2 (e.g., one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2) or a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-2). In some examples, a soluble VEGF receptor includes a portion of an extracellular region of VEGFR-1 and a portion of an extracellular region of VEGFR-2 (e.g., one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1 and one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2) (e.g., aflibercept). In some examples, a soluble VEGF receptor includes a portion of an extracellular region of VEGFR-3 (e.g., a contiguous sequence from wildtype human VEGFR-3 (e.g., one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-3) or a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-3).

[0077] In some examples, a soluble VEGF receptor can further include a stabilizing domain (e.g., a Fc domain, such as an IgG1 Fc domain (e.g., a human wildtype IgG1 Fc domain). In some examples, the soluble VEGF receptor decreases the ability of a VEGF to bind to one or more (e.g., two or three) of VEGFR-1, VEGFR-2, and VEGFR-3. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

BRIEF DESCRIPTION OF DRAWINGS

[0078] FIG. 1A is an exemplary AAV vector of 4474 bp that includes a sequence encoding bevacizumab (Avastin.RTM.).

[0079] FIG. 1B is an exemplary AAV vector of 3814 bp that includes a sequence encoding ranibizumab (Lucentis.RTM.).

[0080] FIG. 1C is an exemplary AAV vector of 4573 bp that includes a sequence encoding ranibizumab and green fluorescent protein (GFP).

[0081] FIG. 1D is an exemplary AAV vector of 3631 bp that includes a sequence encoding aflibercept (Eylea.RTM.).

[0082] FIG. 2 is a Western blot showing HEK cell expression of different anti-VEGF antibodies or antigen-binding antibody fragments, or soluble VEGF receptors using exemplary AAV vectors described herein. Lane 1: pre-stained PageRuler.TM. protein ladder. Lane 2: untransfected/negative control. Lane 3: tranfection with the AAV vector shown in FIG. 1A. Lane 4: transfection with the AAV vector shown in FIG. 1C. Lane 5: transfection with the AAV vector shown in FIG. 1B. Lane 6: transfection with the AAV vector shown in FIG. 1A with an multiplicity of infection (MOI) of 7.5.times.10.sup.4. Lane 7: transfection with the AAV vector shown in FIG. 1A with an MOI of 2.2.times.10.sup.5. Lane 8: transfection with the AAV vector shown in FIG. 1A with an MOI of 5.5.times.10.sup.5. Lane 9: prestained PageRuler.TM. protein ladder. Lane 10: untransfected/negative control. Lane 11: tranfection with the AAV vector shown in FIG. 1A. Lane 12: transfection with the AAV vector shown in FIG. 1C. Lane 13: transfection with the AAV vector shown in FIG. 1B. Lane 14: transfection with the AAV vector shown in FIG. 1A with an multiplicity of infection (MOI) of 7.5.times.10.sup.4. Lane 15: transfection with the AAV vector shown in FIG. 1A with an MOI of 2.2.times.10.sup.5. Lane 16: transfection with the AAV vector shown in FIG. 1A with an MOI of 5.5.times.10.sup.5. Lanes 2-8 contain reduced proteins. Lanes 10-16 contain non-reduced proteins.

[0083] FIG. 3A is a graph showing the affinity of a control mouse anti-human VEGF monoclonal antibody (anti-hVEGF MmAb) in a buffer using recombinant human VEGF as the binding agent, as measured by Octet.RTM. HTX biosensor instrument using the Octet.RTM. analysis software, Data Analysis HT10.0.

[0084] FIG. 3B is a graph showing the affinity of a control anti-hVEGF MmAb in conditioned media (CM) samples using recombinant human VEGF as the binding agent, as measured by Octet.RTM. HTX biosensor instrument using the Octet.RTM. analysis software, Data Analysis HT10.0. *: anti-hVEGF MmAb was prepared in CM at 100 .mu.g/mL, then diluted to a final concentration of 10 .mu.g/mL in 1.times. kinetics buffer.

[0085] FIG. 4A is a graph showing the affinity of conditioned medium using recombinant human VEGF as the binding agent, as measured by Octet.RTM. HTX biosensor instrument using the Octet.RTM. analysis software, Data Analysis HT10.0.

[0086] FIG. 4B is a graph showing the affinity of culture medium from HEK cells transfected with the AAV vector shown in FIG. 1A using recombinant human VEGF as the binding agent, using by Octet.RTM. HTX biosensor instrument using the Octet.RTM. analysis software, Data Analysis HT10.0.

[0087] FIG. 4C is a table showing the equilibrium dissociation constant (K.sub.D) determined from the data shown in FIGS. 3A, 3B, 4A, and 4B (going from the top to the bottom of the table).

DETAILED DESCRIPTION

[0088] Provided herein are methods that include introducing into an inner ear of a mammal a therapeutically effective amount of an adeno-associated virus (AAV) vector that includes a nucleotide sequence encoding: (a) a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide including an antigen-binding antibody fragment (e.g., a Fab or a scFv)operably linked to a signal peptide.

[0089] Also provided herein are methods for increasing the level of an antibody or an antigen-binding antibody fragment in an inner ear of a mammal in need thereof, that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding: (a) a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide including an antigen-binding antibody fragment (e.g., a Fab or a scFv) operably linked to a signal peptide; wherein the introducing results in an increase in the level of the antibody or the antigen-binding antibody fragment in the inner ear of the mammal.

[0090] Also provided are methods for treating an inner ear disorder in a mammal in need thereof that include introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding: (a) a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide; where the introducing results in the treatment of the inner ear disorder in the mammal.

[0091] Also provided herein are methods of reducing VEGF activity in an inner ear of a mammal in need thereof that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding (a) a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide including an antigen-binding antibody fragment (e.g., a Fab or a scFv) operably linked to a signal peptide; wherein the polypeptide of (a) encodes an antibody that binds specifically to VEGF and reduces VEGF activity, the polypeptide of (b) encodes an antigen-binding antibody fragment that binds specifically to VEGF and reduces VEGF activity; and wherein the introducing results in a reduction in VEGF activity in the inner ear of the mammal.

[0092] Also provided herein are methods of treating acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II in an inner ear of a mammal that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding (a) a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide including an antigen-binding antibody fragment (e.g., a Fab or a scFv) operably linked to a signal peptide; wherein the polypeptide of (a) encodes an antibody that binds specifically to VEGF and reduces VEGF activity, the polypeptide of (b) encodes an antigen-binding antibody fragment that binds specifically to VEGF and reduces VEGF activity; and wherein the introducing results in treatment of acoustic neuroma or vestibular schwannoma in the inner ear of the mammal.

[0093] Also provided herein are methods that include introducing into an inner ear of a mammal a therapeutically effective amount of an adeno-associated virus (AAV) vector that include a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide.

[0094] Also provided herein are methods for increasing the level of a soluble vascular endothelial growth factor (VEGF) receptor in an inner ear of a mammal in need thereof that include introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide; where the introducing results in an increase in the level of the soluble VEGF receptor in the inner ear of the mammal.

[0095] Also provided herein are methods for treating an inner ear disorder in a mammal in need thereof that include introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide; where the introducing results in the treatment of the inner ear disorder in the mammal.

[0096] Also provided herein are methods of reducing a VEGF activity in an inner ear of a mammal in need thereof that include introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide; where the introducing results in a reduction in the VEGF activity in the inner ear of the mammal.

[0097] Also provided herein are methods of treating acoustic neuroma, vestibular schwannoma, or neurofibromatosis type 2 in an inner ear of a mammal that include introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide; where the introducing results in treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II, respectively, in the inner ear of the mammal.

[0098] Also provided are kits that include any of the AAV vectors described herein.

[0099] Additional non-limiting aspects of the compositions, kits, and methods are described herein and can be used in any combination without limitation.

Antibodies and Antigen-Binding Antibody Fragments

[0100] In some embodiments, the antibody can be a humanized antibody, a chimeric antibody, or a multivalent antibody. In some embodiments, an antibody or an antigen-binding antibody fragment can be a scFv-Fc, a V.sub.HH domain, a V.sub.NAR domain, a (scFv).sub.2, a minibody, or a BiTE. In some embodiments, an antibody or an antigen-binding antibody fragment can be a DVD-Ig, and a dual-affinity re-targeting antibody (DART), a triomab, kih IgG with a common LC, a crossmab, an ortho-Fab IgG, a 2-in-1-IgG, IgG-ScFv, scFv.sub.2-Fc, a bi-nanobody, tanden antibody, a DART-Fc, a scFv-HAS-scFv, DNL-Fab3, DAF (two-in-one or four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair antibody, Fab-arm exchange antibody, SEEDbody, Triomab, LUZ-Y, Fcab, k.lamda.-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)-IgG, IgG (L,H)-Fc, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, nanobody, nanobody-HSA, a diabody, a TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triple Body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab').sub.2-scFV.sub.2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock and lock bispecific antibody, ImmTAC, HSAbody, scDiabody-HAS, tandem scFv, IgG-IgG, Cov-X-Body, and seFv1-PEG-seFv2.

[0101] Additional examples of an antibody or an antigen-binding antibody fragment include an Fv fragment, a Fab fragment, a F(ab').sub.2 fragment, and a Fab' fragment. Additional examples of an antibody or an antigen-binding antibody fragment include an antigen-binding fragment of an IgG (e.g., an antigen-binding fragment of IgG1, IgG2, IgG3, or IgG4) (e.g., an antigen-binding fragment of a human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4); an antigen-binding fragment of an IgA (e.g., an antigen-binding fragment of IgA1 or IgA2) (e.g., an antigen-binding fragment of a human or humanized IgA, e.g., a human or humanized IgA1 or IgA2); an antigen-binding fragment of an IgD (e.g., an antigen-binding fragment of a human or humanized IgD); an antigen-binding fragment of an IgE (e.g., an antigen-binding fragment of a human or humanized IgE); or an antigen-binding fragment of an IgM (e.g., an antigen-binding fragment of a human or humanized IgM).

[0102] Any of the antibodies or antigen-binding antibody fragments described herein can bind specifically to VEGF.

[0103] A V.sub.HH domain is a single monomeric variable antibody domain that can be found in camelids. A V.sub.NAR domain is a single monomeric variable antibody domain that can be found in cartilaginous fish. Non-limiting aspects of V.sub.HH domains and V.sub.NAR domains are described in, e.g., Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., Trends Biotechnol. 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al., Trends Pharmacol. Sci. 35:247-255, 2014; Muyldermans, J. Biotechnol. 74:277-302, 2001; Muyldermans et al., Trends Biochem. Sci. 26:230-235, 2001; Muyldermans, Ann. Rev. Biochem. 82:775-797, 2013; Rahbarizadeh et al., Immunol. Invest. 40:299-338, 2011; Van Audenhove et al., EBioMedicine 8:40-48, 2016; Van Bockstaele et al., Curr. Opin. Investig. Drugs 10:1212-1224, 2009; Vincke et al., Methods Mol. Biol. 911:15-26, 2012; and Wesolowski et al., Med. Microbiol. Immunol. 198:157-174, 2009.

[0104] A "Fv" fragment includes a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.

[0105] A "Fab" fragment includes, the constant domain of the light chain and the first constant domain (C.sub.H1) of the heavy chain, in addition to the heavy and light chain variable domains of the Fv fragment.

[0106] A "F(ab').sub.2" fragment includes two Fab fragments joined, near the hinge region, by disulfide bonds.

[0107] A "dual variable domain immunoglobulin" or "DVD-Ig" refers to multivalent and multispecific binding proteins as described, e.g., in DiGiammarino et al., Methods Mol. Biol. 899:145-156, 2012; Jakob et al., MABs 5:358-363, 2013; and U.S. Pat. Nos. 7,612,181; 8,258,268; 8,586,714; 8,716,450; 8,722,855; 8,735,546; and 8,822,645, each of which is incorporated by reference in its entirety.

[0108] DARTs are described in, e.g., Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0109] Additional aspects of antibodies and antigen-binding antibody fragments are known in the art.

[0110] In some embodiments, any of the antibodies or antigen-binding antibody fragments described herein has a dissociation constant (K.sub.D) of less than 1.times.10.sup.-5 M (e.g., less than 0.5.times.10.sup.-5 M, less than 1.times.10.sup.-6 M, less than 0.5.times.10.sup.-6 M, less than 1.times.10.sup.-7 M, less than 0.5.times.10.sup.-7 M, less than 1.times.10.sup.-8 M, less than 0.5.times.10.sup.-8 M, less than 1.times.10.sup.-9 M, less than 0.5.times.10.sup.-9 M, less than 1.times.10.sup.-10 M, less than 0.5.times.10.sup.-10 M, less than 1.times.10.sup.-11 M, less than 0.5.times.10.sup.-11 M, or less than 1.times.10.sup.-12 M), e.g., as measured in phosphate buffered saline using surface plasmon resonance (SPR) for a VEGF protein (e.g., any of the VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D).

[0111] In some embodiments, any of the antibodies or antigen-binding antibody fragments described herein has a K.sub.D of about 1.times.10.sup.-12 M to about 1.times.10.sup.-5 M, about 0.5.times.10.sup.-5 M, about 1.times.10.sup.-6 M, about 0.5.times.10.sup.-6 M, about 1.times.10.sup.-7 M, about 0.5.times.10.sup.-7 M, about 1.times.10.sup.-8 M, about 0.5.times.10.sup.-8 M, about 1.times.10.sup.-9 M, about 0.5.times.10.sup.-9 M, about 1.times.10.sup.-10 M, about 0.5.times.10.sup.-10 M, about 1.times.10.sup.-11 M, or about 0.5.times.10.sup.-11 M (inclusive); about 0.5.times.10.sup.-11 M to about 1.times.10.sup.-5 M, about 0.5.times.10.sup.-5 M, about 1.times.10.sup.-6 M, about 0.5.times.10.sup.-6 M, about 1.times.10.sup.-7 M, about 0.5.times.10.sup.-7 M, about 1.times.10.sup.-8 M, about 0.5.times.10.sup.-8 M, about 1.times.10.sup.-9 M, about 0.5.times.10.sup.-9 M, about 1.times.10.sup.-10 M, about 0.5.times.10.sup.-10 M, or about 1.times.10.sup.-11 M (inclusive); about 1.times.10.sup.-11 M to about 1.times.10.sup.-5 M, about 0.5.times.10.sup.-5 M, about 1.times.10.sup.-6 M, about 0.5.times.10.sup.-6 M, about 1.times.10.sup.-7 M, about 0.5.times.10.sup.-7 M, about 1.times.10.sup.-8 M, about 0.5.times.10.sup.-8 M, about 1.times.10.sup.-9 M, about 0.5.times.10.sup.-9 M, about 1.times.10.sup.-10 M, or about 0.5.times.10.sup.-10 M (inclusive); about 0.5.times.10.sup.-10 M to about 1.times.10.sup.-5 M, about 0.5.times.10.sup.-5 M, about 1.times.10.sup.-6 M, about 0.5.times.10.sup.-6 M, about 1.times.10.sup.-7 M, about 0.5.times.10.sup.-7 M, about 1.times.10.sup.-8 M, about 0.5.times.10.sup.-8 M, about 1.times.10.sup.-9 M, about 0.5.times.10.sup.-9 M, or about 1.times.10.sup.-10 M (inclusive); about 1.times.10.sup.-10 M to about 1.times.10.sup.-5 M, about 0.5.times.10.sup.-5 M, about 1.times.10.sup.-6 M, about 0.5.times.10.sup.-6 M, about 1.times.10.sup.-7 M, about 0.5.times.10.sup.-7 M, about 1.times.10.sup.-8 M, about 0.5.times.10.sup.-8 M, about 1.times.10.sup.-9 M, or about 0.5.times.10.sup.-9 M (inclusive); about 0.5.times.10.sup.-9 M to about 1.times.10.sup.-5 M, about 0.5.times.10.sup.-5 M, about 1.times.10.sup.-6 M, about 0.5.times.10.sup.-6 M, about 1.times.10.sup.-7 M, about 0.5.times.10.sup.-7 M, about 1.times.10.sup.-8 M, about 0.5.times.10.sup.-8 M, or about 1.times.10.sup.-9 M (inclusive); about 1.times.10.sup.-9 M to about 1.times.10.sup.-5 M, about 0.5.times.10.sup.-5 M, about 1.times.10.sup.-6 M, about 0.5.times.10.sup.-6 M, about 1.times.10.sup.-7 M, about 0.5.times.10.sup.-7 M, about 1.times.10.sup.-8 M, or about 0.5.times.10.sup.-8 M (inclusive); about 0.5.times.10.sup.-8 M to about 1.times.10.sup.-5 M, about 0.5.times.10.sup.-5 M, about 1.times.10.sup.-6 M, about 0.5.times.10.sup.-6 M, about 1.times.10.sup.-7 M, about 0.5.times.10.sup.--7 M, or about 1.times.10.sup.-8 M (inclusive); about 1.times.10.sup.-8 M to about 1.times.10.sup.-5 M, about 0.5.times.10.sup.-5 M, about 1.times.10.sup.-6 M, about 0.5.times.10.sup.-6 M, about 1.times.10.sup.-7 M, or about 0.5.times.10.sup.-7 M (inclusive); about 0.5.times.10.sup.-7 M to about 1.times.10.sup.-5 M, about 0.5.times.10.sup.-5 M, about 1.times.10.sup.-6 M, about 0.5.times.10.sup.-6 M, or about 1.times.10.sup.-7 M (inclusive); about 1.times.10.sup.-7 M to about 1.times.10.sup.-5 M, about 0.5.times.10.sup.-5 M, about 1.times.10.sup.-6 M, or about 0.5.times.10.sup.-6 M (inclusive); about 0.5.times.10.sup.-6 M to about 1.times.10.sup.-5 M, about 0.5.times.10.sup.-5 M, or about 1.times.10.sup.-6 M (inclusive); about 1.times.10.sup.-6 M to about 1.times.10.sup.-5 M or about 0.5.times.10.sup.-5 M (inclusive); or about 0.5.times.10.sup.-5 M to about 1.times.10.sup.-5 M (inclusive), e.g., as measured in phosphate buffered saline using surface plasmon resonance (SPR), for a VEGF protein (e.g., any of the VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D).

[0112] A variety of different methods known in the art can be used to determine the K.sub.D values of any of the antibodies or antigen-binding antibody fragments described herein (e.g., an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.).

[0113] In some embodiments of any of the antibodies and/or antigen-binding antibody fragments described herein, the half-life of the antibody and/or the antigen-binding antibody fragment in a subject (e.g., a human) is decreased about 0.5-fold to about 4-fold (e.g., about 0.5-fold to about 3.5-fold, about 0.5-fold to about 3-fold, about 0.5-fold to about 2.5-fold, about 0.5-fold to about 2-fold, about 0.5-fold to about 1.5-fold, about 0.5-fold to about 1-fold, about 1-fold to about 4-fold, about 1-fold to about 3.5-fold, about 1-fold to about 3-fold, about 1-fold to about 2.5-fold, about 1-fold to about 2-fold, about 1.5-fold to about 4-fold, about 1.5-fold to about 3.5-fold, about 1.5-fold to about 3-fold, about 1.5-fold to about 2.5-fold, about 1.5-fold to about 2-fold, about 2-fold to about 4-fold, about 2-fold to about 3.5-fold, about 2-fold to about 3-fold, about 2-fold to about 2.5-fold, about 2.5-fold to about 4-fold, about 2.5-fold to about 3.5-fold, about 2.5-fold to about 3-fold, about 3-fold to about 4-fold, about 3-fold to about 3.5-fold, or about 3.5-fold to about 4-fold) as compared to the half-life of a control antibody and/or a control antigen-binding antibody fragment (e.g., any of the control antibodies and control antigen-binding antibody fragments described herein) in a similar subject. See, e.g., Leabman et al., MAbs. 5(6): 896-903, 2013. In some embodiments, an antibody or antigen-binding antibody fragment described herein has one or more amino acid substitutions in the Fc region that decrease its half-life in a mammal, and a control antibody lacks at least one (e.g., lacks all) of these one or more amino acid substiutions in the Fc region.

VEGF

[0114] The VEGF gene encodes vascular endothelial growth factor (VEGF), formerly known as fms-like tyrosine kinase (Flt-1). The VEGF protein is a heparin-biding protein that induces migration and proliferation of vascular endothelial cells.

[0115] Non-limiting examples of protein and nucleotide sequences encoding a wildtype VEGF protein are shown below.

TABLE-US-00001 Human VEGF Transcript Variant 1 Protein Sequence (SEQ ID NO: 1) MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGV ALKLFVQLLGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEE KEEERGPQWRLGARKPGSWTGEAAVCADSAPAARAPQALARASGRGGRVA RRGAEESGPPHSPSRRGSASRAGPGRASETMNFLLSWVHWSLALLLYLHH AKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIE YIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEM SFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGAR CCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLEL NERTCRCDKPRR Human VEGF Transcript Variant 1 cDNA (SEQ ID NO: 2) ct gacggacaga cagacagaca ccgcccccag ccccagctac cacctcctcc ccggccggcg gcggacagtg gacgcggcgg cgagccgcgg gcaggggccg gagcccgcgc ccggaggcgg ggtggagggg gtcggggctc gcggcgtcgc actgaaactt ttcgtccaac ttctgggctg ttctcgcttc ggaggagccg tggtccgcgc gggggaagccgagccgagcg gagccgcgag aagtgctagc tcgggccggg aggagccgca gccggaggag ggggaggagg aagaagagaa ggaagaggag agggggccgc agtggcgact cggcgctcgg aagccgggct catggacggg tgaggcggcg gtgtgcgcag acagtgctcc agccgcgcgc gctccccagg ccctggcccg ggcctcgggc cggggaggaa gagtagctcg ccgaggcgcc gaggagagcg ggccgcccca cagcccgagc cggagaggga gcgcgagccg cgccggcccc ggtcgggcct ccgaaaccat gaactttctg ctgtcttggg tgcattggag ccttgccttgctgctctacc tccaccatgc caagtggtcc caggctgcac ccatggcaga aggaggaggg cagaatcatc acgaagtggt gaagttcatg gatgtctatc agcgcagcta ctgccatcca atcgagaccc tggtggacat cttccaggag taccctgatg agatcgagta catcttcaag ccatcctgtg tgcccctgat gcgatgcggg ggctgctgca atgacgaggg cctggagtgt gtgcccactg aggagtccaa catcaccatg cagattatgc ggatcaaacc tcaccaaggc cagcacatag gagagatgag cttcctacag cacaacaaat gtgaatgcag accaaagaaa gatagagcaa gacaagaaaa aaaatcagtt cgaggaaagg gaaaggggca aaaacgaaag cgcaagaaat cccggtataa gtcctggagc gtgtacgttg gtgcccgctg ctgtctaatg ccctggagcc tccctggccc ccatccctgt gggccttgct cagagcggag aaagcatttg tttgtacaag atccgcagac gtgtaaatgt tcctgcaaaa acacagactc gcgttgcaag gcgaggcagc ttgagttaaa cgaacgtact tgcagatgtg acaagccgag gcggtga Human VEGF Transcript Variant 3 Protein Sequence (SEQ ID NO: 3) MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGV ALKLFVQLLGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEE KEEERGPQWRLGARKPGSWTGEAAVCADSAPAARAPQALARASGRGGRVA RRGAEESGPPHSPSRRGSASRAGPGRASETMNFLLSWVHWSLALLLYLHH AKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIE YIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEM SFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRPCGPCSERRKH LFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR Human VEGF Transcript Variant 3 cDNA (SEQ ID NO: 4) ct gacggacaga cagacagaca ccgcccccag ccccagctac cacctcctcc ccggccggcg gcggacagtg gacgcggcgg cgagccgcgg gcaggggccg gagcccgcgc ccggaggcgg ggtggagggg gtcggggctc gcggcgtcgc actgaaactt ttcgtccaac ttctgggctg ttctcgcttc ggaggagccg tggtccgcgc gggggaagccgagccgagcg gagccgcgag aagtgctagc tcgggccggg aggagccgca gccggaggag ggggaggagg aagaagagaa ggaagaggag agggggccgc agtggcgact cggcgctcgg aagccgggct catggacggg tgaggcggcg gtgtgcgcag acagtgctcc agccgcgcgc gctccccagg ccctggcccg ggcctcgggc cggggaggaa gagtagctcg ccgaggcgcc gaggagagcg ggccgcccca cagcccgagc cggagaggga gcgcgagccg cgccggcccc ggtcgggcct ccgaaaccat gaactttctg ctgtcttggg tgcattggag ccttgccttgctgctctacc tccaccatgc caagtggtcc caggctgcac ccatggcaga aggaggaggg cagaatcatc acgaagtggt gaagttcatg gatgtctatc agcgcagcta ctgccatcca atcgagaccc tggtggacat cttccaggag taccctgatg agatcgagta catcttcaag ccatcctgtg tgcccctgat gcgatgcggg ggctgctgca atgacgaggg cctggagtgt gtgcccactg aggagtccaa catcaccatg cagattatgc ggatcaaacc tcaccaaggc cagcacatag gagagatgag cttcctacag cacaacaaat gtgaatgcag accaaagaaa gatagagcaa gacaagaaaa aaaatcagtt cgaggaaagg gaaaggggca aaaacgaaag cgcaagaaat cccgtccctg tgggccttgc tcagagcgga gaaagcattt gtttgtacaa gatccgcaga cgtgtaaatg ttcctgcaaa aacacagact cgcgttgcaa ggcgaggcag cttgagttaa acgaacgtac ttgcagatgt gacaagccga ggcggtga Mature Human VEGF-A (SEQ ID NO: 13) apma egggqnhhev vkfmdvyqrs ychpietlvd ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem sflghnkcec rpkkdrarqe kksvrgkgkg qkrkrkksry kswsvyvgar cclmpwslpg phpcgpcser rkhlfvqdpq tckcsckntd srckarqlel nertcrcdkp rr Mature Human VEGF-B (SEQ ID NO: 14) pvsqpdapg hqrkvvswid vytratcqpr evvvpltvel mgtvakqlvp scvtvqrcgg ccpddglecv ptgqhqvrmq ilmirypssq lgemsleehs qcecrpkkkd savkpdraat phhrpqprsv pgwdsapgap spadithptp apgpsahaap sttsaltpgp aaaaadaaas svakgga Mature Human VEGF-C (SEQ ID NO: 15) Ahynteilk sidnewrktq cmprevcidv gkefgvatnt ffkppcvsvy rcggccnseg lqcmntstsy lsktifeitv plsqgpkpvt isfanhtscr cmskldvyrq vhsiirr Mature Human VEGF-D (SEQ ID NO: 16) fa atfydietlk videewqrtq cspretcvev aselgkstnt ffkppcvnvf rcggccnees licmntstsy iskqlfeisv pltsvpelvp vkvanhtgck clptaprhpy siirr

[0116] In some examples of any of the antibodies and antigen-binding fragments thereof described herein, the antibody and antigen-binding fragment can bind to a VEGF antigen (e.g., any of the exemplary VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D) (e.g., any of the binding affinities described herein).

[0117] In some embodiments described herein, an antibody or antigen-binding antibody fragment can decrease an activity of a VEGF (e.g., one or more of any of the exemplary VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D). In some embodiments, an antibody or antigen-binding antibody fragment can block a VEGF (e.g., one or more of any of the exemplary VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D) from binding to one or more of its receptors (e.g., one or more VEGF receptors) See, e.g., WO 1998/045331, U.S. Pat. No. 9,079,953, US 2015/0147317, US 2016/0289314, Plotkin et al., Otology & Neurotology 33: 1046-1052 (2012); and Ferrara et al. (2005) Biochem Biophys Res Commun 333(2): 328-335. In some embodiments, an antibody or antigen-binding antibody can decrease downstream signaling (e.g., signaling downstream of a VEGF receptor, e.g., one or more of any of the exemplary VEGF receptors described herein, e.g., one or more of human VEGFR-1, human VEGFR-2, and human VEGFR-3). In some embodiments, a decrease in an activity of a VEGF can be detected indirectly, e.g., through an increase in hearing (e.g., a 1% to about 400% increase (or any of the subranges of this range described herein) in hearing) or a decrease (e.g., a 1% to 99%, a 1% to 95%, a 1% to 90%, a 1% to 85%, a 1% to 80%, a 1% to 75%, a 1% to 70%, a 1% to 65%, a 1% to 60%, a 1% to 55%, a 1% to 50%, a 1% to 45%, a 1% to 40%, a 1% to 35%, a 1% to 30%, a 1% to 25%, a 1% to 20%, a 1% to 15%, a 1% to 10%, a 1% to 5%, a 5% to 99%, a 5% to 95%, a 5% to 90%, a 5% to 85%, a 5% to 80%, a 5% to 75%, a 5% to 70%, a 5% to 65%, a 5% to 60%, a 5% to 55%, a 5% to 50%, a 5% to 45%, a 5% to 40%, a 5% to 35%, a 5% to 30%, a 5% to 25%, a 5% to 20%, a 5% to 15%, a 5% to 10%, a 10% to 99%, a 10% to 95%, a 10% to 90%, a 10% to 85%, a 10% to 80%, a 10% to 75%, a 10% to 70%, a 10% to 65%, a 10% to 60%, a 10% to 55%, a 10% to 50%, a 10% to 45%, a 10% to 40%, a 10% to 35%, a 10% to 30%, a 10% to 25%, a 10% to 20%, a 10% to 15%, a 15% to 99%, a 15% to 95%, a 15% to 90%, a 15% to 85%, a 15% to 80%, a 15% to 75%, a 15% to 70%, a 15% to 65%, a 15% to 60%, a 15% to 55%, a 15% to 50%,a 15% to 45%,a 15% to 40%, a 15% to 35%, a 15% to 30%, a 15% to 25%, a 15% to 20%, a 20% to 99%, a 20% to 95%, a 20% to 90%, a 20% to 85%, a 20% to 80%, a 20% to 75%, a 20% to 70%, a 20% to 65%, a 20% to 60%, a 20% to 55%, a 20% to 50%, a 20% to 45%, a 20% to 40%, a 20% to 35%, a 20% to 30%, a 20% to 25%, a 25% to 99%, a 25% to 95%, a 25% to 90%, a 25% to 85%, a 25% to 80%, a 25% to 75%, a 25% to 70%, a 25% to 65%, a 25% to 60%, a 25% to 55%, a 25% to 50%, a 25% to 45%, a 25% to 40%, a 25% to 35%, a 25% to 30%, a 30% to 99%, a 30% to 95%, a 30% to 90%, a 30% to 85%, a 30% to 80%, a 30% to 75%, a 30% to 70%, a 30% to 65%, a 30% to 60%, a 30% to 55%, a 30% to 50%, a 30% to 45%, a 30% to 40%, a 30% to 35%, a 35% to 99%, a 35% to 95%, a 35% to 90%, a 35% to 85%, a 35% to 80%, a 35% to 75%, a 35% to 70%, a 35% to 65%, a 35% to 60%, a 35% to 55%, a 35% to 50%, a 35% to 45%, a 35% to 40%, a 40% to 99%, a 40% to 95%, a 40% to 90%, a 40% to 85%, a 40% to 80%, a 40% to 75%, a 40% to 70%, a 40% to 65%, a 40% to 60%, a 40% to 55%, a 40% to 50%, a 40% to 45%, a 45% to 99%, a 45% to 95%, a 45% to 90%, a 45% to 85%, a 45% to 80%, a 45% to 75%, a 45% to 70%, a 45% to 65%, a 45% to 60%, a 45% to 55%, a 45% to 50%, a 50% to 99%, a 50% to 95%, a 50% to 90%, a 50% to 85%, a 50% to 80%, a 50% to 75%, a 50% to 70%, a 50% to 65%, a 50% to 60%, a 50% to 55%, a 55% to 99%, a 55% to 95%, a 55% to 90%, a 55% to 85%, a 55% to 80%, a 55% to 75%, a 55% to 70%, a 55% to 65%, a 55% to 60%, a 60% to 99%, a 60% to 95%, a 60% to 90%, a 60% to 85%, a 60% to 80%, a 60% to 75%, a 60% to 70%, a 60% to 65%, a 65% to 99%, a 65% to 95%, a 65% to 90%, a 65% to 85%, a 65% to 80%, a 65% to 75%, a 65% to 70%, a 70% to 99%, a 70% to 95%, a 70% to 90%, a 70% to 85%, a 70% to 80%, a 70% to 75%, a 75% to 99%, a 75% to 95%, a 75% to 90%, a 75% to 85%, a 75% to 80%, a 80% to 99%, a 80% to 95%, a 80% to 90%, a 80% to 85%, a 85% to 99%, a 85% to 95%, a 85% to 90%, a 90% to 99%, a 90% to 95%, or a 95% to 99% decrease) in the size or the severity of one or more symptoms of an acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II in a mammal as compared to the level of hearing or size of an acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II in the mammal, respectively, before administration of any of the AAV vectors described herein. In some embodiments, a decrease in a VEGF activity can be detected in an in vitro assay.

[0118] In some embodiments, the antibody that specifically binds to a VEGF is bevacizumab (Avastatin.RTM.) or an antigen-binding fragment thereof. Bevacizumab (full size antibody.about.150 kDa) inhibits all isoforms of VEGF-A. Bevacizumab received Food and Drug administration (FDA) approval in 2004 for colon cancer for intravenous (IV) dose of 4.0-7.5 mg/kg at 2-3 weeks (plasmatic half life 21 days), for intravetrial (IVT) dose 1.25 mg in 0.05 mL (half-life 5.6 days). Bevacizumab has a KD for VEGF 165 (VEGF-A) of 58 pM. See, e.g., WO 2017/050825. In some embodiments, the antibody that specifically binds to a VEGF is ranibizumab (Lucentis.RTM.), or an antigen-binding fragment thereof. Ranibizumab (.about.50 kDa) inhibits all isoforms of VEGF-A. Ranibizumab received FDA approval in 2006 for ocular use for intravenous (IV) dose of 4.0-7.5 mg/kg at 2-3 weeks (plasma half life of 0.5 days), for intravetrial (IVT) dose 0.5 mg in 0.05 mL (half-life of 3.2 days). Ranibizumab has a KD for VEGF 165 (VEGF-A) of 46 pM. See, e.g., WO 2014/178078. In some embodiments, the antibody that specifically binds to VEGF is sevacizumab (APX003/SIM-BD0801), or an antigen-binding fragment thereof.

TABLE-US-00002 Amino Acid Encoding Light Chain of Bevacizumab (SEQ ID NO: 5) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYF TSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Amino Acid Encoding Heavy Chain of Bevacizumab (SEQ ID NO: 6) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGW INTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYP HYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK

[0119] In some embodiments of the antibodies that specifically bind to VEGF and antigen-binding fragments thereof described herein, the antibody or antigen-binding fragments thereof includes a variable light chain domain that is or includes a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to the variable light chain domain of bevacizumab, and/or includes a variable heavy chain domain that is or includes a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to the variable heavy chain domain of bevacizumab.

[0120] In some embodiments of the antibodies that specifically bind to VEGF and antigen-binding fragments thereof described herein, the antibody or antigen-binding fragments thereof includes a variable light chain domain that is or includes the variable light chain domain of bevacizumab, and/or a variable heavy chain domain that is or includes the variable heavy chain domain of bevacizumab. In some embodiments of the antibodies that specifically bind to VEGF and antigen-binding fragments thereof described herein, the antibody or antigen-binding fragments thereof includes a variable light chain domain that is or includes the sequence of variable light chain domain of bevacizumab, except that it includes one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions, and/or includes a variable heavy chain domain that is or includes the sequence of variable heavy chain of bevacizumab, except that it includes one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions. In some embodiments the first antigen-binding domain includes the three CDRs in the light chain variable domain of bevacizumab, and/or the three CDRs in the heavy chain variable domain of bevacizumab.

TABLE-US-00003 Amino Acid Encoding Light Chain of Ranibizumab (SEQ ID NO: 7) DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYF TSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Amino Acid Encoding Heavy Chain of Ranibizumab (SEQ ID NO: 8) EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGW INTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYP YYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHL

[0121] In some embodiments of the antibodies that specifically bind to VEGF and antigen-binding fragments thereof described herein, the antibody or antigen-binding fragments thereof includes a variable light chain domain that is or includes a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to the variable light chain domain of ranibizumab, and/or includes a variable heavy chain domain that is or includes a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to the variable heavy chain domain of ranibizumab.

[0122] In some embodiments of the antibodies that specifically bind to VEGF and antigen-binding fragments thereof described herein, the antibody or antigen-binding fragments thereof includes a variable light chain domain that is or includes the variable light chain domain of ranibizumab, and/or a variable heavy chain domain that is or includes the variable heavy chain domain of ranibizumab. In some embodiments of the antibodies that specifically bind to VEGF and antigen-binding fragments thereof described herein, the antibody or antigen-binding fragments thereof includes a variable light chain domain that is or includes the sequence of variable light chain domain of ranibizumab, except that it includes one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions, and/or includes a variable heavy chain domain that is or includes the sequence of variable heavy chain of ranibizumab, except that it includes one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions. In some embodiments the first antigen-binding domain includes the three CDRs in the light chain variable domain of ranibizumab, and/or the three CDRs in the heavy chain variable domain of ranibizumab.

Soluble VEGF Receptors

[0123] A soluble VEGF receptor is a polypeptide that includes a portion of an extracellular region of one or more (e.g., two or three) mammalian VEGF receptor(s) (e.g., one or more of VEGFR-1, VEGFR-2, and VEGFR-3) operably linked to a signal peptide (e.g., any of the exemplary signal peptides described herein), where the soluble VEGF receptor is capable of specifically binding to one or more mammalian VEGF protein(s) (e.g., one or more (e.g., two, three, or four) of VEGF-A, VEGF-B, VEGF-C, and VEGF-D, e.g., one or more (e.g., two, three, or four) of human wildtype VEGF-A, human wildtype VEGF-B, human wildtype VEGF-C, and human wildtype VEGF-D).

[0124] In some examples, a soluble VEGF receptor includes a portion (e.g., about 10 amino acids to about 732 amino acids, about 10 amino acids to about 700 amino acids, about 10 amino acids to about 650 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 550 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 450 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, about 10 amino acids to about 300 amino acids, about 10 amino acids to about 250 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 150 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 50 amino acids, about 50 amino acids to about 732 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 250 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 732 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 732 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 250 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 732 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 250 amino acids, about 250 amino acids to about 732 amino acids, about 250 amino acids to about 700 amino acids, about 250 amino acids to about 650 amino acids, about 250 amino acids to about 600 amino acids, about 250 amino acids to about 550 amino acids, about 250 amino acids to about 500 amino acids, about 250 amino acids to about 450 amino acids, about 250 amino acids to about 400 amino acids, about 250 amino acids to about 350 amino acids, about 250 amino acids to about 300 amino acids, about 300 amino acids to about 732 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 350 amino acids, about 350 amino acids to about 732 amino acids, about 350 amino acids to about 700 amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 550 amino acids, about 350 amino acids to about 500 amino acids, about 350 amino acids to about 450 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 732 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 450 amino acids, about 450 amino acids to about 732 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to about 732 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 732 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 732 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 732 amino acids, about 650 amino acids to about 700 amino acids, or about 700 amino acids to about 732 amino acids) of an extracellular region of VEGFR-1 (e.g., a contiguous sequence from wildtype human VEGFR-1 (e.g., a contiguous sequence including one or more (e.g., one, two, three, four, five, six, or seven) immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1 (e.g., SEQ ID NO: 23) or a sequence that is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to a contiguous sequence from wildtype human VEGFR-1, e.g., a sequence that is at least 80% (e.g., least 82%, at least 84%, at least 86%, at least 88%, at least 90%, least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to a contiguous sequence in SEQ ID NO: 23).

[0125] In some examples, a soluble VEGF receptor includes a portion (e.g., about 20 amino acids to about 745 amino acids, or any of the subranges of this range described herein) of an extracellular region of VEGFR-2 (e.g., a contiguous sequence from wildtype human VEGFR-2 (e.g., a contiguous sequence including one or more (e.g., one, two, three, four, five, six, or seven) immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2 (e.g., SEQ ID NO: 26) or a sequence that is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to a contiguous sequence from wildtype human VEGFR-2, e.g., a sequence that is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to a contiguous sequence in SEQ ID NO: 26).

[0126] In some examples, a soluble VEGF receptor includes a portion of an extracellular region of VEGFR-1 (e.g., any of the portions of an extracellular region of VEGFR-1 described herein) and a portion of an extracellular region of VEGFR-2 (e.g., any of the portions of an extracellular region of VEGFR-2 described herein). For example, a soluble VEGF receptor can include one or more (e.g., two, three, four, five, six, or seven) immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1 and one or more (e.g., two, three, four, five, six, or seven) immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2 (e.g., aflibercept).

[0127] In some examples, a soluble VEGF receptor includes a portion (e.g., about 20 amino acids to about 751 amino acids, or any of the subranges of this range described herein) of an extracellular region of VEGFR-3 (e.g., a contiguous sequence from wildtype human VEGFR-3 (e.g., a contiguous sequence including one or more (e.g., one, two, three, four, five, six, or seven) immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-3 (e.g., SEQ ID NO: 29) or a sequence that is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to a contiguous sequence from wildtype human VEGFR-3, e.g., a sequence that is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to a contiguous sequence in SEQ ID NO: 29).

[0128] Non-limiting examples of extracellular regions of different mammalian VEGFR-1, different mammalian VEGFR-2, and different mammalian VEGFR-3 are described herein. Non-limiting examples of protein and nucleotide sequences encoding a wildtype VEGF receptor protein are shown below. As one skilled in the art can appreciate, a substitution in an amino acid that is conserved between species is more likely to result in a change in the function of a protein, while a substitution in an amino acid position that is not converved between species is less likely to have an affect on the function of a protein.

TABLE-US-00004 Human VEGF Receptor 1 Isoform 2 Protein Sequence (SEQ ID NO: 17) MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLH LQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQAN HTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTE GRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYK EIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVL NCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDK MQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGK RSYRLSMKVKAFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKDVTEEDA GNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQ ILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGN RIESITQRMAIIEGKNKMASTLVVADSRISGIYICIASNKVGTVGRNISF YITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTM HYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQK KEITIRGEHCNKKAVFSRISKFKSTRNDCTTQSNVKH Human VEGF Receptor 1 Isoform 2 cDNA (SEQ ID NO: 18) ATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGCTGCTCAGCTG TCTGCTTCTCACAGGATCTAGTTCAGGTTCAAAATTAAAAGATCCTGAAC TGAGTTTAAAAGGCACCCAGCACATCATGCAAGCAGGCCAGACACTGCAT CTCCAATGCAGGGGGGAAGCAGCCCATAAATGGTCTTTGCCTGAAATGGT GAGTAAGGAAAGCGAAAGGCTGAGCATAACTAAATCTGCCTGTGGAAGAA ATGGCAAACAATTCTGCAGTACTTTAACCTTGAACACAGCTCAAGCAAAC CACACTGGCTTCTACAGCTGCAAATATCTAGCTGTACCTACTTCAAAGAA GAAGGAAACAGAATCTGCAATCTATATATTTATTAGTGATACAGGTAGAC CTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAA GGAAGGGAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGT TACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAACGCA TAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAA GAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAA GACAAACTATCTCACACATCGACAAACCAATACAATCATAGATGTCCAAA TAAGCACACCACGCCCAGTCAAATTACTTAGAGGCCATACTCTTGTCCTC AATTGTACTGCTACCACTCCCTTGAACACGAGAGTTCAAATGACCTGGAG TTACCCTGATGAAAAAAATAAGAGAGCTTCCGTAAGGCGACGAATTGACC AAAGCAATTCCCATGCCAACATATTCTACAGTGTTCTTACTATTGACAAA ATGCAGAACAAAGACAAAGGACTTTATACTTGTCGTGTAAGGAGTGGACC ATCATTCAAATCTGTTAACACCTCAGTGCATATATATGATAAAGCATTCA TCACTGTGAAACATCGAAAACAGCAGGTGCTTGAAACCGTAGCTGGCAAG CGGTCTTACCGGCTCTCTATGAAAGTGAAGGCATTTCCCTCGCCGGAAGT TGTATGGTTAAAAGATGGGTTACCTGCGACTGAGAAATCTGCTCGCTATT TGACTCGTGGCTACTCGTTAATTATCAAGGACGTAACTGAAGAGGATGCA GGGAATTATACAATCTTGCTGAGCATAAAACAGTCAAATGTGTTTAAAAA CCTCACTGCCACTCTAATTGTCAATGTGAAACCCCAGATTTACGAAAAGG CCGTGTCATCGTTTCCAGACCCGGCTCTCTACCCACTGGGCAGCAGACAA ATCCTGACTTGTACCGCATATGGTATCCCTCAACCTACAATCAAGTGGTT CTGGCACCCCTGTAACCATAATCATTCCGAAGCAAGGTGTGACTTTTGTT CCAATAATGAAGAGTCCTTTATCCTGGATGCTGACAGCAACATGGGAAAC AGAATTGAGAGCATCACTCAGCGCATGGCAATAATAGAAGGAAAGAATAA GATGGCTAGCACCTTGGTTGTGGCTGACTCTAGAATTTCTGGAATCTACA TTTGCATAGCTTCCAATAAAGTTGGGACTGTGGGAAGAAACATAAGCTTT TATATCACAGATGTGCCAAATGGGTTTCATGTTAACTTGGAAAAAATGCC GACGGAAGGAGAGGACCTGAAACTGTCTTGCACAGTTAACAAGTTCTTAT ACAGAGACGTTACTTGGATTTTACTGCGGACAGTTAATAACAGAACAATG CACTACAGTATTAGCAAGCAAAAAATGGCCATCACTAAGGAGCACTCCAT CACTCTTAATCTTACCATCATGAATGTTTCCCTGCAAGATTCAGGCACCT ATGCCTGCAGAGCCAGGAATGTATACACAGGGGAAGAAATCCTCCAGAAG AAAGAAATTACAATCAGAGGTGAGCACTGCAACAAAAAGGCTGTTTTCTC TCGGATCTCCAAATTTAAAAGCACAAGGAATGATTGTACCACACAAAGTA ATGTAAAACATTAA Human VEGF Receptor 1 Isoform 3 Protein Sequence (sFlt1-14) (SEQ ID NO: 19) MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLH LQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQAN HTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTE GRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYK EIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVL NCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDK MQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGK RSYRLSMKVKAFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKDVTEEDA GNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQ ILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGN RIESITQRMAIIEGKNKMASTLVVADSRISGIYICIASNKVGTVGRNISF YITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTM HYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQK KEITIRDQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHK IQQEPELYTSTSPSSSSSSPLSSSSSSSSSSSS Human VEGF Receptor 1 Isoform 3 cDNA (SEQ ID NO: 20) ATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGCTGCTCAGCTG TCTGCTTCTCACAGGATCTAGTTCAGGTTCAAAATTAAAAGATCCTGAAC TGAGTTTAAAAGGCACCCAGCACATCATGCAAGCAGGCCAGACACTGCAT CTCCAATGCAGGGGGGAAGCAGCCCATAAATGGTCTTTGCCTGAAATGGT GAGTAAGGAAAGCGAAAGGCTGAGCATAACTAAATCTGCCTGTGGAAGAA ATGGCAAACAATTCTGCAGTACTTTAACCTTGAACACAGCTCAAGCAAAC CACACTGGCTTCTACAGCTGCAAATATCTAGCTGTACCTACTTCAAAGAA GAAGGAAACAGAATCTGCAATCTATATATTTATTAGTGATACAGGTAGAC CTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAA GGAAGGGAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGT TACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAACGCA TAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAA GAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAA GACAAACTATCTCACACATCGACAAACCAATACAATCATAGATGTCCAAA TAAGCACACCACGCCCAGTCAAATTACTTAGAGGCCATACTCTTGTCCTC AATTGTACTGCTACCACTCCCTTGAACACGAGAGTTCAAATGACCTGGAG TTACCCTGATGAAAAAAATAAGAGAGCTTCCGTAAGGCGACGAATTGACC AAAGCAATTCCCATGCCAACATATTCTACAGTGTTCTTACTATTGACAAA ATGCAGAACAAAGACAAAGGACTTTATACTTGTCGTGTAAGGAGTGGACC ATCATTCAAATCTGTTAACACCTCAGTGCATATATATGATAAAGCATTCA TCACTGTGAAACATCGAAAACAGCAGGTGCTTGAAACCGTAGCTGGCAAG CGGTCTTACCGGCTCTCTATGAAAGTGAAGGCATTTCCCTCGCCGGAAGT TGTATGGTTAAAAGATGGGTTACCTGCGACTGAGAAATCTGCTCGCTATT TGACTCGTGGCTACTCGTTAATTATCAAGGACGTAACTGAAGAGGATGCA GGGAATTATACAATCTTGCTGAGCATAAAACAGTCAAATGTGTTTAAAAA CCTCACTGCCACTCTAATTGTCAATGTGAAACCCCAGATTTACGAAAAGG CCGTGTCATCGTTTCCAGACCCGGCTCTCTACCCACTGGGCAGCAGACAA ATCCTGACTTGTACCGCATATGGTATCCCTCAACCTACAATCAAGTGGTT CTGGCACCCCTGTAACCATAATCATTCCGAAGCAAGGTGTGACTTTTGTT CCAATAATGAAGAGTCCTTTATCCTGGATGCTGACAGCAACATGGGAAAC AGAATTGAGAGCATCACTCAGCGCATGGCAATAATAGAAGGAAAGAATAA GATGGCTAGCACCTTGGTTGTGGCTGACTCTAGAATTTCTGGAATCTACA TTTGCATAGCTTCCAATAAAGTTGGGACTGTGGGAAGAAACATAAGCTTT TATATCACAGATGTGCCAAATGGGTTTCATGTTAACTTGGAAAAAATGCC GACGGAAGGAGAGGACCTGAAACTGTCTTGCACAGTTAACAAGTTCTTAT ACAGAGACGTTACTTGGATTTTACTGCGGACAGTTAATAACAGAACAATG CACTACAGTATTAGCAAGCAAAAAATGGCCATCACTAAGGAGCACTCCAT CACTCTTAATCTTACCATCATGAATGTTTCCCTGCAAGATTCAGGCACCT ATGCCTGCAGAGCCAGGAATGTATACACAGGGGAAGAAATCCTCCAGAAG AAAGAAATTACAATCAGAGATCAGGAAGCACCATACCTCCTGCGAAACCT CAGTGATCACACAGTGGCCATCAGCAGTTCCACCACTTTAGACTGTCATG CTAATGGTGTCCCCGAGCCTCAGATCACTTGGTTTAAAAACAACCACAAA ATACAACAAGAGCCTGAACTGTATACATCAACGTCACCATCGTCATCGTC ATCATCACCATTGTCATCATCATCATCATCGTCATCATCATCATCATCAT AG Human VEGF Receptor 1 Isoform 4 Protein Sequence (SEQ ID NO: 21) MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLH LQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQAN HTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTE

GRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYK EIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVL NCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDK MQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGK RSYRLSMKVKAFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKDVTEEDA GNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQ ILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGN RIESITQRMAIIEGKNKLPPANSSFMLPPTSFSSNYFHFLP Human VEGF Receptor 1 Isoform 4 cDNA (SEQ ID NO: 22) ATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGCTGCTCAGCTG TCTGCTTCTCACAGGATCTAGTTCAGGTTCAAAATTAAAAGATCCTGAAC TGAGTTTAAAAGGCACCCAGCACATCATGCAAGCAGGCCAGACACTGCAT CTCCAATGCAGGGGGGAAGCAGCCCATAAATGGTCTTTGCCTGAAATGGT GAGTAAGGAAAGCGAAAGGCTGAGCATAACTAAATCTGCCTGTGGAAGAA ATGGCAAACAATTCTGCAGTACTTTAACCTTGAACACAGCTCAAGCAAAC CACACTGGCTTCTACAGCTGCAAATATCTAGCTGTACCTACTTCAAAGAA GAAGGAAACAGAATCTGCAATCTATATATTTATTAGTGATACAGGTAGAC CTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAA GGAAGGGAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGT TACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAACGCA TAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAA GAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAA GACAAACTATCTCACACATCGACAAACCAATACAATCATAGATGTCCAAA TAAGCACACCACGCCCAGTCAAATTACTTAGAGGCCATACTCTTGTCCTC AATTGTACTGCTACCACTCCCTTGAACACGAGAGTTCAAATGACCTGGAG TTACCCTGATGAAAAAAATAAGAGAGCTTCCGTAAGGCGACGAATTGACC AAAGCAATTCCCATGCCAACATATTCTACAGTGTTCTTACTATTGACAAA ATGCAGAACAAAGACAAAGGACTTTATACTTGTCGTGTAAGGAGTGGACC ATCATTCAAATCTGTTAACACCTCAGTGCATATATATGATAAAGCATTCA TCACTGTGAAACATCGAAAACAGCAGGTGCTTGAAACCGTAGCTGGCAAG CGGTCTTACCGGCTCTCTATGAAAGTGAAGGCATTTCCCTCGCCGGAAGT TGTATGGTTAAAAGATGGGTTACCTGCGACTGAGAAATCTGCTCGCTATT TGACTCGTGGCTACTCGTTAATTATCAAGGACGTAACTGAAGAGGATGCA GGGAATTATACAATCTTGCTGAGCATAAAACAGTCAAATGTGTTTAAAAA CCTCACTGCCACTCTAATTGTCAATGTGAAACCCCAGATTTACGAAAAGG CCGTGTCATCGTTTCCAGACCCGGCTCTCTACCCACTGGGCAGCAGACAA ATCCTGACTTGTACCGCATATGGTATCCCTCAACCTACAATCAAGTGGTT CTGGCACCCCTGTAACCATAATCATTCCGAAGCAAGGTGTGACTTTTGTT CCAATAATGAAGAGTCCTTTATCCTGGATGCTGACAGCAACATGGGAAAC AGAATTGAGAGCATCACTCAGCGCATGGCAATAATAGAAGGAAAGAATAA GCTTCCACCAGCTAACAGTTCTTTCATGTTGCCACCTACAAGCTTCTCTT CCAACTACTTCCATTTCCTTCCGTGA Extracellular Region of Wildtype Human VEGFR-1 (the seven Ig-like domains are shown in bold and underlined) (SEQ ID NO: 23) sklk dpelslkgtq himgagqtlh lqcrgeaahk wslpemvske serlsitksa cgrngkqfcs tltlntaqan htgfysckyl avptskkket esaiyifisd tgrpfvemys eipeiihmte grelvipery tspnitvtlk kfpldtlipd gkriiwdsrk gfiisnatyk eiglltceat vnghlyktny lthrqtntii dvqistprpv kllrghtivl nctattpint rvqmtwsypd eknkrasvrr ridqsnshan ifysvltidk mqnkdkglyt crvrsgpsfk svntsvhiyd kafitvkhrk qqvletvagk rsyrlsmkvk afpspevvwl kdglpateks aryltrgysl iikdvteeda gnytillsik qsnvfknita tlivnvkpqi yekayssfpd palvplgsrq iltctaygip qptikwfwhp cnhnhsearc dfcsnneesf ildadsnmgn riesitqrma iiegknkmas tivvadsris giyiciasnk vgtvgrnisf yitdvpngfh vnlekmpteg edlklsctvn kflyrdvtwi llrtvnnrtm hysiskqkma itkehsitln ltimnvslqd sgtyacrarn vytgeeilqk keitirdqea pyllrnlsdh tvaisssttl dchangvpep qitwfknnhk iqqepqiilq pgsstlfier vteedegvyh ckatnqkgsv essayltvqg tsdksnle Extracellular Region of Wildtype Mouse VEGFR-1 (SEQ ID NO: 24) ygsgsklk vpelslkgtq hvmqagqtlf lkcrgeaahs wslpttvsqe dkrlsitpps acgrdnrqfc stltldtaqa nhtglytcry 1ptstskkkk aessiyifvs dagspfiemh tdipklvhmt egrqliiper vtspnvtvtl kkfpfdtltp dgqritwdsr rgfiianaty keigllncea tvnghlyqtn ylthrqtnti ldvqirppsp vrllhgqtiv lnctatteln trvqmswnyp gkatkrasir qridrshshn nvfhsvlkin nvesrdkgly tcrvksgssf qsfntsvhvy ekgfisvkhr kqpvqettag rrsyrlsmkv kafpspeivw lkdgspatlk sarylvhgys liikdvtted agdytillgi kqsrlfknit atlivnvkpq iyeksysslp spplyplgsr qvltctvygi prptitwlwh pchhnhsker ydfctenees fildpssnlg nriesisqrm tviegtnktv stivvadsqt pgiyscrafn kigtvernik fyvtdvpngf hvslekmpae gedlklscvv nkflyrditw illrtvnnrt mhhsiskqkm attqdysitl nlviknvsle dsgtyacrar niytgedilr ktevlvrdse aphllqnlsd yevsisgstt ldcgargvpa pqitwfknnh kiqqepgiil gpgnstlfie rvteedegvy rcratnqkga vesaayltvq gtsdksnle Extracellular Region of Wildtype Rat VEGFR-1 (SEQ ID NO: 25) ycsgsklk gpelslkgtq hvmqagqtlf lkcrgeaahs wslpttvsqe dkklsvtrsa cgrnnrqfcs tltlnmaqan htglyscryl pkstskekkm esaiyifvsd agspfiemhs dipklvhmte greliipery tspnitvtlk kfpfdaltpd gqriawdsrr gfiianatyk eiglltceat vnghlyqtsy lthrqtntil dvqisppspv rflrgqtivl nctvttdlnt rvqmswnypg katkrasirq ridqsnphsn vfhsvlkinn vesrdkglyt crvksgssfr tfntsvhvye kgfisvkhrk qqvqetiagk rshrlsmkvk afpspevvwl kdgvpateks arysvhgysl iikdvtaeda gdytillgik qsklfrnita tlivnvkpqi yeksysslps pplyplgsrq vltctvygip qptikwlwhp chynhskern dfcfgseesf ildsssnign riegitqrmm viegtnktvs tivvadsrtp gsysckafnk igtverdirf yvtdvpngfh vslekipteg edlklscvvs kflyrditwi llrtvnnrtm hhsiskqkma ttqdysitln lviknvsled sgtyacrarn iytgeeilrk tevlvrdlea plllqnlsdh evsisgsttl dcqargvpap qitwfknnhk iqqepgiilg pgnstlfier vteedegvyr cratnqkgvv essayltvqg tsdksnle Extracellular Region of Wildtype Human VEGFR-2 (the seven Ig-like domains are shown in bold and underlined) (SEQ ID NO: 26) asvglpsysld lprlsiqkdi ltikanttlq itcrgqrdld wlwpnngsgs eqrvevtecs dglfcktlti pkvigndtga ykcfyretdl asviyvyvqd yrspfiasys dqhgvvyite nknktvvipc lgsisnlnvs lcarypekrf vpdgnriswd skkgftipsy misyagmvfc eakindesyq simyivvvvg yriydvvlsp shgielsvge klvinctart elnvgidfnw eypsskhqhk klvnrdlktq sgsemkkfls tltidgvtrs dqglytcaas sglmtkknst fvrvhekpfv afgsgmeslv eatvgervri pakylgyppp eikwykngip lesnhtikag hvltimevse rdtgnytvil tnpiskekqs hvvslvvyvp pqiqekslis pvdsvqygtt qtltctvyai ppphhihwyw qleeecanep sqaysvtnpy pceewrsved fqggnkievn knqfaliegk nktvstiviq aanvsalykc eavnkvgrge rvisfhvtrg peitlqpdmq pteqesyslw ctadrstfen ltwyklgpqp lpihvgelpt pvcknldtlw klnatmfsns tndilimelk naslqdqgdy vclaqdrktk krhcvvrqlt vlervaptit qnlengttsi gesievscta sgnpppqimw fkdnetived sgivlkdgnr nltirrvrke deglytcqac svlgcakvea ffiiegaqek tnle

Extracellular Region of Wildtype Mouse VEGFR-2 (SEQ ID NO: 27) asvglpgdflh ppklstqkdi ltilanttlq itcrgqrdld wlwpnaqrds eervlvtecg ggdsifcktl tiprvvgndt gaykcsyrdv diastvyvyv rdyrspfias vsdqhgivyi tenknktvvi pergsisnln vslcarypek rfvpdgnris wdseigftlp symisyagmv fceakindet yqsimyivvv vgyriydvil sppheielsa geklvincta rtelnvgldf twhsppsksh hkkivnrdvk pfpgtvakmf lstltiesvt ksdqgeytcv assgrmikrn rtfvrvhtkp fiafgsgmks lveatvgsqv ripvkylsyp apdikwyrng rpiesnytmi vgdeltimev terdagnytv iltnpismek qshmvslvvn vppqigekal ispmdsyqyg tmqtltctvy anpplhhiqw ywqleeacsy rpgqtspyac kewrhvedfq ggnkievtkn qyaliegknk tvstiviqaa nvsalykcea inkagrgery isfhvirgpe itvqpaaqpt eqesysllct adrntfenit wyklgsqats vhmgesltpv cknldalwkl ngtmfsnstn dilivafqna slqdqgdyvc saqdkktkkr hclvkqliil ermapmitgn lenqtttige tievtcpasg nptphitwfk dnetivedsg ivirdgnrn1 tirrvrkedg glytcqacnv lgcaraetlf iiegaqektn le Extracellular Region of Wildtype Rat VEGFR-2 (SEQ ID NO: 28) asvglpgdslh ppklstqkdi ltilanttlq itcrgqrdld wlwpntprds eervlvtecg dsifcktltv prvvgndtga ykcfyrdtdv ssivyvyvqd hrspfiasys dehgivyite nknktvvipc rgsisnlnvs lcarypekrf vpdgnriswd sekgftipsy misyagmvfc eakindetyq simyivlvvg yriydvvlsp pheielsage klvinctart elnvgldfsw qfpsskhqhk kivnrdvksl pgtvakmfls tltidsvtks dqgeytctay sglmtkknkt fvrvhtkpfi afgsgmkslv eatvgsqvri pvkylsypap dikwyrngrp iesnytmivg deltimevse rdagnytvil tnpismekqs hmvslvvnvp pqigekalis pmdsyqygtm qtltctvyan pplhhiqwyw qleeacsyrp sqtnpytcke wrhvkdfqgg nkievtknqy aliegknktv stiviqaayv salykceain kagrgervis fhvirgpeit vqpatqpter esmsllctad rntfenitwy klgsqatsvh mgesltpvck nldalwklng tvfsnstndi livafqnasl qdqgnyvcsa qdkktkkrhc lvkqlviler mapmitgnle nqtttigeti evvcptsgnp tplitwfkdn etivedsgiv lkdgnrniti rrvrkedggl ytcqacnvlg caraetlfii egvqektnle Extracellular Region of Wildtype Human VEGFR-3 (the seven Ig-like domains are shown in bold) (SEQ ID NO: 29) ysmtpp tlniteeshv idtgdslsis crgqhplewa wpgaqeapat gdkdsedtgv vrdcegtdar pyckvlllhe vhandtgsyv cyykyikari egttaassyv fvrdfeqpfi nkpdtllvnr kdamwvpclv sipglnvtlr sgssvlwpdg qevvwddrrg mlvstpllhd alylqcettw gdgdflsnpf lvhitgnely diqllprksl ellvgeklvl nctvwaefns gvtfdwdypg kqaergkwvp errsqqthte lssiltihnv sqhdlgsyvc kanngiqrfr estevivhen pfisvewlkg pileatagde lvklpvklaa vpppefgwyk dgkalsgrhs phalvlkevt eastgtytla lwnsaaglrr nislelvvnv ppqihekeas spsiysrhsr qaltctaygv plplsiqwhw rpwtpckmfa qrslrrrqqq dlmpqcrdwr avttqdavnp iesldtwtef vegknktvsk lvignanvsa mykcvvsnkv gqderliyfy vttipdgfti eskpseelle ggpvllscqa dsykyehlrw yrinlstlhd ahgnpllldc knvhlfatpl aasleevapg arhatlslsi prvapehegh yvcevqdrrs hdkhchkkyl svgaleaprl tqnitdllvn vsdslemgcl vagahapsiv wykderllee ksgvdladsn qklsiqzvre edagrylcsv cnakgcvnss asvavegsed kgsmeivilv Extracellular Region of Wildtype Mouse VEGFR-3 (SEQ ID NO: 30) ysmtpp tlnitedsyv idtgdslsis crgqhplewt wpgagevltt ggkdsedtry vhdcegtear pyckvillaq thanntgsyh cyykyikari egttaastyv fvrdfkhpfi nkpdtllvnr kdsmwvpclv sipglnitlr sqssalhpdg qevlwddrrg mrvptqllrd alylqcettw gdqnflsnlf vvhitgnely diglypkksm ellvgeklvl nctvwaefds gvtfdwdypg kqaerakwvp errsqqthte lssiltihnv sqndlgpyvc eanngiqrfr estevivhek pfisvewlkg pvleatagde lvklpvklaa ypppefqwyk drkavtgrhn phalvlkevt easagvytla lwnsaaglrq nislelvvnv pphihekeas spsiysrhsr qtltctaygv pqplsvqwhw rpwtpcktfa qrslrrrqqr dgmpqcrdwk evttqdavnp iesldswtef vegknktvsk lviqdanvsa mykcvvvnkv gqderliyfy vttipdgfsi esepsedple gqsvrlscra dnytyehlrw yrinlstlhd aqgnpllldc knvhlfatpl eanleeaepg arhatlslni prvapedegd yvcevqdrrs qdkhchkkyl svgaleaprl tqnitdllvn vsdslemrcp vagahvpsiv wykderllek esgidladsn qrlsiqrvre edagrylcsv cnakgcvnss asvavegsed kgsme Extracellular Region of Wildtype Rat VEGFR-3 (SEQ ID NO: 31) ysmtpp tlnitedsyv idtgdslsis crgqhplewt wrgagevltt ggkdsedtqv vqdcegtear pyckvlslaq thanntgsyy cyykyikari egttaastyv fvrdfeqpfi nkpdtllvnr kdsmwvpclv sipglnitlr sqssvlhpdg qevlwddrrg mrvpt111rd alylqcettw gdqdflsnpf lvhitgnely diglypkks1 ellvgeklvl nctvwaefds gvtfdwdypg kqaerakwvp errsqqthte lssiltihnv sqhdlgpyvc eanngiqqfr estevivhek pfisvewlkg pvleatagde mvklpvklaa ypppefqwyk drkavtgrhn phalvlkevt easagvytla lwnsaaglrq nislelvvnv pphihekeas spsiysrhsr qtltcttygv pqplsvqwhw rpwtpcktfa qrslrrrqpr dgmpqcrdwk evttqdavnp iesldtwtes vegknktvsk lviqdanvsa mykcvvfnkv gqderliyfy vttipdgfsi esepsedple gqsvrlscra dnytyehlrw yrinlstlhd aqgnpllldc knvhlfatpl eanleeaepg arhatlslni prvapedegd yvcevqdrrs qdkhchkkyl svgaleaprl tqnitdllvn vrtslemrcp vagahvpsiv wykderllek esgidladsn qrlsiqrvre edagrylcsv cnakgcvnss asvavegsed kgsme

[0129] In some examples, a soluble VEGF receptor can further include a stabilizing domain (e.g., a Fc domain or a portion of a Fc domain). For example, a stabilizing domain can be an IgG1 Fc domain (e.g., a human wildtype IgG1 Fc domain or a portion thereof). For example, a stalizing domain can be an IgG2 Fc domain (e.g., a human wildtype IgG2 Fc domain or a portion thereof). For example, a stabilizing domain can be an IgG3 Fc domain (e.g., a human wildtype IgG3 domain or a portion thereof).

[0130] Non-limiting examples of human wildtype IgG1 Fc domain, human wildtype IgG2 Fc domain, and human wildtype IgG3 Fc domain are shown below.

TABLE-US-00005 Human Wildtype IgG1 Fe Domain (SEQ ID NO: 32) pcpapellgg psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsrde ltknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw qqgnvfscsv mhealhnhyt qkslslspgk Human Wildtype IgG2 Fe Region (SEQ ID NO: 33) vecppcpapp vagpsvflfp pkpkdtlmis rtpevtcvvv dvshedpevq fnwyvdgvev hnaktkpree qfnstfrvvs vltvvhqdwl ngkeykckvs nkglpapiek tisktkgqpr epqvytlpps reemtknqvs ltclvkgfyp sdiavewesn gqpennyktt ppmldsdgsf flyskltvdk srwqqgnvfs csvmhealhn hytqkslsls pgk Human Wildtype IgG3 Fe Region (SEQ ID NO: 34) tcprcpapel lggpsvflfp pkpkdtlmis rtpevtcvvv dvshedpevq fkwyvdgvev hnaktkpree qfnstfrvvs vltvlhqdwl ngkeykckvs nkalpapiek tisktkgqpr epqvytlpps reemtknqvs ltclvkgfyp sdiavewess gqpennyktt ppmldsdgsf flyskltvdk srwqqgnifs csvmhealhn rftqkslsls pgk

[0131] In some embodiments, the soluble VEGF receptor is aflibercept (Eylea.RTM.). Aflibercept includes portions of human VEGF receptors 1 and 2 extracellular domains fused to the Fc portion of human IgG1 (size.about.115 kDa). Aflibercept inhibits the activity of VEGF-A, VEGF-B, and PIGF. Aflibercept has a K.sub.D for VEGF-A of 0.49 pM. See, e.g., WO 2017/218974.

TABLE-US-00006 Amino Encoding aflibercept (SEQ ID NO: 12) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLI PDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNT IIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKL VNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFV RVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0132] In some embodiments of a soluble VEGF receptor includes a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to SEQ ID NO: 12.

[0133] In some embodiments of the soluble VEGF receptor includes an extracellular domain that is or includes the sequence of SEQ ID NO: 12, except that it includes one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions in the sequence of SEQ ID NO: 12.

[0134] Additional examples of soluble VEGF receptors are described in, e.g., Kendall et al., PNAS 90: 10705-10709, 1993; Kendall et al., Biochem Biophys Res Commun 226: 324-328, 1996; Failla et al., Int J Mol Sci 19(5):pii. E1306, 2018; and Jung et al., PLoS One 7(9): e44572.

Vectors

[0135] Recombinant AAV vectors or "rAAVs" are typically composed of, at a minimum, a transgene or a portion thereof and a regulatory sequence, and optionally 5' and 3' AAV inverted terminal repeats (ITRs). Such a recombinant AAV vector is packaged into a capsid and delivered to a selected target cell (e.g., a cochlear hair cell).

[0136] The AAV sequences of the vector typically comprise the cis-acting 5' and 3' ITR sequences (See, e.g., B. J. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168, 1990). Typical AAV ITR sequences are about 145 nucleotides in length. In some embodiments, at least 75% of a typical ITR sequence (e.g., at least 80%, at least 85%, at least 90%, or at least 95%) is incorporated into the AAV vector. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York, 1989; and K. Fisher et al., J Virol. 70:520 532, 1996). In some embodiments, any of the coding sequences described herein is flanked by 5' and 3' AAV ITR sequences in the AAV vectors. The AAV ITR sequences may be obtained from any known AAV, including presently identified AAV types.

[0137] AAV vectors as described herein may include any of the regulatory elements described herein (e.g., one or more of a promoter, a polyadenylation (poly(A)) signal sequence, and an IRES).

[0138] In some embodiments, the vector(s) is an adenovirus (see, e.g., Dmitriev et al. (1998) J Virol. 72: 9706-9713; and Poulin et al., J. Virol 8: 10074-10086, 2010). In some embodiments, the vector(s) is a retrovirus (see, e.g., Maier et al. (2010) Future Microbiol 5: 1507-23).

[0139] The vectors provided herein can be of different sizes. The choice of vector that is used in any of the compositions, kits, and methods described herein may depend on the size of the vector.

[0140] In some embodiments, the vector(s) can have a total number of nucleotides of up to 10 kb. In some embodiments, the viral vector(s) can have a total number of nucleotides in the range of about 1 kb to about 2 kb, 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 2 kb to about 9 kb, about 2 kb to about 10 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 3 kb to about 9 kb, about 3 kb to about 10 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 4 kb to about 9 kb, about 4 kb to about 10 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 5 kb to about 9 kb, about 5 kb to about 10 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, about 6 kb to about 9 kb, about 6 kb to about 10 kb, about 7 kb to about 8 kb, about 7 kb to about 9 kb, about 7 kb to about 10 kb, about 8 kb to about 9 kb, about 8 kb to about 10 kb, or about 9 kb to about 10 kb.

[0141] In some embodiments, the vector(s) is a lentivirus and can have a total number of nucleotides of up to 8 kb. In some examples, the lentivirus(es) can have a total number of nucleotides of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 8kb, about 6 kb to about 7 kb, or about 7 kb to about 8 kb.

[0142] In some embodiments, the vector(s) is an adenovirus and can have a total number of nucleotides of up to 8 kb. In some embodiments, the adenovirus(es) can have a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 7 kh, about 6 kb to about 8 kb, or about 7 kb to about 8 kb.

[0143] In some embodiments, the vector(s) is an adeno-associated virus (AAV vector) and can include a total number of nucleotides of up to 5 kb. In some embodiments, the AAV vector(s) can include a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.

[0144] A variety of different methods known in the art can be used to introduce any of vectors disclosed herein into a mammalian cell (e.g., an inner ear cell, a cochlear inner hair cell). Non-limiting examples of methods for introducing nucleic acid into a mammalian cell include: lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transfection, and nucleofection.

[0145] Any of the vectors described herein can further include a control sequence, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (polyA) signal, and a Kozak consensus sequence. Non-limiting examples of these control sequences are described herein. In some embodiments, a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and/or a tissue-specific promoter.

Promoters

[0146] The term "promoter" means a DNA sequence recognized by enzymes/proteins in a mammalian cell required to initiate the transcription of a specific gene. A promoter typically refers to, e.g., a nucleotide sequence to which an RNA polymerase and/or any associated factor binds and at which transcription is initiated. Non-limiting examples of promoters are described herein. Additional examples of promoters are known in the art.

[0147] In some embodiments, a vector (e.g., an adeno-associated virus (AAV) vector) encoding an antibody (e.g., an antibody that binds specifically to VEGF or an antigen-binding antibody fragment thereof,) can include a promoter and/or an enhancer. The vector encoding the antibody or antigen-binding antibody fragment can include any of the promoters and/or enhancers described herein or known in the art.

[0148] In some embodiments, the promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissue-specific promoter, or any other type of promoter known in the art. In some embodiments, the promoter is a RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter. In some embodiments, the promoter is a RNA polymerase III promoter, including, but not limited to, a H1 promoter, a human U6 promoter, a mouse U6 promoter, or a swine U6 promoter. The promoter will generally be one that is able to promote transcription in an inner hair cell In some examples, the promoter is a cochlea-specific promoter or a cochlea-oriented promoter.

[0149] A variety of promoters are known in the art that can be used herein. Non-limiting examples of promoters that can be used herein include: human EF1a, human cytomegalovirus (CMV) (U.S. Pat. No. 5,168,062), human ubiquitin C (UBC), mouse phosphoglycerate kinase 1, polyoma adenovirus, simian virus 40 (SV40), .beta.-globin, .beta.-actin, .alpha.-fetoprotein, .gamma.-globin, .beta.-interferon, .gamma.-glutamyl transferase, mouse mammary tumor virus (MMTV), Rous sarcoma virus, rat insulin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein II (MT II), amylase, cathepsin, MI muscarinic receptor, retroviral LTR (e.g. human T-cell leukemia virus HTLV), AAV ITR, interleukin-2, collagenase, platelet-derived growth factor, adenovirus 5 E2, stromelysin, murine MX gene, glucose regulated proteins (GRP78 and GRP94), .alpha.-2-macroglobulin, vimentin, MHC class I gene H-2.kappa. b, HSP70, proliferin, tumor necrosis factor, thyroid stimulating hormone a gene, immunoglobulin light chain, T-cell receptor, HLA DQ.alpha. and DQ.beta., interleukin-2 receptor, MHC class II, MHC class II HLA-DR.alpha., muscle creatine kinase, prealbumin (transthyretin), elastase I, albumin gene, c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), H2B (TH2B) histone, rat growth hormone, human serum amyloid (SAA), troponin I (TN I), duchenne muscular dystrophy, human immunodeficiency virus, and Gibbon Ape Leukemia Virus (GALV) promoters. Additional examples of promoters are known in the art. See, e.g., Lodish, Molecular Cell Biology, Freeman and Company, New York 2007. In some embodiments, the promoter is the CMV immediate early promoter. In some embodiments, the promoter is a CAG promoter or a CAG/CBA promoter.

[0150] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked with a nucleic acid encoding a protein (e.g., an antibody or an antigen-binding antibody fragment), causes RNA to be transcribed from the nucleic acid in a mammalian cell under most or all physiological conditions.

[0151] Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al, Cell 41:521-530, 1985), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1-alpha promoter (Invitrogen).

[0152] Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Additional examples of inducible promoters are known in the art.

[0153] Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. U.S.A. 93:3346-3351, 1996), the tetracycline-repressible system (Gossen et al, Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551, 1992), the tetracycline-inducible system (Gossen et al, Science 268:1766-1769, 1995, see also Harvey et al, Curr. Opin. Chem. Biol. 2:512-518, 1998), the RU486-inducible system (Wang et al, Nat. Biotech. 15:239-243, 1997) and Wang et al, Gene Ther. 4:432-441, 1997), and the rapamycin-inducible system (Magari et al. J. Clin. Invest. 100:2865-2872, 1997).

[0154] The term "tissue-specific" promoter refers to a promoter that is active only in certain specific cell types and/or tissues (e.g., transcription of a specific gene occurs only within cells expressing transcription regulatory proteins that bind to the tissue-specific promoter).

[0155] In some embodiments, the regulatory sequences impart tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner.

[0156] Exemplary tissue-specific promoters include but are not limited to the following: a liver-specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, an alpha-myosin heavy chain (a-MHC) promoter, and a cardiac Troponin T (cTnT) promoter. Additional exemplary promoters include Beta-actin promoter, hepatitis B virus core promoter (Sandig et al., Gene Ther. 3:1002-1009, 1996), alpha-fetoprotein (AFP) promoter (Arbuthnot et al., Hum. Gene Ther. 7:1503-1514, 1996), bone osteocalcin promoter (Stein et al., Mol. Biol. Rep. 24:185-196, 1997); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res. 11:654-664, 1996), CD2 promoter (Hansal et al., J. Immunol. 161:1063-1068, 1998); immunoglobulin heavy chain promoter; T cell receptor alpha-chain promoter, neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol. 13:503-515, 1993), neurofilament light-chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. U.S.A. 88:5611-5615, 1991), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron 15:373-384, 1995).

[0157] In some embodiments, the tissue-specific promoter is a cochlea-specific promoter. In some embodiments, the tissue-specific promoter is a cochlear hair cell-specific promoter. Non-limiting examples of cochlear hair cell-specific promoters include but are not limited to: a ATOH1 promoter, a POU4F3 promoter, a LHX3 promoter, a MYO7A promoter, a MYO6 promoter, a .alpha.9ACHR promoter, and a .alpha.10ACHR promoter. In some embodiments, the promoter is an cochlear hair cell-specific promoter such as a PRESTIN promoter or an ONCOMOD promoter. See, e.g., Zheng et al., Nature 405:149-155, 2000; Tian et al. Dev. Dyn. 231:199-203, 2004; and Ryan et al., Adv. Otorhinolaryngol. 66: 99-115, 2009.

Enhancers

[0158] In some instances, a vector (e.g., an AAV vector) can include an enhancer sequence. The term "enhancer" refers to a nucleotide sequence that can increase the level of transcription of a nucleic acid encoding a protein of interest (e.g., an antibody that binds specifically to VEGF or an antigen-binding antibody fragment thereof, or a soluble VEGF receptor). Enhancer sequences (50-1500 basepairs in length) generally increase the level of transcription by providing additional binding sites for transcription-associated proteins (e.g., transcription factors). In some embodiments, an enhancer sequence is found within an intronic sequence. Unlike promoter sequences, enhancer sequences can act at much larger distance away from the transcription start site (e.g., as compared to a promoter). Non-limiting examples of enhancers include a RSV enhancer, a CMV enhancer, and a SV40 enhancer.

Poly(A) Signal Sequence

[0159] In some embodiments, any of the vectors provided herein (e.g., an AAV vector) can include a polyadenylation (poly(A)) signal sequence. Most nascent eukaryotic mRNAs possess a poly(A) tail at their 3' end which is added during a complex process that includes cleavage of the primary transcript and a coupled polyadenylation reaction driven by the poly(A) signal sequence (see, e.g., Proudfoot et al., Cell 108:501-512, 2002). The poly(A) tail confers mRNA stability and transferability (Molecular Biology of the Cell, Third Edition by B. Alberts et al., Garland Publishing, 1994). In some embodiments, the poly(A) signal sequence is positioned 3' to the nucleic acid sequence encoding the antibody heavy chain, the antibody light chain, the antigen-binding antibody fragment, or the soluble VEGF receptor.

[0160] As used herein, "polyadenylation" refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation (or poly(A)) signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but also can occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end at the cleavage site.

[0161] As used herein, a "poly(A) signal sequence" or "polyadenylation signal sequence" is a sequence that triggers the endonuclease cleavage of an mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.

[0162] There are several poly(A) signal sequences that can be used, including those derived from bovine growth hormone (bgh) (Woychik et al., Proc. Natl. Acad. Sci. U.S.A. 81(13):3944-3948, 1984; U.S. Pat. No. 5,122,458), mouse-.beta.-globin, mouse-.alpha.-globin (Orkin et al., EMBO J. 4(2):453-456, 1985; Thein et al., Blood 71(2):313-319, 1988), human collagen, polyoma virus (Batt et al., Mol. Cell Biol. 15(9):4783-4790, 1995), the Herpes simplex virus thymidine kinase gene (HSV TK), IgG heavy-chain gene polyadenylation signal (US 2006/0040354), human growth hormone (hGH) (Szymanski et al., Mol. Therapy 15(7):1340-1347, 2007), the group consisting of SV40 poly(A) site, such as the SV40 late and early poly(A) site (Schek et al., Mol. Cell Biol. 12(12):5386-5393, 1992).

[0163] The poly(A) signal sequence can be AATAAA. The AATAAA sequence may be substituted with other hexanucleotide sequences with homology to AATAAA and that are capable of signaling polyadenylation, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, or AATAAG (see, e.g., WO 06/12414).

[0164] In some embodiments, the poly(A) signal sequence can be a synthetic polyadenylation site (see, e.g., the pCl-neo expression vector of Promega that is based on Levitt el al, Genes Dev. 3(7):1019-1025, 1989). In some embodiments, the poly(A) signal sequence is the polyadenylation signal of soluble neuropilin-1 (sNRP) (AAATAAAATACGAAATG; SEQ ID NO: 11) (see, e.g., WO 05/073384). Additional examples of poly(A) signal sequences are known in the art.

Internal Ribosome Entry Site (IRES)

[0165] In some embodiments, a vector (e.g., an adeno-associated virus (AAV) vector) encoding an antibody (e.g., an antibody heavy chain and an antibody light chain), an antigen-binding antibody fragment, or a soluble VEGF receptor can include a polynucleotide internal ribosome entry site (IRES). An IRES sequence is used to produce more than one polypeptide from a single gene transcript. An IRES forms a complex secondary structure that allows translation initiation to occur from any position with an mRNA immediately downstream from where the IRES is located (see, e.g., Pelletier and Sonenberg, Mol. Cell. Biol. 8(3):1103-1112, 1988).

[0166] There are several IRES sequences known to those in skilled in the art, including those from, e.g., foot and mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), human rhinovirus (HRV), cricket paralysis virus, human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis C virus (HCV), and poliovirus (PV). See e.g., Alberts, Molecular Biology of the Cell, Garland Science, 2002; and Hellen et al., Genes Dev. 15(13):1593-612, 2001.

[0167] In some embodiments, the IRES sequence that is incorporated into the AAV vector is the foot and mouth diseause virus (FMDV) 2A sequence. The Foot and Mouth Disease Virus 2A sequence is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (Ryan, M D et al., EMBO 4:928-933, 1994; Mattion et al., J. Virology 70:8124-8127, 1996; Furler et al., Gene Therapy 8:864-873, 2001; and Halpin et al., Plant Journal 4:453-459, 1999). The cleavage activity of the 2A sequence has previously been demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan et al., EMBO 4:928-933, 1994; Mattion et al., J. Virology 70:8124-8127, 1996; Furler et al., Gene Therapy 8:864-873, 2001; and Halpin et al., Plant Journal 4:453-459, 1999; de Felipe et al., Gene Therapy 6:198-208, 1999; de Felipe et al., Human Gene Therapy 11:1921-1931, 2000; and Klump et al., Gene Therapy 8:811-817, 2001).

Reporter Sequences

[0168] Any of the AAVs provided herein can optionally include a sequence encoding a reporter protein ("a reporter sequence"). Non-limiting examples of reporter sequences include DNA sequences encoding: a beta-lactamase, a beta-galactosidase (LacZ), an alkaline phosphatase, a thymidine kinase, a green fluorescent protein (GFP), a red fluorescent protein, an mCherry fluorescent protein, a yellow fluorescent protein, a chloramphenicol acetyltransferase (CAT), and a luciferase. Additional examples of reporter sequences are known in the art. When associated with regulatory elements which drive their expression, the reporter sequence can provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectrographic assays; fluorescent activating cell sorting (FACS) assays; immunological assays (e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).

[0169] In some embodiments, the reporter sequence is the LacZ gene, and the presence of a vector carrying the LacZ gene in a mammalian cell (e.g., a cochlear hair cell) is detected by assays for beta-galactosidase activity. When the reporter is a fluorescent protein (e.g., green fluorescent protein) or luciferase, the presence of a vector carrying the fluorescent protein or luciferase in a mammalian cell (e.g., a cochlear hair cell) may be measured by fluorescent techniques (e.g., fluorescent microscopy or FACS) or light production in a luminometer (e.g., a spectrophotometer or an IVIS imaging instrument). In some embodiments, the reporter sequence can be used to verify the tissue-specific targeting capabilities and tissue-specific promoter regulatory activity of any of the vectors described herein.

Flanking Regions Untranslated Regions (UTRs)

[0170] In some embodiments, any of the adeno-associated virus (AAV) vectors can include an untranslated region, such as a 5' UTR or a 3' UTR.

[0171] Untranslated regions (UTRs) of a gene are transcribed but not translated. The 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon. The 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into any of the vectors, compositions, kits, or methods as described herein to enhance the expression of an antibody (e.g., an antibody that binds specifically to VEGF), an antigen-binding antibody fragment (e.g., an antigen-binding fragment that binds specifically to VEGF), or a soluble VEGF receptor.

[0172] Natural 5' UTRs include a sequence that plays a role in translation initiation. They harbor signatures like Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus sequence CCR(A/G)CCAUGG, where R is a purine (A or G) three bases upstream of the start codon (AUG), and the start codon is followed by another "G". The 5' UTRs have also been known to form secondary structures that are involved in elongation factor binding.

[0173] In some embodiments, a 5' UTR is included in any of the vectors described herein. Non-limiting examples of 5' UTRs, including those from the following genes: albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, and Factor VIII, can be used to enhance expression of a nucleic acid molecule, such as a mRNA.

[0174] In some embodiments, a 5' UTR from a mRNA that is transcribed by a cell in the cochlea can be included in any of the vectors, compositions, kits, and methods described herein.

[0175] 3' UTRs are known to have stretches of adenosines and uridines (in the RNA form) or thymidines (in the DNA form) embedded in them. These AU-rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU-rich elements (AREs) can be separated into three classes (Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995; Chen et al., Mol. Cell Biol. 15:2010-2018, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. For example, c-Myc and MyoD mRNAs contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A) (U/A) nonamers. GM-CSF and TNF-alpha mRNAs are examples that contain class II AREs. Class III AREs are less well defined. These U-rich regions do not contain an AUUUA motif. Two well-studied examples of this class are c-Jun and myogenin mRNAs.

[0176] Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.

[0177] In some embodiments, the introduction, removal, or modification of 3' UTR AREs can be used to modulate the stability of an mRNA encoding a protein of interest (e.g., any antibody described herein, any antigen-binding antibody fragment described herein, or any soluble VEGF receptor described herein). In other embodiments, AREs can be removed or mutated to increase the intracellular stability and thus increase translation and production of a protein of interest (e.g., any antibody described herein, any antigen-binding antibody fragment described herein, or any soluble VEGF receptor described herein).

[0178] In other embodiments, non-ARE sequences may be incorporated into the 5' or 3' UTRs. In some embodiments, introns or portions of intron sequences may be incorporated into the flanking regions of the polynucleotides in any of the vectors, compositions, kits, and methods provided herein. Incorporation of intronic sequences may increase protein production as well as mRNA levels.

Fc Mutations that Decrease the Half-Life of an Antibody, Antigen-Binding Antibody Fragment, or a Soluble VEGF Receptor in a Mammal

[0179] Any of the antibodies, antigen-binding antibody fragments, or soluble VEGF receptors described herein can include one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid substitutions in the Fc region that decrease the half-life of the antibody, the antigen-binding antibody fragment, or soluble VEGF receptor in a mammal, e.g., as compared to the half-life of an otherwise identical antibody, antigen-binding antibody fragment, or soluble VEGF receptor not including at least one of the one or more amino acid substituions in the Fc region. Methods for determining the half-life of an antibody, antigen-binding antibody fragment, or soluble VEGF receptor in a mammal are well-known in the art.

[0180] Non-limiting examples of point mutations in a Fc mutation that can decrease the half-life of an antibody, an antigen-binding antibody fragment, or soluble VEGF receptor are described in Leabman et al., MAbs 5(6):896-903, 2013.

Methods

[0181] Also provided herein are methods that include introducing into an inner ear of a mammal a therapeutically effective amount of an adeno-associated virus (AAV) vector that includes a nucleotide sequence encoding (a) a polypeptide including an antibody heavy chain variable domain (e.g., any of the exemplary antibody heavy chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein) and a polypeptide including an antibody light chain variable domain (e.g., any of the exemplary antibody light chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein); (b) a polypeptide including an antigen-binding antibody fragment (e.g., a scFv) (e.g., any of the exemplary antigen-binding antibody fragments described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) a soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein). Also provided herein are methods for increasing the level of an antibody or an antigen-binding antibody fragment in an inner ear of a mammal in need thereof, that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding (a) a polypeptide including an antibody heavy chain variable domain (e.g., any of the antibody heavy chain variable domains described herein) operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain (e.g., any of the antibody light chain variable domains described herein) operably linked to a signal peptide; or (b) a polypeptide including an antigen-binding antibody fragment (e.g., a scFv) (e.g., any of the exemplary antigen-binding antibody fragments described herein) operably linked to a signal peptide (e.g., any of the exemplary signal peptides described herein); where the introducing results in an increase (e.g., a 1% to 400% increase (or any of the subranges of this range described herein), or at least a 1%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, at least a 100%, at least a 150%, at least a 200%, at least a 250%, at least a 300%, at least a 350%, at least a 400%, at least a 450%, at least a 500%, at least a 550%, at least a 600%, at least a 650%, at least a 700% , at least a 750%, at least a 800%, at least a 850%, at least a 900%, at least a 950%, at least a 1000%, at least a 1100%, at least a 1200%, at least a 1300%, at least a 1400%, at least a 1500%, at least a 1600%, at least a 1700%, at least a 1800%, at least a 1900%, or at least a 2000% increase) in the level of the antibody or the antigen-binding antibody fragment in the inner ear of the mammal, e.g., as compared to the level of the antibody or the antigen-binding antibody fragment in the inner ear of the mammal prior to the administration.

[0182] Also provided herein are methods for increasing the level of a soluble VEGF receptor that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein); where the introducing results in an increase (e.g., a 1% to 400% increase (or any of the subranges of this range described herein), or at least a 1%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, at least a 100%, at least a 150%, at least a 200%, at least a 250%, at least a 300%, at least a 350%, at least a 400%, at least a 450%, at least a 500%, at least a 550%, at least a 600%, at least a 650%, at least a 700% , at least a 750%, at least a 800%, at least a 850%, at least a 900%, at least a 950%, at least a 1000%, at least a 1100%, at least a 1200%, at least a 1300%, at least a 1400%, at least a 1500%, at least a 1600%, at least a 1700%, at least a 1800%, at least a 1900%, or at least a 2000% increase) in the level of the soluble VEGF receptor in the inner ear of the mammal, e.g., as compared to the level of the soluble VEGF receptor in the inner ear of the mammal prior to the administration.

[0183] Also provided herein are methods for treating an inner ear disorder in a mammal in need thereof that include introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding: (a) a polypeptide including an antibody heavy chain variable domain (e.g., e.g., any of the antibody heavy chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein) and a polypeptide comprising an antibody light chain variable domain (e.g., any of the antibody light chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein); (b) a polypeptide including an antigen-binding antibody fragment (e.g., any of the exemplary antigen-binding antibody fragments described herein) linked to a signal peptide (e.g., any of the signal peptides described herein); or (c) a soluble VEGF receptor (e.g., any of the soluble VEGR receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), where the introducing results in the treatment of the inner ear disorder in the mammal. In some embodiments, treatment of an inner ear disorder results in a reduction (e.g., a 1% to 100% reduction, or any of the subranges of this range described herein) in the severity, frequency, or number of symptoms of an inner ear disorder in a mammal following the introducing as compared to before the introducing. In some embodiments, treatment of any inner ear disorder results in an increase (e.g., a 1% to 400% increase, or any of the subranges of this range described herein) in the hearing (e.g., one or more metrics of hearing) of the mammal following the introducing as compared to before the introducing.

[0184] In some embodiments of any of these methods, the antibody or the antigen-binding antibody fragment, or the soluble VEGF receptor, binds specifically to a vascular endothelial growth factor (VEGF) (e.g., one of more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D, e.g., one or more of human VEGF-A, human VEGF-B, human VEGF-C, and human VEGF-D). In some embodiments of any of these methods, the AAV vector further includes one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment. In some embodiments wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of these methods, the AAV vector further includes a polyadenylation signal sequence. In some embodiments of any of these methods, the mammal is a human. In some embodiments of any of these methods, the mammal (e.g., the human) has been identified as having an inner ear disorder. In some embodiments of any of these methods, the mammal (e.g., the human) has previously been diagnosed as having an inner ear disorder. In some embodiments of any of these methods, the vector includes a nucleic acid sequence encoding a polypeptide comprising an antibody heavy chain and an antibody light chain. In some embodiments of any of these methods, the vector includes a nucleic acid sequence encoding an antigen-binding antibody fragment. In some embodiments of any of these methods, the vector include a nucleic acid sequence encoding a soluble VEGF receptor operably linked to a signal peptide.

[0185] Also provided herein are methods of reducing a VEGF activity (e.g., one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D, e.g., one or more of human VEGF-A, human VEGF-B, human VEGF-C, and human VEGF-D) in an inner ear of a mammal in need thereof that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding (a) a polypeptide including an antibody heavy chain variable domain (e.g., any of the antibody heavy chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein) and a polypeptide including an antibody light chain variable domain (e.g., any of the antibody light chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein); (b) a polypeptide including an antigen-binding antibody fragment (e.g., a Fab or a scFv) (e.g., any of the antigen-binding antibody fragments described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein); or (c) a soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), where the polypeptide of (a) includes an antibody that binds specifically to a VEGF and reduces a VEGF activity, the polypeptide of (b) includes an antigen-binding antibody fragment that binds specifically to a VEGF and reduces a VEGF activity, or the soluble VEGF receptor of (c) binds specifically to one or more VEGF proteins and reduces the activity of the one or more VEGF proteins; and where the introducing results in a reduction (e.g., a 1% to 100% reduction, or any of the subranges of this range described herein) in a VEGF activity (e.g., an activity of one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D, e.g., one or more human VEGF-A, human VEGF-B, human VEGF-C, and human VEGF-D) in the inner ear of the mammal, e.g., as compared to the VEGF activity in the mammal prior to the introducing. A reduction in a VEGF activity in a mammal can be detected using any of the exemplary methods described herein.

[0186] Also provided herein are methods of treating acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II (NF2) in an inner ear of a mammal that include: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that includes a nucleotide sequence encoding (a) a polypeptide including an antibody heavy chain variable domain (e.g., any of the antibody heavy chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein) and a polypeptide including an antibody light chain variable domain (e.g., any of the antibody light chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein); (b) a polypeptide including an antigen-binding antibody fragment (e.g., a Fab or a scFv) (e.g., any of the antigen-binding antibody fragments described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) a soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein); where the polypeptide of (a) encodes an antibody that binds specifically to a VEGF (e.g., one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D, e.g., one or more of human VEGF-A, human VEGF-B, human VEGF-C, and human VEGF-D) and reduces the VEGF activity, the polypeptide of (b) encodes an antigen-binding antibody fragment that binds specifically to a VEGF (e.g., one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D, e.g., one or more of human VEGF-A, human VEGF-B, human VEGF-C, and human VEGF-D) and reduces the VEGF activity, or the soluble VEGF receptor of (c) binds to specifically to one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D (e.g., one or more of human VEGF-A, human VEGF-B, human VEGF-C, and human VEGF-D) and where the introducing results in treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II (NF2) in the inner ear of the mammal. As described herein, successful treatment of one or more of an acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II can be detected by observing a reduction (e.g., a 1% to 100% decrease, or any of the subranges of this range described herein) in the number, severity, or frequency of one or more symptoms of an acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II, respectively, in the mammal, e.g., as compared to before the introducing step.

[0187] In some embodiments of any of these methods, the vector includes a nucleic acid sequence encoding a polypeptide encoding an antibody heavy chain variable domain (e.g., any of the antibody heavy chains described herein) and an antibody light chain variable domain (e.g., any of the antibody light chain variable domains described herein). In some embodiments of any of these methods, the vector includes a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment (e.g., any of the antigen-binding antibody fragments described herein). In some embodiments of any of these methods, the vector includes a nucleic acid sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein). In some embodiments of any of these methods, the AAV vector further includes one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment. In some embodiments, the AAV vector comprises a promoter, where the promoter is selected from the group consisting of: an inducible promoter, a constitutive promoter, or a tissue-specific promoter. In some embodiments, the AAV vector further includes a polyadenylation signal sequence. In some embodiments of any of these methods, the mammal is a human. In some embodiments of any of these methods, the mammal (e.g., the human) has been identified as having an inner ear disorder. In some embodiments of any of these methods, the mammal (e.g., the human) has previously been diagnosed as having an inner ear disorder. In some embodiments of any of these methods, the mammal (e.g., the human) has been identified or diagnosed as having drug-induced hearing loss. In some embodiments of any of these methods, the mammal (e.g., the human) has been identified or diagnosed as having age-related hearing loss.

[0188] In some embodiments, the antibody or antigen-binding fragment thereof includes a Fc region that includes one or more point mutations that decrease the half-life of the antibody or antigen-binding antibody fragment in vivo.

[0189] In some embodiments of any of these methods, two or more doses of any of the adeno-associated virus (AAV) vectors described herein are introduced or administered into the inner ear of the mammal or subject. Some embodiments of any of these methods can include introducing or administering a first dose of the adeno-associated virus (AAV) vectors into the inner ear of the mammal or subject, assessing hearing function of the mammal or subject following the introducing or the administering of the first dose, and administering an additional dose of the adeno-associated virus (AAV) vector into the inner ear of the mammal or subject found not to have a hearing function within a normal range (e.g., as determined using any test for hearing known in the art).

[0190] In some embodiments of any of the methods described herein, the adeno-associated virus (AAV) vectors can be formulated for intra-cochlear administration. In some embodiments of any of the methods described herein, the adeno-associated virus (AAV) vectors described herein can be administered via intra-cochlear administration or local administration. In some embodiments of any of the methods described herein, the adeno-associated virus (AAV) vectors are administered through the use of a medical device (e.g., any of the exemplary medical devices described herein).

[0191] In some embodiments, intra-cochlear administration can be performed using any of the methods described herein or known in the art. For example, an adeno-associated virus (AAV) vector can be administered or introduced into the cochlea using the following surgical technique: first using visualization with a 0 degree, 2.5-mm rigid endoscope, the external auditory canal is cleared and a round knife is used to sharply delineate an approximately 5-mm tympanomeatal flap. The tympanomeatal flap is then elevated and the middle ear is entered posteriorly. The chorda tympani nerve is identified and divided, and a currette is used to remove the scutal bone, exposing the round window membrane. To enhance apical distribution of the administered or introduced adeno-associated virus (AAV) vector, a surgical laser may be used to make a small 2-mm fenestration in the oval window to allow for perilymph displacement during trans-round window membrane infusion of the adeno-associated virus (AAV) vectors. The microinfusion device is then primed and brought into the surgical field. The device is maneuvered to the round window, and the tip is seated within the bony round window overhang to allow for penetration of the membrane by the microneedle(s). The footpedal is engaged to allow for a measured, steady infusion of the adeno-associated virus (AAV) vectors. The device is then withdrawn and the round window and stapes foot plate are sealed with a gelfoam patch.

[0192] In some embodiments of any of the methods described herein, the subject or mammal is a rodent, a non-human primate, or a human. In some embodiments of any of the methods described herein, the subject or mammal is an adult, a teenager, a juvenile, a child, a toddler, an infant, or a newborn. In some embodiments of any of the methods described herein, the subject or mammal is 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 2-5, 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-110, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-110, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 40-60, 40-70, 40-80, 40-90, 40-100, 50-70, 50-80, 50-90, 50-100, 60-80, 60-90, 60-100, 70-90, 70-100, 70-110, 80-100, 80-110, or 90-110 years of age. In some embodiments of any of the methods described herein, the subject or mammal is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months of age.

[0193] In some embodiments of any of the methods described herein, the subject or mammal has or is at risk of developing hearing loss (e.g., drug-induced hearing loss). In some embodiments of any of the methods described herein, the subject or mammal has been previously identified as having a mutation in a VEGF gene.

[0194] In some embodiments, successful treatment of hearing loss (e.g., drug-induced hearing loss) can be determined in a subject using any of the conventional functional hearing tests known in the art. Non-limiting examples of functional hearing tests are various types of audiometric assays (e.g., pure-tone testing, speech testing, test of the middle ear, auditory brainstem response, and otoacoustic emissions).

[0195] Methods for introducing any of the adeno-associated virus (AAV) vectors described herein into a mammalian cell are known in the art (e.g., via lipofection or through the use of a viral vector, e.g., any of the viral vectors described herein).

Pharmaceutical Compositions and Kits

[0196] In some embodiments, any of the compositions described herein can further include one or more agents that promote the entry of a nucleic acid or any of the vectors described herein into a mammalian cell (e.g., a liposome or cationic lipid). In some embodiments, any of the vectors described herein can be formulated using natural and/or synthetic polymers. Non-limiting examples of polymers that may be included in any of the compositions described herein can include, but are not limited to, DYNAMIC POLYCONJUGATE.RTM. (Arrowhead Research Corp., Pasadena, Calif.), formulations from Mirus Bio (Madison, Wis.) and Roche Madison (Madison, Wis.), PhaseRX polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGY.RTM. (PhaseRX, Seattle, Wash.), DMRI/DOPE, poloxamer, VAXFECTIN.RTM. adjuvant from Vical (San Diego, Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimers and poly (lactic-co-glycolic acid) (PLGA) polymers, RONDEL.TM. (RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research Corporation, Pasadena, Calif.), and pH responsive co-block polymers, such as, but not limited to, those produced by PhaseRX (Seattle, Wash.). Many of these polymers have demonstrated efficacy in delivering oligonucleotides in vivo into a mammalian cell (see, e.g., deFougerolles, Human Gene Ther. 19:125-132, 2008; Rozema et al., Proc. Natl. Acad. Sci. U.S.A. 104:12982-12887, 2007; Rozema et al., Proc. Natl. Acad. Sci. U.S.A. 104:12982-12887, 2007; Hu-Lieskovan et al., Cancer Res. 65:8984-8982, 2005; Heidel et al., Proc. Natl. Acad. Sci. U.S.A. 104:5715-5721, 2007).

[0197] Any of the compositions described herein can be, e.g., a pharmaceutical composition. A pharmaceutical composition can include any of the compositions described herein and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise one or more buffers, such as neutral-buffered saline, phosphate-buffered saline, and the like; one or more carbohydrates, such as glucose, mannose, sucrose, and dextran; mannitol; one or more proteins, polypeptides, or amino acids, such as glycine; one or more antioxidants; one or more chelating agents, such as EDTA or glutathione; and/or one or more preservatives.

[0198] In some embodiments, the composition includes a pharmaceutically acceptable carrier (e.g., phosphate buffered saline, saline, or bacteriostatic water). Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, injectable gels, drug-release capsules, and the like.

[0199] As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial agents, antifungal agents, and the like that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into any of the compostions described herein.

[0200] In some embodiments, a single dose of any of the compositions described herein can include a total sum amount of the at least two different vectors of at least 1 ng, at least 2 ng, at least 4 ng, about 6 ng, about 8 ng, at least 10 ng, at least 20 ng, at least 30 ng, at least 40 ng, at least 50 ng, at least 60 ng, at least 70 ng, at least 80 ng, at least 90 ng, at least 100 ng, at least 200 ng, at least 300 ng, at least 400 ng, at least 500 ng, at least 1 .mu.g, at least 2 .mu.g, at least 4 .mu.g, at least 6 .mu.g, at least 8 .mu.g, at least 10 .mu.g, at least 12 .mu.g, at least 14 .mu.g, at least 16 .mu.g, at least 18 .mu.g, at least 20 .mu.g, at least 22 .mu.g, at least 24 .mu.g, at least 26 .mu.g, at least 28 .mu.g, at least 30 pg at least 32 .mu.g, at least 34 .mu.g, at least 36 .mu.g, at least 38 .mu.g, at least 40 .mu.g, at least 42 .mu.g, at least 44 .mu.g, at least 46 .mu.g, at least 48 .mu.g, at least 50 .mu.g, at least 52 .mu.g, at least 54 .mu.g, at least 56 .mu.g, at least 58 .mu.g, at least 60 .mu.g, at least 62 .mu.g, at least 64 .mu.g, at least 66 .mu.g, at least 68 .mu.g, at least 70 .mu.g, at least 72 .mu.g, at least 74 .mu.g, at least 76 .mu.g, at least 78 .mu.g, at least 80 .mu.g, at least 82 .mu.g, at least 84 .mu.g, at least 86 .mu.g, at least 88 .mu.g, at least 90 .mu.g, at least 92 .mu.g, at least 94 .mu.g, at least 96 .mu.g, at least 98 .mu.g, at least 100 .mu.g, at least 102 .mu.g, at least 104 .mu.g, at least 106 .mu.g, at least 108 .mu.g, at least 110 .mu.g, at least 112 .mu.g, at least 114 .mu.g, at least 116 .mu.g, at least 118 .mu.g, at least 120 .mu.g, at least 122 .mu.g, at least 124 .mu.g, at least 126 .mu.g, at least 128 .mu.g, at least 130 pg at least 132 .mu.g, at least 134 .mu.g, at least 136 .mu.g, at least 138 .mu.g, at least 140 .mu.g, at least 142 .mu.g, at least 144 .mu.g, at least 146 .mu.g, at least 148 .mu.g, at least 150 .mu.g, at least 152 .mu.g, at least 154 .mu.g, at least 156 .mu.g, at least 158 .mu.g, at least 160 .mu.g, at least 162 .mu.g, at least 164 .mu.g, at least 166 .mu.g, at least 168 .mu.g, at least 170 .mu.g, at least 172 .mu.g, at least 174 .mu.g, at least 176 .mu.g, at least 178 .mu.g, at least 180 .mu.g, at least 182 .mu.g, at least 184 .mu.g, at least 186 .mu.g, at least 188 .mu.g, at least 190 .mu.g, at least 192 .mu.g, at least 194 .mu.g, at least 196 .mu.g, at least 198 .mu.g, or at least 200 .mu.g, e.g., in a buffered solution.

[0201] The compositions provided herein can be, e.g., formulated to be compatible with their intended route of administration. A non-limiting example of an intended route of administration is local administration (e.g., intra-cochlear administration). In some embodiments, the therapeutic compositions are formulated to include a lipid nanoparticle. In some embodiments, the therapeutic compositions are formulated to include a polymeric nanoparticle. In some embodiments, the therapeutic compositions are formulated to comprise a mini-circle DNA. In some embodiments, the therapeutic compositions are formulated to comprise a CELiD DNA. In some embodiments, the therapeutic compositions are formulated to comprise a synthetic perilymph solution. An exemplary synthetic perilymph solution includes 20-200 mM NaCl; 1-5 mM KCl; 0.1-10 mM CaCl.sub.2; 1-10 mM glucose; 2-50 mM HEPES, having a pH of between about 6 and about 9.

[0202] Also provided are kits including any of the compositions described herein. In some embodiments, a kit can include a solid composition (e.g., a lyophilized composition including the at least two different vectors described herein) and a liquid for solubilizing the lyophilized composition. In some embodiments, a kit can include a pre-loaded syringe including any of the compositions described herein.

[0203] In some embodiments, the kit includes a vial comprising any of the compositions described herein (e.g., formulated as an aqueous composition, e.g., an aqueous pharmaceutical composition).

[0204] In some embodiments, the kits can include instructions for performing any of the methods described herein.

Devices and Surgical Methods

[0205] Provided herein are therapeutic delivery systems for treating hearing loss (e.g., acoustic neuromas/vestibular schwannomas and associated-hearing loss). In one aspect, the therapeutic delivery systems include i) a medical device capable of creating one or a plurality of incisions in a round window membrane of an inner ear of a human subject in need thereof, and ii) an effective dose of a composition (e.g., any of the compositions described herein). In some embodiments, the medical device includes a plurality of micro-needles.

[0206] Also provided herein are surgical methods for treatment of hearing loss (e.g., acoustic neuromas/vestibular schwannomas and associated-hearing loss). In some embodiments, the methods include the steps of: introducing into a cochlea of a human subject a first incision at a first incision point; and administering intra-cochlearly a therapeutically effective amount of any of the compositions provided herein. In some embodiments, the composition is administered to the subject at the first incision point. In some embodiments, the composition is administered to the subject into or through the first incision.

[0207] In some embodiments of any of the methods described herein, any of the compositions described herein is administered to the subject into or through the cochlea oval window membrane. In some embodiments of any of the methods described herein, any of the compositions described herein is administered to the subject into or through the cochlea round window membrane. In some embodiments of any of the methods described herein, the composition is administered using a medical device capable of creating a plurality of incisions in the round window membrane. In some embodiments, the medical device includes a plurality of micro-needles. In some embodiments, the medical device includes a plurality of micro-needles including a generally circular first aspect, where each micro-needle has a diameter of at least about 10 microns. In some embodiments, the medical device includes a base and/or a reservoir capable of holding the composition. In some embodiments, the medical device includes a plurality of hollow micro-needles individually including a lumen capable of transferring the composition. In some embodiments, the medical device includes a means for generating at least a partial vacuum.

[0208] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations that become evident as a result of the teaching provided herein.

[0209] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compositions of the present invention and practice the claimed methods. The following working examples specifically point out various aspects of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLES

Example 1

Construction of Viral Vectors

[0210] Four different recombinant AAV vectors were generated and are shown in FIGS. 1A-D.

[0211] The vector in FIG. 1A is an exemplary AAV vector of 4474 bp (SEQ ID NO: 35) that includes the following sub-sequences going in the 5' to 3' direction:

TABLE-US-00007 CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCG GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG GAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGT(5' ITR;SEQ ID NO: 36); GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTA GTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT GGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTAC ATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGT TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTAT TTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGG GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGG CGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCC TTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGC GGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCC GCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGT GAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTT AATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTC CGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGT GTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTG AGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGG GGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGA ACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTG GGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTG CTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCG GGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGG GGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCC CCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTT TTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTG TGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCG GGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTT CGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTG TCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCG GCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCAT GCCTTCTTCTTTTTCCTACAG(CBA sequence;SEQ ID NO: 37); CTCCTGGGCAACGTGCTGGTTATTGTGACCGGTGCCACC(spacer; SEQ ID NO: 38); ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGTCTCTGGCCCTGGT CACCAATTCT (IL-2 secretion signal sequence;SEQ ID NO: 39); GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTTGTTCAACCTGGCGGCTC TCTGAGACTGAGCTGTGCCGCTTCTGGCTACACCTTCACCAACTACGGCA TGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGATGG ATCAACACCTACACCGGCGAGCCAACATACGCCGCCGACTTCAAGCGGAG ATTCACCTTCAGCCTGGACACCAGCAAGAGCACCGCCTACCTGCAGATGA ACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAAGTATCCC CACTACTACGGCAGCAGCCACTGGTACTTTGACGTGTGGGGACAGGGCAC ACTGGTCACAGTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTTTTCCCAC TGGCTCCTAGCAGCAAGTCTACCAGCGGAGGAACAGCCGCTCTGGGCTGT CTGGTCAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATTCTGG CGCTCTGACAAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAAAGCAGCG GCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCAAGCAGCTCTCTGGGC ACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAACACCAAGGT GGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCTC CATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCT CCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTG CGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGT ACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAA CAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCA GGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCC TGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGG GAACCCCAGGTTTACACACTGCCTCCAAGCCGGGAAGAGATGACCAAGAA CCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCTTCCGATATCG CCGTGGAATGGGAGAGCAATGGCCAGCCAGAGAACAACTACAAGACAACC CCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGAC AGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGA TGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGTCT CCTGGCAAG(sequence encoding heavy chain of bevacizumab;SEQ ID NO: 40); CGGAAGAGAAGA(linker sequence;SEQ ID NO: 41); GGCTCTGGCGAAGGCAGAGGCAGCCTGCTTACATGTGGCGACGTGGAAGA GAACCCCGGACCT(T2A sequence;SEQ ID NO: 42); ATGTATAGAATGCAGCTCCTGTCCTGCATTGCCCTGAGCCTGGCTCTCGT GACCAACAGC(IL-2 secretion signal sequence;SEQ ID NO: 43); GACATCCAGATGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGGGAGA CAGAGTGACCATCACCTGTAGCGCCAGCCAGGACATCTCCAACTACCTGA ACTGGTATCAGCAAAAGCCCGGCAAGGCCCCTAAGGTGCTGATCTACTTC ACAAGCAGCCTGCACTCCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAG CGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCG CCACCTACTACTGCCAGCAGTACAGCACCGTGCCTTGGACATTTGGCCAG GGCACAAAGGTGGAAATCAAGCGGACTGTGGCCGCTCCTAGCGTGTTCAT CTTTCCACCTAGCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGT GCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAAGTG GACAATGCCCTGCAGAGCGGCAACAGCCAAGAGAGCGTGACAGAGCAGGA CTCCAAGGATAGCACCTATAGCCTGAGCAGCACCCTGACACTGAGCAAGG CCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGC CTTTCTAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAATGTTAA (sequence encoding light chain of bevacizumab;SEQ ID NO: 44); GAGCTCGCTGATCAGCCTCGA(linker sequence;SEQ ID NO: 45); CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCT TCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA GGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT GCTGGGGATGCGGTGGGCTCTATGG(bovine growth hormone polyA tail sequence;SEQ ID NO: 46); AAGCTTGAATTCAGCTGACGTGCCTCGGACCGCT(linker sequence;SEQ ID NO: 47); and AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG CTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCG GGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG(3' ITR; SEQ ID NO: 48).

[0212] The IL-2 signal sequence encoded by each of SEQ ID NOs: 39 and 43 is MYRMQLLSCIALSLALVTNS (SEQ ID NO: 49). The T2A sequence encoded by SEQ ID NO: 42 is GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 50). SEQ ID NO: 40 encodes the heavy chain of bevacizumab (SEQ ID NO: 6). SEQ ID NO: 44 encodes the light chain of bevacizumab (SEQ ID NO: 5). The last three nucleotides in SEQ ID NO: 44 are a stop codon.

[0213] The vector in FIG. 1B is an exemplary AAV vector of 3814 bp (SEQ ID NO: 51) that includes the following sub-sequences going in the 5' to 3' direction:

TABLE-US-00008 CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCG GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG GAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGT(3' ITR;SEQ ID NO: 36); GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTA GTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT GGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTAC ATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGT TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTAT TTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGG GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGG CGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCC TTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGC GGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCC GCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGT GAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTT AATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTC CGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGT GTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTG AGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGG GGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGA ACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTG GGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTG CTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCG GGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGG GGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCC CCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTT TTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTG TGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCG GGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTT CGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTG TCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCG GCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCAT GCCTTCTTCTTTTTCCTACAG(CBA sequence;SEQ ID NO: 37); CTCCTGGGCAACGTGCTGGTTATTGTGACCGGTGCCACC(linker sequence;SEQ ID NO: 38); ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGTCTCTGGCCCTGGT CACCAATTCT(IL-2 secretion signal sequence;SEQ ID NO: 39); GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTTGTTCAACCTGGCGGCTC TCTGAGACTGAGCTGTGCCGCTTCTGGCTACGACTTCACCCACTACGGCA TGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGATGG ATCAACACCTACACCGGCGAGCCAACATACGCCGCCGACTTCAAGCGGAG ATTCACCTTCAGCCTGGACACCAGCAAGAGCACCGCCTACCTGCAGATGA ACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAAGTATCCC TACTACTACGGCACCAGCCACTGGTACTTTGACGTGTGGGGACAGGGCAC ACTGGTCACAGTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTTTTCCCAC TGGCTCCTAGCAGCAAGTCTACCAGCGGAGGAACAGCCGCTCTGGGCTGT CTGGTCAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATTCTGG CGCTCTGACAAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAAAGCAGCG GCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCAAGCAGCTCTCTGGGC ACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAACACCAAGGT GGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCGGCAAG (sequence encoding ranibizumab heavy chain;SEQ ID NO: 52); CGGAAGAGAAGA(linker sequence;SEQ ID NO: 41); GGCTCTGGCGAAGGCAGAGGCAGCCTGCTTACATGTGGCGACGTGGAAGA GAACCCCGGACCT(T2A sequence;SEQ ID NO: 42); ATGTATAGAATGCAGCTCCTGTCCTGCATTGCCCTGAGCCTGGCTCTCGT GACCAACAGC(IL-2 signal secretion sequence;SEQ ID NO: 43); GACATCCAGCTGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGGGAGA CAGAGTGACCATCACCTGTAGCGCCAGCCAGGACATCTCCAACTACCTGA ACTGGTATCAGCAAAAGCCCGGCAAGGCCCCTAAGGTGCTGATCTACTTC ACAAGCAGCCTGCACTCCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAG CGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCG CCACCTACTACTGCCAGCAGTACAGCACCGTGCCTTGGACATTTGGCCAG GGCACAAAGGTGGAAATCAAGCGGACTGTGGCCGCTCCTAGCGTGTTCAT CTTTCCACCTAGCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGT GCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAAGTG GACAATGCCCTGCAGAGCGGCAACAGCCAAGAGAGCGTGACAGAGCAGGA CTCCAAGGATAGCACCTATAGCCTGAGCAGCACCCTGACACTGAGCAAGG CCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGC CTTTCTAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAATGTTAA (sequence encoding ranibizumab light chain;SEQ ID NO: 53). GAGCTCGCTGATCAGCCTCGA(linker sequence;SEQ ID NO: 45); CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCT TCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA GGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT GCTGGGGATGCGGTGGGCTCTATGG(bovine growth hormone polyA tail sequence;SEQ ID NO: 46); and AAGCTTGAATTCAGCTGACGTGCCTCGGACCGCT(linker;SEQ ID NO: 47); AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG CTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCG GGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG(SEQ ID NO: 48).

[0214] The IL-2 signal sequence encoded by each of SEQ ID NOs: 39 and 43 is MYRMQLLSCIALSLALVTNS (SEQ ID NO: 49). The T2A sequence encoded by SEQ ID NO: 42 is GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 50). SEQ ID NO: 52 encodes the heavy chain of ranibizumab

TABLE-US-00009 SEQ ID NO: 53 (EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVG WINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKY PYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGK;SEQ ID NO: 54).

encodes the light chain of bevacizumab (SEQ ID NO: 7). The last three nucleotides in SEQ ID NO: 53 are a stop codon.

[0215] FIG. 1C is an exemplary AAV vector of 4573 bp (SEQ ID NO: 55) that includes the following sub-sequences going in the 5' to 3' direction:

TABLE-US-00010 CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCG GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG GAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGT(5' ITR;SEQ ID NO: 36); GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTA GTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT GGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTAC ATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGT TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTAT TTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGG GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGG CGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCC TTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGC GGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCC GCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGT GAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTT AATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTC CGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGT GTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTG AGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGG GGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGA ACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTG GGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTG CTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCG GGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGG GGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCC CCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTT TTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTG TGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCG GGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTT CGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTG TCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCG GCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCAT GCCTTCTTCTTTTTCCTACAG(CBA sequence;SEQ ID NO: 37); CTCCTGGGCAACGTGCTGGTTATTGTGACCGGTGCCACC(linker sequence;SEQ ID NO: 38); ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGTCTCTGGCCCTGGT CACCAATTCT(IL-2 secretion signal sequence;SEQ ID NO: 39); GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTTGTTCAACCTGGCGGCTC TCTGAGACTGAGCTGTGCCGCTTCTGGCTACGACTTCACCCACTACGGCA TGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGATGG ATCAACACCTACACCGGCGAGCCAACATACGCCGCCGACTTCAAGCGGAG ATTCACCTTCAGCCTGGACACCAGCAAGAGCACCGCCTACCTGCAGATGA ACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAAGTATCCC TACTACTACGGCACCAGCCACTGGTACTTTGACGTGTGGGGACAGGGCAC ACTGGTCACAGTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTTTTCCCAC TGGCTCCTAGCAGCAAGTCTACCAGCGGAGGAACAGCCGCTCTGGGCTGT CTGGTCAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATTCTGG CGCTCTGACAAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAAAGCAGCG GCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCAAGCAGCTCTCTGGGC ACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAACACCAAGGT GGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCGGCAAG (sequence encoding ranibizumab heavy chain;SEQ ID NO: 52); CGGAAGAGAAGA(linker sequence; SEQ ID NO: 41); GGCTCTGGCGAAGGCAGAGGCAGCCTGCTTACATGTGGCGACGTGGAAGA GAACCCCGGACCT(T2A sequence;SEQ ID NO: 42); ATGTATAGAATGCAGCTCCTGTCCTGCATTGCCCTGAGCCTGGCTCTCGT GACCAACAGC(IL-2 signal secretion sequence;SEQ ID NO: 43); GACATCCAGCTGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGGGAGA CAGAGTGACCATCACCTGTAGCGCCAGCCAGGACATCTCCAACTACCTGA ACTGGTATCAGCAAAAGCCCGGCAAGGCCCCTAAGGTGCTGATCTACTTC ACAAGCAGCCTGCACTCCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAG CGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCG CCACCTACTACTGCCAGCAGTACAGCACCGTGCCTTGGACATTTGGCCAG GGCACAAAGGTGGAAATCAAGCGGACTGTGGCCGCTCCTAGCGTGTTCAT CTTTCCACCTAGCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGT GCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAAGTG GACAATGCCCTGCAGAGCGGCAACAGCCAAGAGAGCGTGACAGAGCAGGA CTCCAAGGATAGCACCTATAGCCTGAGCAGCACCCTGACACTGAGCAAGG CCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGC CTTTCTAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAATGT (sequence encoding ranibizumab light chain;SEQ ID NO: 56); GGCTCCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGA GAATCCTGGCCCA(linker sequence; SEQ ID NO: 57); ATGGAGAGCGACGAGAGCGGCCTGCCCGCCATGGAGATCGAGTGCCGCAT CACCGGCACCCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGAGG GCACCCCCGAGCAGGGCCGCATGACCAACAAGATGAAGAGCACCAAAGGC GCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGCTT CTACCACTTCGGCACCTACCCCAGCGGCTACGAGAACCCCTTCCTGCACG CCATCAACAACGGCGGCTACACCAACACCCGCATCGAGAAGTACGAGGAC GGCGGCGTGCTGCACGTGAGCTTCAGCTACCGCTACGAGGCCGGCCGCGT GATCGGCGACTTCAAGGTGATGGGCACCGGCTTCCCCGAGGACAGCGTGA TCTTCACCGACAAGATCATCCGCAGCAACGCCACCGTGGAGCACCTGCAC CCCATGGGCGATAACGATCTGGATGGCAGCTTCACCCGCACCTTCAGCCT GCGCGACGGCGGCTACTACAGCTCCGTGGTGGACAGCCACATGCACTTCA AGAGCGCCATCCACCCCAGCATCCTGCAGAACGGGGGCCCCATGTTCGCC TTCCGCCGCGTGGAGGAGGATCACAGCAACACCGAGCTGGGCATCGTGGA GTACCAGCACGCCTTCAAGACCCCGGATGCAGATGCCGGTGAAGAATAA (sequence encoding Turbo GFP;SEQ ID NO: 58); GAGCTCGCTGATCAGCCTCGA(linker sequence;SEQ ID NO: 45); CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCT TCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA GGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT GCTGGGGATGCGGTGGGCTCTATGG(bovine growth hormone polyA tail sequence;SEQ ID NO: 46); AAGCTTGAATTCAGCTGACGTGCCTCGGACCGCT(linker sequence; SEQ ID NO: 47); and AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG CTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCG GGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG(3' ITR; SEQ ID NO: 48).

[0216] The IL-2 signal sequence encoded by each of SEQ ID NOs: 39 and 43 is MYRMQLLSCIALSLALVTNS (SEQ ID NO: 49). The T2A sequence encoded by SEQ ID NO: 42 is GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 50). SEQ ID NO: 52 encodes the heavy chain of ranibizumab (SEQ ID NO: 54). SEQ ID NO: 56 encodes the light chain of bevacizumab (SEQ ID NO: 7). SEQ ID NO: 58 encodes TurboGFP (MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGA LTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGV LHVSFSYRYEAGRVIGDFKVMGTGFPEDSVIFTDKIIRSNATVEHLHPMGDND LDGSFTRTFSLRDGGYYSSVVDSHMHFKSAIHPSILQNGGPMFAFRRVEEDHS NTELGIVEYQHAFKTPDADAGEE; SEQ ID NO: 59). The last three nucleotides in SEQ ID NO: 58 is a stop codon.

[0217] FIG. 1D is an exemplary AAV vector of 3631 bp (SEQ ID NO: 60) that includes the following sub-sequences going in the 5' to 3' direction:

TABLE-US-00011 CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCG GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG GAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGT(5' ITR;SEQ ID NO: 36); GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTA GTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT GGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTAC ATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGT TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTAT TTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGG GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGG CGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCC TTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGC GGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCC GCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGT GAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTT AATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTC CGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGT GTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTG AGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGG GGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGA ACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTG GGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTG CTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCG GGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGG GGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCC CCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTT TTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTG TGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCG GGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTT CGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTG TCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCG GCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCAT GCCTTCTTCTTTTTCCTACAG(CBA sequence;SEQ ID NO: 37); CTCCTGGGCAACGTGCTGGTTATTGTGACCGGTGCCACC(spacer; SEQ ID NO:38); ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGTCTCTGGCCCTGGT CACCAATTCT(IL-2 secretion signal sequence; SEQ ID NO: 39); AGCGATACCGGCAGACCCTTCGTGGAAATGTACAGCGAGATCCCCGAGAT CATCCACATGACCGAGGGCAGAGAGCTGGTCATCCCCTGCAGAGTGACAA GCCCCAACATCACCGTGACTCTGAAGAAGTTCCCTCTGGACACACTGATC CCCGACGGCAAGAGAATCATCTGGGACAGCCGGAAGGGCTTCATCATCAG CAACGCCACCTACAAAGAGATCGGCCTGCTGACCTGTGAAGCCACCGTGA ATGGCCACCTGTACAAGACCAACTACCTGACACACAGACAGACCAACACC ATCATCGACGTGGTGCTGAGCCCTAGCCACGGCATTGAACTGTCTGTGGG CGAGAAGCTGGTGCTGAACTGTACCGCCAGAACCGAGCTGAACGTGGGCA TCGACTTCAACTGGGAGTACCCCAGCAGCAAGCACCAGCACAAGAAACTG GTCAACCGGGACCTGAAAACCCAGAGCGGCAGCGAGATGAAGAAATTCCT GAGCACCCTGACCATCGACGGCGTGACCAGATCTGACCAGGGCCTGTACA CATGTGCCGCCAGCTCTGGCCTGATGACCAAGAAAAACAGCACCTTCGTG CGGGTGCACGAGAAGGACAAGACCCACACCTGTCCTCCATGTCCTGCTCC AGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGG ACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGAT GTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGT GGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAATAGCA CCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAAC GGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTAT CGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTT ACACACTGCCTCCAAGCAGGGACGAGCTGACAAAGAACCAGGTGTCCCTG ACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGA GAGCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGG ACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGC AGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCT GCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGATAA (sequence encoding a flibercept;SEQ ID NO: 61); GAGCTCGCTGATCAGCCTCGA(linker sequence;SEQ ID NO: 45); CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCT TCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA GGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT GCTGGGGATGCGGTGGGCTCTATGG(bovine growth hormone polyA tail sequence; SEQ ID NO: 46); AAGCTTGAATTCAGCTGACGTGCCTCGGACCGCT(linker sequence; SEQ ID NO: 47); and AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG CTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCG GGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG(3' ITR; SEQ ID NO: 48).

[0218] The IL-2 signal sequence encoded by SEQ ID NO: 39 is MYRMQLLSCIALSLALVTNS (SEQ ID NO: 49). SEQ ID NO: 61 encodes aflibercept (SEQ ID NO: 12). The last three nucleotides in SEQ ID NO: 61 is a stop codon.

[0219] To determine protein expression driven by the AAV vectors shown in FIGS. 1A-1C, HEK293FT cells were seeded overnight at 7.times.10.sup.4 cells/well (400 .mu.L per well) in wells of a 24-well plate. HEK293FT cells were transfected at .about.800 ng with the AAV vectors shown in FIGS. 1A-1D using a Jetprime Polypus reagent (used to generate the data in Lanes 2-5 and 10-13 of FIG. 2). HEK293FT cells were also seeded for six hours at 4.times.10.sup.4 cells/well (50 .mu.L per well) in wells of a 96-well plate in the presence of 2 .mu.M etoposide (used to generate the data in Lanes 6-8 and 14-16 of FIG. 2). The AAV vector shown in FIG. 1A was added into the media with a multiplicity of infection (MOI) of 7.5.times.10.sup.4, 2.2.times.10.sup.5, or 5.5.times.10.sup.5. The supernatant was harvested at 72 hours post-treatment from well and was loaded onto a 4-12% Bolt protein gel in reducing (lanes 2-8 of FIG. 2) and non-reducing conditions (lanes 10-16 of FIG. 2). An anti-ranibizumab antibody detecting the Fab region was used as a primary antibody, and anti-human IgG was used as the second antibody.

[0220] As shown in FIG. 2, the heavy chain and light chain ranibizumab were detected in Lanes 3 and 6-8, and intact ranibizumab (heterodimer) was detected in lanes 11 and 14-16.

Example 2

Binding Activity of Anti-Human VEGF Monoclonal Antibodies

[0221] A set of experiments were performed to determine the binding activity of bevacizumab produced in HEK293FT cells following transfection with the AAV vector shown in FIG. 1A. A first set of control experiments were performed to calibrate the plasmon surface resonance instrumentation (using a mouse anti-human VEGF monoclonal antibody (anti-hVEGF MmAb; R&D, MAB293-100) in buffer or in conditioned medium (FIGS. 3A and 3B, respectivey) using recombinant human VEGF as the binding agent. A second set of experiments were performed to determine the human VEGF-binding activity of control conditioned medium and conditioned medium from HEK293TF cells following transfection with the AAV vector shown in FIG. 1A (FIGS. 4A and 4B, respectively).

[0222] The samples, bevacizumab in medium from HEK293TF cells transfected with the AAV vector shown in FIG. 1A or conditioned medium), were prepared by diluting 1:10 in lx kinetics buffer (Fortebio, 18-1105) into a 384-well sample plate. Anti-hVEGF MmAb (R&D, MAB293-100) was diluted at a concentration of 10 .mu.g/mL as a positive control. The capture agent, recombinant human VEGF (R&D, 293-VE-010) was diluted in a series of 1:2 dilution ratio from 200 nM to 3.125 nM.

[0223] The binding affinities of the conditioned medium samples and mouse anti-human VEGF antibody (R&D) samples were measured in 1.times. kinetics buffer in Octet.RTM. HTX biosensor instrument. The binding features and K.sub.D values were generated by the Octet.RTM. analysis software, Data Analysis HT10.0. As shown in FIGS. 3A-B, the K.sub.D of anti-hVEGF MmAb in buffer was <1.0.times.10.sup.-12 M, and the anti-hVEGF MmAb in conditioned medium was <1.0.times.10.sup.-12 M. The conditioned medium itself had no binding affinity and very low intensity (background signal only) (FIG. 4A). In contrast, the conditioned medium including bevacizumab produced by HEK293TF cells transfected with the AAV vector shown in FIG. 1A had high binding affinity, but low intensity (FIG. 4B; K.sub.D<1.0.times.10.sup.-12 M). FIG. 4C shows a table of the loading samples and the respective K.sub.D, K.sub.D errors, equilibrium association constant (k.sub.a), and the dissociation (k.sub.dis), and k.sub.dis error.

[0224] In summary, the anti-hVEGF mouse antibody (R&D) showed high binding affinity (K.sub.D was lower than measurable range of 1.0.times.10.sup.-12 M). The bevacizumab conditioned medium sample showed high binding affinity (K.sub.D was lower than measurable range). No K.sub.D value could be extrapolated from the binding data of control conditioned medium sample.

[0225] In sum, these data show that the AAV vectors provided herein can result in expression and secretion of anti-VEGF antibodies and can be used to express anti-VEGF antibodies in the inner ear of a mammal.

Other Embodiments

[0226] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0227] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Section headings and any descriptions of materials, methods, and examples are illustrative only and not intended to be limiting.

Sequence CWU 1

1

611412PRTHuman 1Met Thr Asp Arg Gln Thr Asp Thr Ala Pro Ser Pro Ser Tyr His Leu1 5 10 15Leu Pro Gly Arg Arg Arg Thr Val Asp Ala Ala Ala Ser Arg Gly Gln 20 25 30Gly Pro Glu Pro Ala Pro Gly Gly Gly Val Glu Gly Val Gly Ala Arg 35 40 45Gly Val Ala Leu Lys Leu Phe Val Gln Leu Leu Gly Cys Ser Arg Phe 50 55 60Gly Gly Ala Val Val Arg Ala Gly Glu Ala Glu Pro Ser Gly Ala Ala65 70 75 80Arg Ser Ala Ser Ser Gly Arg Glu Glu Pro Gln Pro Glu Glu Gly Glu 85 90 95Glu Glu Glu Glu Lys Glu Glu Glu Arg Gly Pro Gln Trp Arg Leu Gly 100 105 110Ala Arg Lys Pro Gly Ser Trp Thr Gly Glu Ala Ala Val Cys Ala Asp 115 120 125Ser Ala Pro Ala Ala Arg Ala Pro Gln Ala Leu Ala Arg Ala Ser Gly 130 135 140Arg Gly Gly Arg Val Ala Arg Arg Gly Ala Glu Glu Ser Gly Pro Pro145 150 155 160His Ser Pro Ser Arg Arg Gly Ser Ala Ser Arg Ala Gly Pro Gly Arg 165 170 175Ala Ser Glu Thr Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 180 185 190Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro 195 200 205Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met 210 215 220Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp225 230 235 240Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser 245 250 255Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu 260 265 270Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg 275 280 285Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln 290 295 300His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu305 310 315 320Lys Lys Ser Val Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys 325 330 335Lys Ser Arg Tyr Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys 340 345 350Leu Met Pro Trp Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser 355 360 365Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys 370 375 380Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu385 390 395 400Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg 405 41021239DNAHuman 2ctgacggaca gacagacaga caccgccccc agccccagct accacctcct ccccggccgg 60cggcggacag tggacgcggc ggcgagccgc gggcaggggc cggagcccgc gcccggaggc 120ggggtggagg gggtcggggc tcgcggcgtc gcactgaaac ttttcgtcca acttctgggc 180tgttctcgct tcggaggagc cgtggtccgc gcgggggaag ccgagccgag cggagccgcg 240agaagtgcta gctcgggccg ggaggagccg cagccggagg agggggagga ggaagaagag 300aaggaagagg agagggggcc gcagtggcga ctcggcgctc ggaagccggg ctcatggacg 360ggtgaggcgg cggtgtgcgc agacagtgct ccagccgcgc gcgctcccca ggccctggcc 420cgggcctcgg gccggggagg aagagtagct cgccgaggcg ccgaggagag cgggccgccc 480cacagcccga gccggagagg gagcgcgagc cgcgccggcc ccggtcgggc ctccgaaacc 540atgaactttc tgctgtcttg ggtgcattgg agccttgcct tgctgctcta cctccaccat 600gccaagtggt cccaggctgc acccatggca gaaggaggag ggcagaatca tcacgaagtg 660gtgaagttca tggatgtcta tcagcgcagc tactgccatc caatcgagac cctggtggac 720atcttccagg agtaccctga tgagatcgag tacatcttca agccatcctg tgtgcccctg 780atgcgatgcg ggggctgctg caatgacgag ggcctggagt gtgtgcccac tgaggagtcc 840aacatcacca tgcagattat gcggatcaaa cctcaccaag gccagcacat aggagagatg 900agcttcctac agcacaacaa atgtgaatgc agaccaaaga aagatagagc aagacaagaa 960aaaaaatcag ttcgaggaaa gggaaagggg caaaaacgaa agcgcaagaa atcccggtat 1020aagtcctgga gcgtgtacgt tggtgcccgc tgctgtctaa tgccctggag cctccctggc 1080ccccatccct gtgggccttg ctcagagcgg agaaagcatt tgtttgtaca agatccgcag 1140acgtgtaaat gttcctgcaa aaacacagac tcgcgttgca aggcgaggca gcttgagtta 1200aacgaacgta cttgcagatg tgacaagccg aggcggtga 12393389PRTHuman 3Met Thr Asp Arg Gln Thr Asp Thr Ala Pro Ser Pro Ser Tyr His Leu1 5 10 15Leu Pro Gly Arg Arg Arg Thr Val Asp Ala Ala Ala Ser Arg Gly Gln 20 25 30Gly Pro Glu Pro Ala Pro Gly Gly Gly Val Glu Gly Val Gly Ala Arg 35 40 45Gly Val Ala Leu Lys Leu Phe Val Gln Leu Leu Gly Cys Ser Arg Phe 50 55 60Gly Gly Ala Val Val Arg Ala Gly Glu Ala Glu Pro Ser Gly Ala Ala65 70 75 80Arg Ser Ala Ser Ser Gly Arg Glu Glu Pro Gln Pro Glu Glu Gly Glu 85 90 95Glu Glu Glu Glu Lys Glu Glu Glu Arg Gly Pro Gln Trp Arg Leu Gly 100 105 110Ala Arg Lys Pro Gly Ser Trp Thr Gly Glu Ala Ala Val Cys Ala Asp 115 120 125Ser Ala Pro Ala Ala Arg Ala Pro Gln Ala Leu Ala Arg Ala Ser Gly 130 135 140Arg Gly Gly Arg Val Ala Arg Arg Gly Ala Glu Glu Ser Gly Pro Pro145 150 155 160His Ser Pro Ser Arg Arg Gly Ser Ala Ser Arg Ala Gly Pro Gly Arg 165 170 175Ala Ser Glu Thr Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 180 185 190Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro 195 200 205Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met 210 215 220Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp225 230 235 240Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser 245 250 255Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu 260 265 270Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg 275 280 285Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln 290 295 300His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu305 310 315 320Lys Lys Ser Val Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys 325 330 335Lys Ser Arg Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe 340 345 350Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser 355 360 365Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys 370 375 380Asp Lys Pro Arg Arg38541170DNAHuman 4ctgacggaca gacagacaga caccgccccc agccccagct accacctcct ccccggccgg 60cggcggacag tggacgcggc ggcgagccgc gggcaggggc cggagcccgc gcccggaggc 120ggggtggagg gggtcggggc tcgcggcgtc gcactgaaac ttttcgtcca acttctgggc 180tgttctcgct tcggaggagc cgtggtccgc gcgggggaag ccgagccgag cggagccgcg 240agaagtgcta gctcgggccg ggaggagccg cagccggagg agggggagga ggaagaagag 300aaggaagagg agagggggcc gcagtggcga ctcggcgctc ggaagccggg ctcatggacg 360ggtgaggcgg cggtgtgcgc agacagtgct ccagccgcgc gcgctcccca ggccctggcc 420cgggcctcgg gccggggagg aagagtagct cgccgaggcg ccgaggagag cgggccgccc 480cacagcccga gccggagagg gagcgcgagc cgcgccggcc ccggtcgggc ctccgaaacc 540atgaactttc tgctgtcttg ggtgcattgg agccttgcct tgctgctcta cctccaccat 600gccaagtggt cccaggctgc acccatggca gaaggaggag ggcagaatca tcacgaagtg 660gtgaagttca tggatgtcta tcagcgcagc tactgccatc caatcgagac cctggtggac 720atcttccagg agtaccctga tgagatcgag tacatcttca agccatcctg tgtgcccctg 780atgcgatgcg ggggctgctg caatgacgag ggcctggagt gtgtgcccac tgaggagtcc 840aacatcacca tgcagattat gcggatcaaa cctcaccaag gccagcacat aggagagatg 900agcttcctac agcacaacaa atgtgaatgc agaccaaaga aagatagagc aagacaagaa 960aaaaaatcag ttcgaggaaa gggaaagggg caaaaacgaa agcgcaagaa atcccgtccc 1020tgtgggcctt gctcagagcg gagaaagcat ttgtttgtac aagatccgca gacgtgtaaa 1080tgttcctgca aaaacacaga ctcgcgttgc aaggcgaggc agcttgagtt aaacgaacgt 1140acttgcagat gtgacaagcc gaggcggtga 11705214PRTArtificialBevacizumab light chain variable domain 5Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 2106453PRTArtificialBevacizumab heavy chain variable domain 6Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu225 230 235 240Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu305 310 315 320Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr385 390 395 400Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445Leu Ser Pro Gly Lys 4507214PRTArtificialRanibizumab light chain variable domain 7Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 2108231PRTArtificialRanibizumab heavy chain variable domain 8Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp Lys Thr His Leu225 230922PRTArtificialSignal peptide 9Met Glu Phe Phe Lys Lys Thr Ala Leu Ala Ala Leu Val Met Gly Phe1 5 10 15Ser Gly Ala Ala Leu Ala 201022PRTArtificialSignal peptide 10Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala 201117DNAHuman 11aaataaaata cgaaatg 1712431PRTArtificialAflibercept 12Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu1 5 10 15Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys

Gly Phe 50 55 60Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu65 70 75 80Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile 100 105 110Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120 125Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135 140His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly145 150 155 160Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr 165 170 175Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180 185 190Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr 195 200 205His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 210 215 220Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg225 230 235 240Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 245 250 255Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 260 265 270Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 275 280 285Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 290 295 300Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr305 310 315 320Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 325 330 335Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 340 345 350Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 355 360 365Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 370 375 380Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser385 390 395 400Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 405 410 415Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 420 425 43013206PRTHuman 13Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys1 5 10 15Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu 20 25 30Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35 40 45Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu 50 55 60Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile65 70 75 80Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg 100 105 110Gln Glu Lys Lys Ser Val Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys 115 120 125Arg Lys Lys Ser Arg Tyr Lys Ser Trp Ser Val Tyr Val Gly Ala Arg 130 135 140Cys Cys Leu Met Pro Trp Ser Leu Pro Gly Pro His Pro Cys Gly Pro145 150 155 160Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr Cys 165 170 175Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu 180 185 190Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg 195 200 20514186PRTHuman 14Pro Val Ser Gln Pro Asp Ala Pro Gly His Gln Arg Lys Val Val Ser1 5 10 15Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln Pro Arg Glu Val Val 20 25 30Val Pro Leu Thr Val Glu Leu Met Gly Thr Val Ala Lys Gln Leu Val 35 40 45Pro Ser Cys Val Thr Val Gln Arg Cys Gly Gly Cys Cys Pro Asp Asp 50 55 60Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln Val Arg Met Gln Ile65 70 75 80Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly Glu Met Ser Leu Glu 85 90 95Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys Lys Asp Ser Ala Val 100 105 110Lys Pro Asp Arg Ala Ala Thr Pro His His Arg Pro Gln Pro Arg Ser 115 120 125Val Pro Gly Trp Asp Ser Ala Pro Gly Ala Pro Ser Pro Ala Asp Ile 130 135 140Thr His Pro Thr Pro Ala Pro Gly Pro Ser Ala His Ala Ala Pro Ser145 150 155 160Thr Thr Ser Ala Leu Thr Pro Gly Pro Ala Ala Ala Ala Ala Asp Ala 165 170 175Ala Ala Ser Ser Val Ala Lys Gly Gly Ala 180 18515116PRTHuman 15Ala His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg1 5 10 15Lys Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu 20 25 30Phe Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val 35 40 45Tyr Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn 50 55 60Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro65 70 75 80Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr 85 90 95Ser Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser 100 105 110Ile Ile Arg Arg 11516117PRTHuman 16Phe Ala Ala Thr Phe Tyr Asp Ile Glu Thr Leu Lys Val Ile Asp Glu1 5 10 15Glu Trp Gln Arg Thr Gln Cys Ser Pro Arg Glu Thr Cys Val Glu Val 20 25 30Ala Ser Glu Leu Gly Lys Ser Thr Asn Thr Phe Phe Lys Pro Pro Cys 35 40 45Val Asn Val Phe Arg Cys Gly Gly Cys Cys Asn Glu Glu Ser Leu Ile 50 55 60Cys Met Asn Thr Ser Thr Ser Tyr Ile Ser Lys Gln Leu Phe Glu Ile65 70 75 80Ser Val Pro Leu Thr Ser Val Pro Glu Leu Val Pro Val Lys Val Ala 85 90 95Asn His Thr Gly Cys Lys Cys Leu Pro Thr Ala Pro Arg His Pro Tyr 100 105 110Ser Ile Ile Arg Arg 11517687PRTHuman 17Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser1 5 10 15Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr 35 40 45Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala65 70 75 80Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85 90 95Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val 100 105 110Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115 120 125Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135 140Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val145 150 155 160Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185 190Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200 205Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 210 215 220Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val225 230 235 240Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245 250 255Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys 260 265 270Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His 275 280 285Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys305 310 315 320Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325 330 335Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser 340 345 350Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355 360 365Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370 375 380Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala385 390 395 400Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420 425 430Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440 445Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455 460Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys465 470 475 480Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser 485 490 495Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile 500 505 510Glu Gly Lys Asn Lys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg 515 520 525Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly Thr Val 530 535 540Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His545 550 555 560Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565 570 575Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile Leu Leu 580 585 590Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln Lys 595 600 605Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met 610 615 620Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn625 630 635 640Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg 645 650 655Gly Glu His Cys Asn Lys Lys Ala Val Phe Ser Arg Ile Ser Lys Phe 660 665 670Lys Ser Thr Arg Asn Asp Cys Thr Thr Gln Ser Asn Val Lys His 675 680 685182064DNAHuman 18atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc 60acaggatcta gttcaggttc aaaattaaaa gatcctgaac tgagtttaaa aggcacccag 120cacatcatgc aagcaggcca gacactgcat ctccaatgca ggggggaagc agcccataaa 180tggtctttgc ctgaaatggt gagtaaggaa agcgaaaggc tgagcataac taaatctgcc 240tgtggaagaa atggcaaaca attctgcagt actttaacct tgaacacagc tcaagcaaac 300cacactggct tctacagctg caaatatcta gctgtaccta cttcaaagaa gaaggaaaca 360gaatctgcaa tctatatatt tattagtgat acaggtagac ctttcgtaga gatgtacagt 420gaaatccccg aaattataca catgactgaa ggaagggagc tcgtcattcc ctgccgggtt 480acgtcaccta acatcactgt tactttaaaa aagtttccac ttgacacttt gatccctgat 540ggaaaacgca taatctggga cagtagaaag ggcttcatca tatcaaatgc aacgtacaaa 600gaaatagggc ttctgacctg tgaagcaaca gtcaatgggc atttgtataa gacaaactat 660ctcacacatc gacaaaccaa tacaatcata gatgtccaaa taagcacacc acgcccagtc 720aaattactta gaggccatac tcttgtcctc aattgtactg ctaccactcc cttgaacacg 780agagttcaaa tgacctggag ttaccctgat gaaaaaaata agagagcttc cgtaaggcga 840cgaattgacc aaagcaattc ccatgccaac atattctaca gtgttcttac tattgacaaa 900atgcagaaca aagacaaagg actttatact tgtcgtgtaa ggagtggacc atcattcaaa 960tctgttaaca cctcagtgca tatatatgat aaagcattca tcactgtgaa acatcgaaaa 1020cagcaggtgc ttgaaaccgt agctggcaag cggtcttacc ggctctctat gaaagtgaag 1080gcatttccct cgccggaagt tgtatggtta aaagatgggt tacctgcgac tgagaaatct 1140gctcgctatt tgactcgtgg ctactcgtta attatcaagg acgtaactga agaggatgca 1200gggaattata caatcttgct gagcataaaa cagtcaaatg tgtttaaaaa cctcactgcc 1260actctaattg tcaatgtgaa accccagatt tacgaaaagg ccgtgtcatc gtttccagac 1320ccggctctct acccactggg cagcagacaa atcctgactt gtaccgcata tggtatccct 1380caacctacaa tcaagtggtt ctggcacccc tgtaaccata atcattccga agcaaggtgt 1440gacttttgtt ccaataatga agagtccttt atcctggatg ctgacagcaa catgggaaac 1500agaattgaga gcatcactca gcgcatggca ataatagaag gaaagaataa gatggctagc 1560accttggttg tggctgactc tagaatttct ggaatctaca tttgcatagc ttccaataaa 1620gttgggactg tgggaagaaa cataagcttt tatatcacag atgtgccaaa tgggtttcat 1680gttaacttgg aaaaaatgcc gacggaagga gaggacctga aactgtcttg cacagttaac 1740aagttcttat acagagacgt tacttggatt ttactgcgga cagttaataa cagaacaatg 1800cactacagta ttagcaagca aaaaatggcc atcactaagg agcactccat cactcttaat 1860cttaccatca tgaatgtttc cctgcaagat tcaggcacct atgcctgcag agccaggaat 1920gtatacacag gggaagaaat cctccagaag aaagaaatta caatcagagg tgagcactgc 1980aacaaaaagg ctgttttctc tcggatctcc aaatttaaaa gcacaaggaa tgattgtacc 2040acacaaagta atgtaaaaca ttaa 206419733PRTHuman 19Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser1 5 10 15Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr 35 40 45Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala65 70 75 80Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85 90 95Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val 100 105 110Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115 120 125Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135 140Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val145 150 155 160Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185 190Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200 205Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 210 215 220Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val225 230 235 240Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245 250 255Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys 260 265 270Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His 275 280 285Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys305 310 315 320Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325 330 335Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser 340 345 350Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355 360 365Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370 375 380Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala385 390 395 400Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420

425 430Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440 445Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455 460Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys465 470 475 480Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser 485 490 495Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile 500 505 510Glu Gly Lys Asn Lys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg 515 520 525Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly Thr Val 530 535 540Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His545 550 555 560Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565 570 575Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile Leu Leu 580 585 590Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln Lys 595 600 605Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met 610 615 620Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn625 630 635 640Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg 645 650 655Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp His Thr Val 660 665 670Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val Pro 675 680 685Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn His Lys Ile Gln Gln Glu 690 695 700Pro Glu Leu Tyr Thr Ser Thr Ser Pro Ser Ser Ser Ser Ser Ser Pro705 710 715 720Leu Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 725 730202202DNAHuman 20atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc 60acaggatcta gttcaggttc aaaattaaaa gatcctgaac tgagtttaaa aggcacccag 120cacatcatgc aagcaggcca gacactgcat ctccaatgca ggggggaagc agcccataaa 180tggtctttgc ctgaaatggt gagtaaggaa agcgaaaggc tgagcataac taaatctgcc 240tgtggaagaa atggcaaaca attctgcagt actttaacct tgaacacagc tcaagcaaac 300cacactggct tctacagctg caaatatcta gctgtaccta cttcaaagaa gaaggaaaca 360gaatctgcaa tctatatatt tattagtgat acaggtagac ctttcgtaga gatgtacagt 420gaaatccccg aaattataca catgactgaa ggaagggagc tcgtcattcc ctgccgggtt 480acgtcaccta acatcactgt tactttaaaa aagtttccac ttgacacttt gatccctgat 540ggaaaacgca taatctggga cagtagaaag ggcttcatca tatcaaatgc aacgtacaaa 600gaaatagggc ttctgacctg tgaagcaaca gtcaatgggc atttgtataa gacaaactat 660ctcacacatc gacaaaccaa tacaatcata gatgtccaaa taagcacacc acgcccagtc 720aaattactta gaggccatac tcttgtcctc aattgtactg ctaccactcc cttgaacacg 780agagttcaaa tgacctggag ttaccctgat gaaaaaaata agagagcttc cgtaaggcga 840cgaattgacc aaagcaattc ccatgccaac atattctaca gtgttcttac tattgacaaa 900atgcagaaca aagacaaagg actttatact tgtcgtgtaa ggagtggacc atcattcaaa 960tctgttaaca cctcagtgca tatatatgat aaagcattca tcactgtgaa acatcgaaaa 1020cagcaggtgc ttgaaaccgt agctggcaag cggtcttacc ggctctctat gaaagtgaag 1080gcatttccct cgccggaagt tgtatggtta aaagatgggt tacctgcgac tgagaaatct 1140gctcgctatt tgactcgtgg ctactcgtta attatcaagg acgtaactga agaggatgca 1200gggaattata caatcttgct gagcataaaa cagtcaaatg tgtttaaaaa cctcactgcc 1260actctaattg tcaatgtgaa accccagatt tacgaaaagg ccgtgtcatc gtttccagac 1320ccggctctct acccactggg cagcagacaa atcctgactt gtaccgcata tggtatccct 1380caacctacaa tcaagtggtt ctggcacccc tgtaaccata atcattccga agcaaggtgt 1440gacttttgtt ccaataatga agagtccttt atcctggatg ctgacagcaa catgggaaac 1500agaattgaga gcatcactca gcgcatggca ataatagaag gaaagaataa gatggctagc 1560accttggttg tggctgactc tagaatttct ggaatctaca tttgcatagc ttccaataaa 1620gttgggactg tgggaagaaa cataagcttt tatatcacag atgtgccaaa tgggtttcat 1680gttaacttgg aaaaaatgcc gacggaagga gaggacctga aactgtcttg cacagttaac 1740aagttcttat acagagacgt tacttggatt ttactgcgga cagttaataa cagaacaatg 1800cactacagta ttagcaagca aaaaatggcc atcactaagg agcactccat cactcttaat 1860cttaccatca tgaatgtttc cctgcaagat tcaggcacct atgcctgcag agccaggaat 1920gtatacacag gggaagaaat cctccagaag aaagaaatta caatcagaga tcaggaagca 1980ccatacctcc tgcgaaacct cagtgatcac acagtggcca tcagcagttc caccacttta 2040gactgtcatg ctaatggtgt ccccgagcct cagatcactt ggtttaaaaa caaccacaaa 2100atacaacaag agcctgaact gtatacatca acgtcaccat cgtcatcgtc atcatcacca 2160ttgtcatcat catcatcatc gtcatcatca tcatcatcat ag 220221541PRTHuman 21Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser1 5 10 15Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr 35 40 45Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala65 70 75 80Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85 90 95Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val 100 105 110Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115 120 125Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135 140Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val145 150 155 160Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185 190Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200 205Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 210 215 220Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val225 230 235 240Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245 250 255Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys 260 265 270Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His 275 280 285Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys305 310 315 320Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325 330 335Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser 340 345 350Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355 360 365Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370 375 380Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala385 390 395 400Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420 425 430Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440 445Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455 460Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys465 470 475 480Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser 485 490 495Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile 500 505 510Glu Gly Lys Asn Lys Leu Pro Pro Ala Asn Ser Ser Phe Met Leu Pro 515 520 525Pro Thr Ser Phe Ser Ser Asn Tyr Phe His Phe Leu Pro 530 535 540221626DNAHuman 22atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc 60acaggatcta gttcaggttc aaaattaaaa gatcctgaac tgagtttaaa aggcacccag 120cacatcatgc aagcaggcca gacactgcat ctccaatgca ggggggaagc agcccataaa 180tggtctttgc ctgaaatggt gagtaaggaa agcgaaaggc tgagcataac taaatctgcc 240tgtggaagaa atggcaaaca attctgcagt actttaacct tgaacacagc tcaagcaaac 300cacactggct tctacagctg caaatatcta gctgtaccta cttcaaagaa gaaggaaaca 360gaatctgcaa tctatatatt tattagtgat acaggtagac ctttcgtaga gatgtacagt 420gaaatccccg aaattataca catgactgaa ggaagggagc tcgtcattcc ctgccgggtt 480acgtcaccta acatcactgt tactttaaaa aagtttccac ttgacacttt gatccctgat 540ggaaaacgca taatctggga cagtagaaag ggcttcatca tatcaaatgc aacgtacaaa 600gaaatagggc ttctgacctg tgaagcaaca gtcaatgggc atttgtataa gacaaactat 660ctcacacatc gacaaaccaa tacaatcata gatgtccaaa taagcacacc acgcccagtc 720aaattactta gaggccatac tcttgtcctc aattgtactg ctaccactcc cttgaacacg 780agagttcaaa tgacctggag ttaccctgat gaaaaaaata agagagcttc cgtaaggcga 840cgaattgacc aaagcaattc ccatgccaac atattctaca gtgttcttac tattgacaaa 900atgcagaaca aagacaaagg actttatact tgtcgtgtaa ggagtggacc atcattcaaa 960tctgttaaca cctcagtgca tatatatgat aaagcattca tcactgtgaa acatcgaaaa 1020cagcaggtgc ttgaaaccgt agctggcaag cggtcttacc ggctctctat gaaagtgaag 1080gcatttccct cgccggaagt tgtatggtta aaagatgggt tacctgcgac tgagaaatct 1140gctcgctatt tgactcgtgg ctactcgtta attatcaagg acgtaactga agaggatgca 1200gggaattata caatcttgct gagcataaaa cagtcaaatg tgtttaaaaa cctcactgcc 1260actctaattg tcaatgtgaa accccagatt tacgaaaagg ccgtgtcatc gtttccagac 1320ccggctctct acccactggg cagcagacaa atcctgactt gtaccgcata tggtatccct 1380caacctacaa tcaagtggtt ctggcacccc tgtaaccata atcattccga agcaaggtgt 1440gacttttgtt ccaataatga agagtccttt atcctggatg ctgacagcaa catgggaaac 1500agaattgaga gcatcactca gcgcatggca ataatagaag gaaagaataa gcttccacca 1560gctaacagtt ctttcatgtt gccacctaca agcttctctt ccaactactt ccatttcctt 1620ccgtga 162623732PRTHuman 23Ser Lys Leu Lys Asp Pro Glu Leu Ser Leu Lys Gly Thr Gln His Ile1 5 10 15Met Gln Ala Gly Gln Thr Leu His Leu Gln Cys Arg Gly Glu Ala Ala 20 25 30His Lys Trp Ser Leu Pro Glu Met Val Ser Lys Glu Ser Glu Arg Leu 35 40 45Ser Ile Thr Lys Ser Ala Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser 50 55 60Thr Leu Thr Leu Asn Thr Ala Gln Ala Asn His Thr Gly Phe Tyr Ser65 70 75 80Cys Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser 85 90 95Ala Ile Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met 100 105 110Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu 115 120 125Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys 130 135 140Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp145 150 155 160Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile 165 170 175Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr 180 185 190Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Gln Ile 195 200 205Ser Thr Pro Arg Pro Val Lys Leu Leu Arg Gly His Thr Leu Val Leu 210 215 220Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr Arg Val Gln Met Thr Trp225 230 235 240Ser Tyr Pro Asp Glu Lys Asn Lys Arg Ala Ser Val Arg Arg Arg Ile 245 250 255Asp Gln Ser Asn Ser His Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile 260 265 270Asp Lys Met Gln Asn Lys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg 275 280 285Ser Gly Pro Ser Phe Lys Ser Val Asn Thr Ser Val His Ile Tyr Asp 290 295 300Lys Ala Phe Ile Thr Val Lys His Arg Lys Gln Gln Val Leu Glu Thr305 310 315 320Val Ala Gly Lys Arg Ser Tyr Arg Leu Ser Met Lys Val Lys Ala Phe 325 330 335Pro Ser Pro Glu Val Val Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu 340 345 350Lys Ser Ala Arg Tyr Leu Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp 355 360 365Val Thr Glu Glu Asp Ala Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys 370 375 380Gln Ser Asn Val Phe Lys Asn Leu Thr Ala Thr Leu Ile Val Asn Val385 390 395 400Lys Pro Gln Ile Tyr Glu Lys Ala Val Ser Ser Phe Pro Asp Pro Ala 405 410 415Leu Tyr Pro Leu Gly Ser Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly 420 425 430Ile Pro Gln Pro Thr Ile Lys Trp Phe Trp His Pro Cys Asn His Asn 435 440 445His Ser Glu Ala Arg Cys Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe 450 455 460Ile Leu Asp Ala Asp Ser Asn Met Gly Asn Arg Ile Glu Ser Ile Thr465 470 475 480Gln Arg Met Ala Ile Ile Glu Gly Lys Asn Lys Met Ala Ser Thr Leu 485 490 495Val Val Ala Asp Ser Arg Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser 500 505 510Asn Lys Val Gly Thr Val Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp 515 520 525Val Pro Asn Gly Phe His Val Asn Leu Glu Lys Met Pro Thr Glu Gly 530 535 540Glu Asp Leu Lys Leu Ser Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp545 550 555 560Val Thr Trp Ile Leu Leu Arg Thr Val Asn Asn Arg Thr Met His Tyr 565 570 575Ser Ile Ser Lys Gln Lys Met Ala Ile Thr Lys Glu His Ser Ile Thr 580 585 590Leu Asn Leu Thr Ile Met Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr 595 600 605Ala Cys Arg Ala Arg Asn Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys 610 615 620Lys Glu Ile Thr Ile Arg Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn625 630 635 640Leu Ser Asp His Thr Val Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys 645 650 655His Ala Asn Gly Val Pro Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn 660 665 670His Lys Ile Gln Gln Glu Pro Gly Ile Ile Leu Gly Pro Gly Ser Ser 675 680 685Thr Leu Phe Ile Glu Arg Val Thr Glu Glu Asp Glu Gly Val Tyr His 690 695 700Cys Lys Ala Thr Asn Gln Lys Gly Ser Val Glu Ser Ser Ala Tyr Leu705 710 715 720Thr Val Gln Gly Thr Ser Asp Lys Ser Asn Leu Glu 725 73024737PRTMouse 24Tyr Gly Ser Gly Ser Lys Leu Lys Val Pro Glu Leu Ser Leu Lys Gly1 5 10 15Thr Gln His Val Met Gln Ala Gly Gln Thr Leu Phe Leu Lys Cys Arg 20 25 30Gly Glu Ala Ala His Ser Trp Ser Leu Pro Thr Thr Val Ser Gln Glu 35 40 45Asp Lys Arg Leu Ser Ile Thr Pro Pro Ser Ala Cys Gly Arg Asp Asn 50 55 60Arg Gln Phe Cys Ser Thr Leu Thr Leu Asp Thr Ala Gln Ala Asn His65 70 75 80Thr Gly Leu Tyr Thr Cys Arg Tyr Leu Pro Thr Ser Thr Ser Lys Lys 85 90 95Lys Lys Ala Glu Ser Ser Ile Tyr Ile Phe Val Ser Asp Ala Gly Ser 100 105 110Pro Phe Ile Glu Met His Thr Asp Ile Pro Lys Leu Val His Met Thr 115 120 125Glu Gly Arg Gln Leu Ile Ile Pro Cys Arg Val Thr Ser Pro Asn Val 130 135 140Thr Val Thr Leu Lys Lys Phe Pro Phe Asp Thr Leu Thr Pro Asp Gly145 150 155 160Gln Arg Ile Thr Trp Asp Ser Arg Arg Gly Phe Ile Ile Ala Asn Ala 165 170 175Thr Tyr Lys Glu Ile Gly Leu Leu Asn Cys Glu Ala Thr Val Asn Gly 180 185 190His Leu Tyr Gln Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile 195 200 205Leu Asp Val Gln Ile Arg Pro Pro Ser Pro Val Arg Leu Leu His Gly 210 215 220Gln Thr Leu Val Leu Asn Cys Thr Ala Thr Thr Glu Leu Asn Thr Arg225 230 235 240Val Gln Met Ser Trp Asn Tyr Pro Gly Lys Ala Thr Lys Arg Ala Ser

245 250 255Ile Arg Gln Arg Ile Asp Arg Ser His Ser His Asn Asn Val Phe His 260 265 270Ser Val Leu Lys Ile Asn Asn Val Glu Ser Arg Asp Lys Gly Leu Tyr 275 280 285Thr Cys Arg Val Lys Ser Gly Ser Ser Phe Gln Ser Phe Asn Thr Ser 290 295 300Val His Val Tyr Glu Lys Gly Phe Ile Ser Val Lys His Arg Lys Gln305 310 315 320Pro Val Gln Glu Thr Thr Ala Gly Arg Arg Ser Tyr Arg Leu Ser Met 325 330 335Lys Val Lys Ala Phe Pro Ser Pro Glu Ile Val Trp Leu Lys Asp Gly 340 345 350Ser Pro Ala Thr Leu Lys Ser Ala Arg Tyr Leu Val His Gly Tyr Ser 355 360 365Leu Ile Ile Lys Asp Val Thr Thr Glu Asp Ala Gly Asp Tyr Thr Ile 370 375 380Leu Leu Gly Ile Lys Gln Ser Arg Leu Phe Lys Asn Leu Thr Ala Thr385 390 395 400Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu Lys Ser Val Ser Ser 405 410 415Leu Pro Ser Pro Pro Leu Tyr Pro Leu Gly Ser Arg Gln Val Leu Thr 420 425 430Cys Thr Val Tyr Gly Ile Pro Arg Pro Thr Ile Thr Trp Leu Trp His 435 440 445Pro Cys His His Asn His Ser Lys Glu Arg Tyr Asp Phe Cys Thr Glu 450 455 460Asn Glu Glu Ser Phe Ile Leu Asp Pro Ser Ser Asn Leu Gly Asn Arg465 470 475 480Ile Glu Ser Ile Ser Gln Arg Met Thr Val Ile Glu Gly Thr Asn Lys 485 490 495Thr Val Ser Thr Leu Val Val Ala Asp Ser Gln Thr Pro Gly Ile Tyr 500 505 510Ser Cys Arg Ala Phe Asn Lys Ile Gly Thr Val Glu Arg Asn Ile Lys 515 520 525Phe Tyr Val Thr Asp Val Pro Asn Gly Phe His Val Ser Leu Glu Lys 530 535 540Met Pro Ala Glu Gly Glu Asp Leu Lys Leu Ser Cys Val Val Asn Lys545 550 555 560Phe Leu Tyr Arg Asp Ile Thr Trp Ile Leu Leu Arg Thr Val Asn Asn 565 570 575Arg Thr Met His His Ser Ile Ser Lys Gln Lys Met Ala Thr Thr Gln 580 585 590Asp Tyr Ser Ile Thr Leu Asn Leu Val Ile Lys Asn Val Ser Leu Glu 595 600 605Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn Ile Tyr Thr Gly Glu 610 615 620Asp Ile Leu Arg Lys Thr Glu Val Leu Val Arg Asp Ser Glu Ala Pro625 630 635 640His Leu Leu Gln Asn Leu Ser Asp Tyr Glu Val Ser Ile Ser Gly Ser 645 650 655Thr Thr Leu Asp Cys Gln Ala Arg Gly Val Pro Ala Pro Gln Ile Thr 660 665 670Trp Phe Lys Asn Asn His Lys Ile Gln Gln Glu Pro Gly Ile Ile Leu 675 680 685Gly Pro Gly Asn Ser Thr Leu Phe Ile Glu Arg Val Thr Glu Glu Asp 690 695 700Glu Gly Val Tyr Arg Cys Arg Ala Thr Asn Gln Lys Gly Ala Val Glu705 710 715 720Ser Ala Ala Tyr Leu Thr Val Gln Gly Thr Ser Asp Lys Ser Asn Leu 725 730 735Glu25736PRTRat 25Tyr Cys Ser Gly Ser Lys Leu Lys Gly Pro Glu Leu Ser Leu Lys Gly1 5 10 15Thr Gln His Val Met Gln Ala Gly Gln Thr Leu Phe Leu Lys Cys Arg 20 25 30Gly Glu Ala Ala His Ser Trp Ser Leu Pro Thr Thr Val Ser Gln Glu 35 40 45Asp Lys Lys Leu Ser Val Thr Arg Ser Ala Cys Gly Arg Asn Asn Arg 50 55 60Gln Phe Cys Ser Thr Leu Thr Leu Asn Met Ala Gln Ala Asn His Thr65 70 75 80Gly Leu Tyr Ser Cys Arg Tyr Leu Pro Lys Ser Thr Ser Lys Glu Lys 85 90 95Lys Met Glu Ser Ala Ile Tyr Ile Phe Val Ser Asp Ala Gly Ser Pro 100 105 110Phe Ile Glu Met His Ser Asp Ile Pro Lys Leu Val His Met Thr Glu 115 120 125Gly Arg Glu Leu Ile Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr 130 135 140Val Thr Leu Lys Lys Phe Pro Phe Asp Ala Leu Thr Pro Asp Gly Gln145 150 155 160Arg Ile Ala Trp Asp Ser Arg Arg Gly Phe Ile Ile Ala Asn Ala Thr 165 170 175Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His 180 185 190Leu Tyr Gln Thr Ser Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Leu 195 200 205Asp Val Gln Ile Ser Pro Pro Ser Pro Val Arg Phe Leu Arg Gly Gln 210 215 220Thr Leu Val Leu Asn Cys Thr Val Thr Thr Asp Leu Asn Thr Arg Val225 230 235 240Gln Met Ser Trp Asn Tyr Pro Gly Lys Ala Thr Lys Arg Ala Ser Ile 245 250 255Arg Gln Arg Ile Asp Gln Ser Asn Pro His Ser Asn Val Phe His Ser 260 265 270Val Leu Lys Ile Asn Asn Val Glu Ser Arg Asp Lys Gly Leu Tyr Thr 275 280 285Cys Arg Val Lys Ser Gly Ser Ser Phe Arg Thr Phe Asn Thr Ser Val 290 295 300His Val Tyr Glu Lys Gly Phe Ile Ser Val Lys His Arg Lys Gln Gln305 310 315 320Val Gln Glu Thr Ile Ala Gly Lys Arg Ser His Arg Leu Ser Met Lys 325 330 335Val Lys Ala Phe Pro Ser Pro Glu Val Val Trp Leu Lys Asp Gly Val 340 345 350Pro Ala Thr Glu Lys Ser Ala Arg Tyr Ser Val His Gly Tyr Ser Leu 355 360 365Ile Ile Lys Asp Val Thr Ala Glu Asp Ala Gly Asp Tyr Thr Ile Leu 370 375 380Leu Gly Ile Lys Gln Ser Lys Leu Phe Arg Asn Leu Thr Ala Thr Leu385 390 395 400Ile Val Asn Val Lys Pro Gln Ile Tyr Glu Lys Ser Val Ser Ser Leu 405 410 415Pro Ser Pro Pro Leu Tyr Pro Leu Gly Ser Arg Gln Val Leu Thr Cys 420 425 430Thr Val Tyr Gly Ile Pro Gln Pro Thr Ile Lys Trp Leu Trp His Pro 435 440 445Cys His Tyr Asn His Ser Lys Glu Arg Asn Asp Phe Cys Phe Gly Ser 450 455 460Glu Glu Ser Phe Ile Leu Asp Ser Ser Ser Asn Ile Gly Asn Arg Ile465 470 475 480Glu Gly Ile Thr Gln Arg Met Met Val Ile Glu Gly Thr Asn Lys Thr 485 490 495Val Ser Thr Leu Val Val Ala Asp Ser Arg Thr Pro Gly Ser Tyr Ser 500 505 510Cys Lys Ala Phe Asn Lys Ile Gly Thr Val Glu Arg Asp Ile Arg Phe 515 520 525Tyr Val Thr Asp Val Pro Asn Gly Phe His Val Ser Leu Glu Lys Ile 530 535 540Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser Cys Val Val Ser Lys Phe545 550 555 560Leu Tyr Arg Asp Ile Thr Trp Ile Leu Leu Arg Thr Val Asn Asn Arg 565 570 575Thr Met His His Ser Ile Ser Lys Gln Lys Met Ala Thr Thr Gln Asp 580 585 590Tyr Ser Ile Thr Leu Asn Leu Val Ile Lys Asn Val Ser Leu Glu Asp 595 600 605Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn Ile Tyr Thr Gly Glu Glu 610 615 620Ile Leu Arg Lys Thr Glu Val Leu Val Arg Asp Leu Glu Ala Pro Leu625 630 635 640Leu Leu Gln Asn Leu Ser Asp His Glu Val Ser Ile Ser Gly Ser Thr 645 650 655Thr Leu Asp Cys Gln Ala Arg Gly Val Pro Ala Pro Gln Ile Thr Trp 660 665 670Phe Lys Asn Asn His Lys Ile Gln Gln Glu Pro Gly Ile Ile Leu Gly 675 680 685Pro Gly Asn Ser Thr Leu Phe Ile Glu Arg Val Thr Glu Glu Asp Glu 690 695 700Gly Val Tyr Arg Cys Arg Ala Thr Asn Gln Lys Gly Val Val Glu Ser705 710 715 720Ser Ala Tyr Leu Thr Val Gln Gly Thr Ser Asp Lys Ser Asn Leu Glu 725 730 73526745PRTHuman 26Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro Arg Leu Ser1 5 10 15Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr Leu Gln Ile 20 25 30Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro Asn Asn Gln 35 40 45Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser Asp Gly Leu 50 55 60Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn Asp Thr Gly65 70 75 80Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser Val Ile Tyr 85 90 95Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp 100 105 110Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val 115 120 125Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala 130 135 140Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp145 150 155 160Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala 165 170 175Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser 180 185 190Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val 195 200 205Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val 210 215 220Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn225 230 235 240Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg 245 250 255Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr 260 265 270Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys 275 280 285Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg 290 295 300Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu305 310 315 320Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu 325 330 335Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro Leu 340 345 350Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr Ile Met Glu 355 360 365Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu Thr Asn Pro 370 375 380Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val Val Tyr Val385 390 395 400Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val Asp Ser Tyr 405 410 415Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr Ala Ile Pro 420 425 430Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu Glu Cys Ala 435 440 445Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr Pro Cys Glu 450 455 460Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys Ile Glu Val465 470 475 480Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys Thr Val Ser 485 490 495Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr Lys Cys Glu 500 505 510Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser Phe His Val 515 520 525Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln Pro Thr Glu 530 535 540Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser Thr Phe Glu545 550 555 560Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro Ile His Val 565 570 575Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr Leu Trp Lys 580 585 590Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile Met 595 600 605Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr Val Cys Leu 610 615 620Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val Arg Gln Leu625 630 635 640Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn Leu Glu Asn 645 650 655Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys Thr Ala Ser 660 665 670Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn Glu Thr Leu 675 680 685Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg Asn Leu Thr 690 695 700Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr Cys Gln Ala705 710 715 720Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe Ile Ile Glu 725 730 735Gly Ala Gln Glu Lys Thr Asn Leu Glu 740 74527743PRTMouse 27Ala Ser Val Gly Leu Pro Gly Asp Phe Leu His Pro Pro Lys Leu Ser1 5 10 15Thr Gln Lys Asp Ile Leu Thr Ile Leu Ala Asn Thr Thr Leu Gln Ile 20 25 30Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro Asn Ala Gln 35 40 45Arg Asp Ser Glu Glu Arg Val Leu Val Thr Glu Cys Gly Gly Gly Asp 50 55 60Ser Ile Phe Cys Lys Thr Leu Thr Ile Pro Arg Val Val Gly Asn Asp65 70 75 80Thr Gly Ala Tyr Lys Cys Ser Tyr Arg Asp Val Asp Ile Ala Ser Thr 85 90 95Val Tyr Val Tyr Val Arg Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val 100 105 110Ser Asp Gln His Gly Ile Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr 115 120 125Val Val Ile Pro Cys Arg Gly Ser Ile Ser Asn Leu Asn Val Ser Leu 130 135 140Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile145 150 155 160Ser Trp Asp Ser Glu Ile Gly Phe Thr Leu Pro Ser Tyr Met Ile Ser 165 170 175Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Thr Tyr 180 185 190Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp 195 200 205Val Ile Leu Ser Pro Pro His Glu Ile Glu Leu Ser Ala Gly Glu Lys 210 215 220Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Leu Asp225 230 235 240Phe Thr Trp His Ser Pro Pro Ser Lys Ser His His Lys Lys Ile Val 245 250 255Asn Arg Asp Val Lys Pro Phe Pro Gly Thr Val Ala Lys Met Phe Leu 260 265 270Ser Thr Leu Thr Ile Glu Ser Val Thr Lys Ser Asp Gln Gly Glu Tyr 275 280 285Thr Cys Val Ala Ser Ser Gly Arg Met Ile Lys Arg Asn Arg Thr Phe 290 295 300Val Arg Val His Thr Lys Pro Phe Ile Ala Phe Gly Ser Gly Met Lys305 310 315 320Ser Leu Val Glu Ala Thr Val Gly Ser Gln Val Arg Ile Pro Val Lys 325 330 335Tyr Leu Ser Tyr Pro Ala Pro Asp Ile Lys Trp Tyr Arg Asn Gly Arg 340 345 350Pro Ile Glu Ser Asn Tyr Thr Met Ile Val Gly Asp Glu Leu Thr Ile 355 360 365Met Glu Val Thr Glu Arg Asp Ala Gly Asn Tyr Thr Val Ile Leu Thr 370 375 380Asn Pro Ile Ser Met Glu Lys Gln Ser His Met Val Ser Leu Val Val385 390 395 400Asn Val Pro Pro Gln Ile Gly Glu Lys Ala Leu Ile Ser Pro Met Asp 405 410 415Ser Tyr Gln Tyr Gly Thr Met Gln Thr Leu Thr Cys Thr Val Tyr Ala 420 425 430Asn Pro Pro Leu His His Ile Gln Trp Tyr Trp Gln Leu Glu Glu Ala 435 440 445Cys Ser Tyr Arg Pro Gly Gln Thr Ser Pro Tyr Ala Cys Lys Glu Trp 450 455 460Arg His Val Glu Asp Phe Gln Gly Gly Asn Lys Ile Glu Val Thr Lys465 470 475 480Asn Gln Tyr Ala Leu Ile Glu Gly Lys Asn Lys Thr Val Ser Thr Leu 485 490 495Val Ile Gln

Ala Ala Asn Val Ser Ala Leu Tyr Lys Cys Glu Ala Ile 500 505 510Asn Lys Ala Gly Arg Gly Glu Arg Val Ile Ser Phe His Val Ile Arg 515 520 525Gly Pro Glu Ile Thr Val Gln Pro Ala Ala Gln Pro Thr Glu Gln Glu 530 535 540Ser Val Ser Leu Leu Cys Thr Ala Asp Arg Asn Thr Phe Glu Asn Leu545 550 555 560Thr Trp Tyr Lys Leu Gly Ser Gln Ala Thr Ser Val His Met Gly Glu 565 570 575Ser Leu Thr Pro Val Cys Lys Asn Leu Asp Ala Leu Trp Lys Leu Asn 580 585 590Gly Thr Met Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile Val Ala Phe 595 600 605Gln Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr Val Cys Ser Ala Gln 610 615 620Asp Lys Lys Thr Lys Lys Arg His Cys Leu Val Lys Gln Leu Ile Ile625 630 635 640Leu Glu Arg Met Ala Pro Met Ile Thr Gly Asn Leu Glu Asn Gln Thr 645 650 655Thr Thr Ile Gly Glu Thr Ile Glu Val Thr Cys Pro Ala Ser Gly Asn 660 665 670Pro Thr Pro His Ile Thr Trp Phe Lys Asp Asn Glu Thr Leu Val Glu 675 680 685Asp Ser Gly Ile Val Leu Arg Asp Gly Asn Arg Asn Leu Thr Ile Arg 690 695 700Arg Val Arg Lys Glu Asp Gly Gly Leu Tyr Thr Cys Gln Ala Cys Asn705 710 715 720Val Leu Gly Cys Ala Arg Ala Glu Thr Leu Phe Ile Ile Glu Gly Ala 725 730 735Gln Glu Lys Thr Asn Leu Glu 74028741PRTRat 28Ala Ser Val Gly Leu Pro Gly Asp Ser Leu His Pro Pro Lys Leu Ser1 5 10 15Thr Gln Lys Asp Ile Leu Thr Ile Leu Ala Asn Thr Thr Leu Gln Ile 20 25 30Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro Asn Thr Pro 35 40 45Arg Asp Ser Glu Glu Arg Val Leu Val Thr Glu Cys Gly Asp Ser Ile 50 55 60Phe Cys Lys Thr Leu Thr Val Pro Arg Val Val Gly Asn Asp Thr Gly65 70 75 80Ala Tyr Lys Cys Phe Tyr Arg Asp Thr Asp Val Ser Ser Ile Val Tyr 85 90 95Val Tyr Val Gln Asp His Arg Ser Pro Phe Ile Ala Ser Val Ser Asp 100 105 110Glu His Gly Ile Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val 115 120 125Ile Pro Cys Arg Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala 130 135 140Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp145 150 155 160Asp Ser Glu Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala 165 170 175Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Thr Tyr Gln Ser 180 185 190Ile Met Tyr Ile Val Leu Val Val Gly Tyr Arg Ile Tyr Asp Val Val 195 200 205Leu Ser Pro Pro His Glu Ile Glu Leu Ser Ala Gly Glu Lys Leu Val 210 215 220Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Leu Asp Phe Ser225 230 235 240Trp Gln Phe Pro Ser Ser Lys His Gln His Lys Lys Ile Val Asn Arg 245 250 255Asp Val Lys Ser Leu Pro Gly Thr Val Ala Lys Met Phe Leu Ser Thr 260 265 270Leu Thr Ile Asp Ser Val Thr Lys Ser Asp Gln Gly Glu Tyr Thr Cys 275 280 285Thr Ala Tyr Ser Gly Leu Met Thr Lys Lys Asn Lys Thr Phe Val Arg 290 295 300Val His Thr Lys Pro Phe Ile Ala Phe Gly Ser Gly Met Lys Ser Leu305 310 315 320Val Glu Ala Thr Val Gly Ser Gln Val Arg Ile Pro Val Lys Tyr Leu 325 330 335Ser Tyr Pro Ala Pro Asp Ile Lys Trp Tyr Arg Asn Gly Arg Pro Ile 340 345 350Glu Ser Asn Tyr Thr Met Ile Val Gly Asp Glu Leu Thr Ile Met Glu 355 360 365Val Ser Glu Arg Asp Ala Gly Asn Tyr Thr Val Ile Leu Thr Asn Pro 370 375 380Ile Ser Met Glu Lys Gln Ser His Met Val Ser Leu Val Val Asn Val385 390 395 400Pro Pro Gln Ile Gly Glu Lys Ala Leu Ile Ser Pro Met Asp Ser Tyr 405 410 415Gln Tyr Gly Thr Met Gln Thr Leu Thr Cys Thr Val Tyr Ala Asn Pro 420 425 430Pro Leu His His Ile Gln Trp Tyr Trp Gln Leu Glu Glu Ala Cys Ser 435 440 445Tyr Arg Pro Ser Gln Thr Asn Pro Tyr Thr Cys Lys Glu Trp Arg His 450 455 460Val Lys Asp Phe Gln Gly Gly Asn Lys Ile Glu Val Thr Lys Asn Gln465 470 475 480Tyr Ala Leu Ile Glu Gly Lys Asn Lys Thr Val Ser Thr Leu Val Ile 485 490 495Gln Ala Ala Tyr Val Ser Ala Leu Tyr Lys Cys Glu Ala Ile Asn Lys 500 505 510Ala Gly Arg Gly Glu Arg Val Ile Ser Phe His Val Ile Arg Gly Pro 515 520 525Glu Ile Thr Val Gln Pro Ala Thr Gln Pro Thr Glu Arg Glu Ser Met 530 535 540Ser Leu Leu Cys Thr Ala Asp Arg Asn Thr Phe Glu Asn Leu Thr Trp545 550 555 560Tyr Lys Leu Gly Ser Gln Ala Thr Ser Val His Met Gly Glu Ser Leu 565 570 575Thr Pro Val Cys Lys Asn Leu Asp Ala Leu Trp Lys Leu Asn Gly Thr 580 585 590Val Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile Val Ala Phe Gln Asn 595 600 605Ala Ser Leu Gln Asp Gln Gly Asn Tyr Val Cys Ser Ala Gln Asp Lys 610 615 620Lys Thr Lys Lys Arg His Cys Leu Val Lys Gln Leu Val Ile Leu Glu625 630 635 640Arg Met Ala Pro Met Ile Thr Gly Asn Leu Glu Asn Gln Thr Thr Thr 645 650 655Ile Gly Glu Thr Ile Glu Val Val Cys Pro Thr Ser Gly Asn Pro Thr 660 665 670Pro Leu Ile Thr Trp Phe Lys Asp Asn Glu Thr Leu Val Glu Asp Ser 675 680 685Gly Ile Val Leu Lys Asp Gly Asn Arg Asn Leu Thr Ile Arg Arg Val 690 695 700Arg Lys Glu Asp Gly Gly Leu Tyr Thr Cys Gln Ala Cys Asn Val Leu705 710 715 720Gly Cys Ala Arg Ala Glu Thr Leu Phe Ile Ile Glu Gly Val Gln Glu 725 730 735Lys Thr Asn Leu Glu 74029756PRTHuman 29Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro 20 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp 35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp 50 55 60Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn65 70 75 80Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile 85 90 95Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu 100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp 115 120 125Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr 130 135 140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp 165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu 180 185 190Ser Asn Pro Phe Leu Val His Ile Thr Gly Asn Glu Leu Tyr Asp Ile 195 200 205Gln Leu Leu Pro Arg Lys Ser Leu Glu Leu Leu Val Gly Glu Lys Leu 210 215 220Val Leu Asn Cys Thr Val Trp Ala Glu Phe Asn Ser Gly Val Thr Phe225 230 235 240Asp Trp Asp Tyr Pro Gly Lys Gln Ala Glu Arg Gly Lys Trp Val Pro 245 250 255Glu Arg Arg Ser Gln Gln Thr His Thr Glu Leu Ser Ser Ile Leu Thr 260 265 270Ile His Asn Val Ser Gln His Asp Leu Gly Ser Tyr Val Cys Lys Ala 275 280 285Asn Asn Gly Ile Gln Arg Phe Arg Glu Ser Thr Glu Val Ile Val His 290 295 300Glu Asn Pro Phe Ile Ser Val Glu Trp Leu Lys Gly Pro Ile Leu Glu305 310 315 320Ala Thr Ala Gly Asp Glu Leu Val Lys Leu Pro Val Lys Leu Ala Ala 325 330 335Tyr Pro Pro Pro Glu Phe Gln Trp Tyr Lys Asp Gly Lys Ala Leu Ser 340 345 350Gly Arg His Ser Pro His Ala Leu Val Leu Lys Glu Val Thr Glu Ala 355 360 365Ser Thr Gly Thr Tyr Thr Leu Ala Leu Trp Asn Ser Ala Ala Gly Leu 370 375 380Arg Arg Asn Ile Ser Leu Glu Leu Val Val Asn Val Pro Pro Gln Ile385 390 395 400His Glu Lys Glu Ala Ser Ser Pro Ser Ile Tyr Ser Arg His Ser Arg 405 410 415Gln Ala Leu Thr Cys Thr Ala Tyr Gly Val Pro Leu Pro Leu Ser Ile 420 425 430Gln Trp His Trp Arg Pro Trp Thr Pro Cys Lys Met Phe Ala Gln Arg 435 440 445Ser Leu Arg Arg Arg Gln Gln Gln Asp Leu Met Pro Gln Cys Arg Asp 450 455 460Trp Arg Ala Val Thr Thr Gln Asp Ala Val Asn Pro Ile Glu Ser Leu465 470 475 480Asp Thr Trp Thr Glu Phe Val Glu Gly Lys Asn Lys Thr Val Ser Lys 485 490 495Leu Val Ile Gln Asn Ala Asn Val Ser Ala Met Tyr Lys Cys Val Val 500 505 510Ser Asn Lys Val Gly Gln Asp Glu Arg Leu Ile Tyr Phe Tyr Val Thr 515 520 525Thr Ile Pro Asp Gly Phe Thr Ile Glu Ser Lys Pro Ser Glu Glu Leu 530 535 540Leu Glu Gly Gln Pro Val Leu Leu Ser Cys Gln Ala Asp Ser Tyr Lys545 550 555 560Tyr Glu His Leu Arg Trp Tyr Arg Leu Asn Leu Ser Thr Leu His Asp 565 570 575Ala His Gly Asn Pro Leu Leu Leu Asp Cys Lys Asn Val His Leu Phe 580 585 590Ala Thr Pro Leu Ala Ala Ser Leu Glu Glu Val Ala Pro Gly Ala Arg 595 600 605His Ala Thr Leu Ser Leu Ser Ile Pro Arg Val Ala Pro Glu His Glu 610 615 620Gly His Tyr Val Cys Glu Val Gln Asp Arg Arg Ser His Asp Lys His625 630 635 640Cys His Lys Lys Tyr Leu Ser Val Gln Ala Leu Glu Ala Pro Arg Leu 645 650 655Thr Gln Asn Leu Thr Asp Leu Leu Val Asn Val Ser Asp Ser Leu Glu 660 665 670Met Gln Cys Leu Val Ala Gly Ala His Ala Pro Ser Ile Val Trp Tyr 675 680 685Lys Asp Glu Arg Leu Leu Glu Glu Lys Ser Gly Val Asp Leu Ala Asp 690 695 700Ser Asn Gln Lys Leu Ser Ile Gln Arg Val Arg Glu Glu Asp Ala Gly705 710 715 720Arg Tyr Leu Cys Ser Val Cys Asn Ala Lys Gly Cys Val Asn Ser Ser 725 730 735Ala Ser Val Ala Val Glu Gly Ser Glu Asp Lys Gly Ser Met Glu Ile 740 745 750Val Ile Leu Val 75530751PRTMouse 30Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Asp Ser Tyr Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro 20 25 30Leu Glu Trp Thr Trp Pro Gly Ala Gln Glu Val Leu Thr Thr Gly Gly 35 40 45Lys Asp Ser Glu Asp Thr Arg Val Val His Asp Cys Glu Gly Thr Glu 50 55 60Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu Ala Gln Thr His Ala Asn65 70 75 80Asn Thr Gly Ser Tyr His Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile 85 90 95Glu Gly Thr Thr Ala Ala Ser Thr Tyr Val Phe Val Arg Asp Phe Lys 100 105 110His Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp 115 120 125Ser Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Ile Thr 130 135 140Leu Arg Ser Gln Ser Ser Ala Leu His Pro Asp Gly Gln Glu Val Leu145 150 155 160Trp Asp Asp Arg Arg Gly Met Arg Val Pro Thr Gln Leu Leu Arg Asp 165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asn Phe Leu 180 185 190Ser Asn Leu Phe Val Val His Ile Thr Gly Asn Glu Leu Tyr Asp Ile 195 200 205Gln Leu Tyr Pro Lys Lys Ser Met Glu Leu Leu Val Gly Glu Lys Leu 210 215 220Val Leu Asn Cys Thr Val Trp Ala Glu Phe Asp Ser Gly Val Thr Phe225 230 235 240Asp Trp Asp Tyr Pro Gly Lys Gln Ala Glu Arg Ala Lys Trp Val Pro 245 250 255Glu Arg Arg Ser Gln Gln Thr His Thr Glu Leu Ser Ser Ile Leu Thr 260 265 270Ile His Asn Val Ser Gln Asn Asp Leu Gly Pro Tyr Val Cys Glu Ala 275 280 285Asn Asn Gly Ile Gln Arg Phe Arg Glu Ser Thr Glu Val Ile Val His 290 295 300Glu Lys Pro Phe Ile Ser Val Glu Trp Leu Lys Gly Pro Val Leu Glu305 310 315 320Ala Thr Ala Gly Asp Glu Leu Val Lys Leu Pro Val Lys Leu Ala Ala 325 330 335Tyr Pro Pro Pro Glu Phe Gln Trp Tyr Lys Asp Arg Lys Ala Val Thr 340 345 350Gly Arg His Asn Pro His Ala Leu Val Leu Lys Glu Val Thr Glu Ala 355 360 365Ser Ala Gly Val Tyr Thr Leu Ala Leu Trp Asn Ser Ala Ala Gly Leu 370 375 380Arg Gln Asn Ile Ser Leu Glu Leu Val Val Asn Val Pro Pro His Ile385 390 395 400His Glu Lys Glu Ala Ser Ser Pro Ser Ile Tyr Ser Arg His Ser Arg 405 410 415Gln Thr Leu Thr Cys Thr Ala Tyr Gly Val Pro Gln Pro Leu Ser Val 420 425 430Gln Trp His Trp Arg Pro Trp Thr Pro Cys Lys Thr Phe Ala Gln Arg 435 440 445Ser Leu Arg Arg Arg Gln Gln Arg Asp Gly Met Pro Gln Cys Arg Asp 450 455 460Trp Lys Glu Val Thr Thr Gln Asp Ala Val Asn Pro Ile Glu Ser Leu465 470 475 480Asp Ser Trp Thr Glu Phe Val Glu Gly Lys Asn Lys Thr Val Ser Lys 485 490 495Leu Val Ile Gln Asp Ala Asn Val Ser Ala Met Tyr Lys Cys Val Val 500 505 510Val Asn Lys Val Gly Gln Asp Glu Arg Leu Ile Tyr Phe Tyr Val Thr 515 520 525Thr Ile Pro Asp Gly Phe Ser Ile Glu Ser Glu Pro Ser Glu Asp Pro 530 535 540Leu Glu Gly Gln Ser Val Arg Leu Ser Cys Arg Ala Asp Asn Tyr Thr545 550 555 560Tyr Glu His Leu Arg Trp Tyr Arg Leu Asn Leu Ser Thr Leu His Asp 565 570 575Ala Gln Gly Asn Pro Leu Leu Leu Asp Cys Lys Asn Val His Leu Phe 580 585 590Ala Thr Pro Leu Glu Ala Asn Leu Glu Glu Ala Glu Pro Gly Ala Arg 595 600 605His Ala Thr Leu Ser Leu Asn Ile Pro Arg Val Ala Pro Glu Asp Glu 610 615 620Gly Asp Tyr Val Cys Glu Val Gln Asp Arg Arg Ser Gln Asp Lys His625 630 635 640Cys His Lys Lys Tyr Leu Ser Val Gln Ala Leu Glu Ala Pro Arg Leu 645 650 655Thr Gln Asn Leu Thr Asp Leu Leu Val Asn Val Ser Asp Ser Leu Glu 660 665 670Met Arg Cys Pro Val Ala Gly Ala His Val Pro Ser Ile Val Trp Tyr 675 680 685Lys Asp Glu Arg Leu Leu Glu Lys Glu Ser Gly Ile Asp Leu Ala Asp 690 695 700Ser Asn Gln Arg Leu Ser Ile Gln Arg Val Arg Glu Glu Asp Ala Gly705 710 715 720Arg Tyr Leu Cys Ser Val Cys Asn Ala Lys Gly Cys Val Asn Ser

Ser 725 730 735Ala Ser Val Ala Val Glu Gly Ser Glu Asp Lys Gly Ser Met Glu 740 745 75031751PRTRat 31Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Asp Ser Tyr Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro 20 25 30Leu Glu Trp Thr Trp Arg Gly Ala Gln Glu Val Leu Thr Thr Gly Gly 35 40 45Lys Asp Ser Glu Asp Thr Gln Val Val Gln Asp Cys Glu Gly Thr Glu 50 55 60Ala Arg Pro Tyr Cys Lys Val Leu Ser Leu Ala Gln Thr His Ala Asn65 70 75 80Asn Thr Gly Ser Tyr Tyr Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile 85 90 95Glu Gly Thr Thr Ala Ala Ser Thr Tyr Val Phe Val Arg Asp Phe Glu 100 105 110Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp 115 120 125Ser Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Ile Thr 130 135 140Leu Arg Ser Gln Ser Ser Val Leu His Pro Asp Gly Gln Glu Val Leu145 150 155 160Trp Asp Asp Arg Arg Gly Met Arg Val Pro Thr Leu Leu Leu Arg Asp 165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu 180 185 190Ser Asn Pro Phe Leu Val His Ile Thr Gly Asn Glu Leu Tyr Asp Ile 195 200 205Gln Leu Tyr Pro Lys Lys Ser Leu Glu Leu Leu Val Gly Glu Lys Leu 210 215 220Val Leu Asn Cys Thr Val Trp Ala Glu Phe Asp Ser Gly Val Thr Phe225 230 235 240Asp Trp Asp Tyr Pro Gly Lys Gln Ala Glu Arg Ala Lys Trp Val Pro 245 250 255Glu Arg Arg Ser Gln Gln Thr His Thr Glu Leu Ser Ser Ile Leu Thr 260 265 270Ile His Asn Val Ser Gln His Asp Leu Gly Pro Tyr Val Cys Glu Ala 275 280 285Asn Asn Gly Ile Gln Gln Phe Arg Glu Ser Thr Glu Val Ile Val His 290 295 300Glu Lys Pro Phe Ile Ser Val Glu Trp Leu Lys Gly Pro Val Leu Glu305 310 315 320Ala Thr Ala Gly Asp Glu Met Val Lys Leu Pro Val Lys Leu Ala Ala 325 330 335Tyr Pro Pro Pro Glu Phe Gln Trp Tyr Lys Asp Arg Lys Ala Val Thr 340 345 350Gly Arg His Asn Pro His Ala Leu Val Leu Lys Glu Val Thr Glu Ala 355 360 365Ser Ala Gly Val Tyr Thr Leu Ala Leu Trp Asn Ser Ala Ala Gly Leu 370 375 380Arg Gln Asn Ile Ser Leu Glu Leu Val Val Asn Val Pro Pro His Ile385 390 395 400His Glu Lys Glu Ala Ser Ser Pro Ser Ile Tyr Ser Arg His Ser Arg 405 410 415Gln Thr Leu Thr Cys Thr Thr Tyr Gly Val Pro Gln Pro Leu Ser Val 420 425 430Gln Trp His Trp Arg Pro Trp Thr Pro Cys Lys Thr Phe Ala Gln Arg 435 440 445Ser Leu Arg Arg Arg Gln Pro Arg Asp Gly Met Pro Gln Cys Arg Asp 450 455 460Trp Lys Glu Val Thr Thr Gln Asp Ala Val Asn Pro Ile Glu Ser Leu465 470 475 480Asp Thr Trp Thr Glu Ser Val Glu Gly Lys Asn Lys Thr Val Ser Lys 485 490 495Leu Val Ile Gln Asp Ala Asn Val Ser Ala Met Tyr Lys Cys Val Val 500 505 510Phe Asn Lys Val Gly Gln Asp Glu Arg Leu Ile Tyr Phe Tyr Val Thr 515 520 525Thr Ile Pro Asp Gly Phe Ser Ile Glu Ser Glu Pro Ser Glu Asp Pro 530 535 540Leu Glu Gly Gln Ser Val Arg Leu Ser Cys Arg Ala Asp Asn Tyr Thr545 550 555 560Tyr Glu His Leu Arg Trp Tyr Arg Leu Asn Leu Ser Thr Leu His Asp 565 570 575Ala Gln Gly Asn Pro Leu Leu Leu Asp Cys Lys Asn Val His Leu Phe 580 585 590Ala Thr Pro Leu Glu Ala Asn Leu Glu Glu Ala Glu Pro Gly Ala Arg 595 600 605His Ala Thr Leu Ser Leu Asn Ile Pro Arg Val Ala Pro Glu Asp Glu 610 615 620Gly Asp Tyr Val Cys Glu Val Gln Asp Arg Arg Ser Gln Asp Lys His625 630 635 640Cys His Lys Lys Tyr Leu Ser Val Gln Ala Leu Glu Ala Pro Arg Leu 645 650 655Thr Gln Asn Leu Thr Asp Leu Leu Val Asn Val Arg Thr Ser Leu Glu 660 665 670Met Arg Cys Pro Val Ala Gly Ala His Val Pro Ser Ile Val Trp Tyr 675 680 685Lys Asp Glu Arg Leu Leu Glu Lys Glu Ser Gly Ile Asp Leu Ala Asp 690 695 700Ser Asn Gln Arg Leu Ser Ile Gln Arg Val Arg Glu Glu Asp Ala Gly705 710 715 720Arg Tyr Leu Cys Ser Val Cys Asn Ala Lys Gly Cys Val Asn Ser Ser 725 730 735Ala Ser Val Ala Val Glu Gly Ser Glu Asp Lys Gly Ser Met Glu 740 745 75032220PRTHuman 32Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe1 5 10 15Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 20 25 30Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 35 40 45Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 50 55 60Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr65 70 75 80Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 85 90 95Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 100 105 110Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 115 120 125Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 130 135 140Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro145 150 155 160Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 165 170 175Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 180 185 190Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 195 200 205Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 22033223PRTHuman 33Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val1 5 10 15Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25 30Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 35 40 45Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 50 55 60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser65 70 75 80Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile 100 105 110Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120 125Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 130 135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn145 150 155 160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser 165 170 175Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 180 185 190Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 195 200 205His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 22034223PRTHuman 34Thr Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val1 5 10 15Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25 30Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 35 40 45Val Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 50 55 60Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser65 70 75 80Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 100 105 110Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120 125Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 130 135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser145 150 155 160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser 165 170 175Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 180 185 190Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu 195 200 205His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220354474DNAArtificial SequencepITR.CBA.Bevacizumab 35cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct gcggccgcac gcgtgacatt gattattgac tagttattaa tagtaatcaa 180ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 240atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 300ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt 360aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 420tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 480ctacttggca gtacatctac gtattagtca tcgctattac catgggtcga ggtgagcccc 540acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt 600attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggcgcg cgccaggcgg 660ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 720gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 780aaagcgaagc gcgcggcggg cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg 840cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg 900gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggctcgtttc 960ttttctgtgg ctgcgtgaaa gccttaaagg gctccgggag ggccctttgt gcggggggga 1020gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct 1080gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcgtgtgcgc 1140gaggggagcg cggccggggg cggtgccccg cggtgcgggg gggctgcgag gggaacaaag 1200gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc 1260tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg 1320gggctccgtg cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg 1380ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc 1440ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg 1500taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct 1560gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg 1620aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc 1680cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg 1740ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc 1800ttctttttcc tacagctcct gggcaacgtg ctggttattg tgaccggtgc caccatgtac 1860cggatgcagc tgctgagctg tatcgccctg tctctggccc tggtcaccaa ttctgaggtg 1920cagctggtgg aatctggcgg cggacttgtt caacctggcg gctctctgag actgagctgt 1980gccgcttctg gctacacctt caccaactac ggcatgaact gggtccgaca ggcccctggc 2040aaaggccttg aatgggtcgg atggatcaac acctacaccg gcgagccaac atacgccgcc 2100gacttcaagc ggagattcac cttcagcctg gacaccagca agagcaccgc ctacctgcag 2160atgaacagcc tgagagccga ggacaccgcc gtgtactact gcgccaagta tccccactac 2220tacggcagca gccactggta ctttgacgtg tggggacagg gcacactggt cacagtgtct 2280agcgcctcta caaagggccc cagcgttttc ccactggctc ctagcagcaa gtctaccagc 2340ggaggaacag ccgctctggg ctgtctggtc aaggactact ttcccgagcc tgtgaccgtg 2400tcctggaatt ctggcgctct gacaagcggc gtgcacacct ttccagctgt gctgcaaagc 2460agcggcctgt actctctgag cagcgtcgtg acagtgccaa gcagctctct gggcacccag 2520acctacatct gcaatgtgaa ccacaagcct agcaacacca aggtggacaa gaaggtggaa 2580cccaagagct gcgacaagac ccacacctgt cctccatgtc ctgctccaga actgctcggc 2640ggaccttccg tgttcctgtt tcctccaaag cctaaggaca ccctgatgat cagcagaacc 2700cctgaagtga cctgcgtggt ggtggatgtg tcccacgagg atcccgaagt gaagttcaat 2760tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 2820aacagcacct acagagtggt gtccgtgctg accgtgctgc accaggattg gctgaacggc 2880aaagagtaca agtgcaaggt gtccaacaag gccctgcctg ctcctatcga gaaaaccatc 2940agcaaggcca agggccagcc tagggaaccc caggtttaca cactgcctcc aagccgggaa 3000gagatgacca agaaccaggt gtccctgacc tgcctcgtga agggcttcta cccttccgat 3060atcgccgtgg aatgggagag caatggccag ccagagaaca actacaagac aacccctcct 3120gtgctggaca gcgacggctc attcttcctg tacagcaagc tgacagtgga caagtccaga 3180tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 3240acccagaagt ctctgagcct gtctcctggc aagcggaaga gaagaggctc tggcgaaggc 3300agaggcagcc tgcttacatg tggcgacgtg gaagagaacc ccggacctat gtatagaatg 3360cagctcctgt cctgcattgc cctgagcctg gctctcgtga ccaacagcga catccagatg 3420acacagagcc ccagcagcct gtctgcctct gtgggagaca gagtgaccat cacctgtagc 3480gccagccagg acatctccaa ctacctgaac tggtatcagc aaaagcccgg caaggcccct 3540aaggtgctga tctacttcac aagcagcctg cactccggcg tgcccagcag attttctggc 3600tctggcagcg gcaccgactt caccctgacc atatctagcc tgcagcctga ggacttcgcc 3660acctactact gccagcagta cagcaccgtg ccttggacat ttggccaggg cacaaaggtg 3720gaaatcaagc ggactgtggc cgctcctagc gtgttcatct ttccacctag cgacgagcag 3780ctgaagtctg gcacagcctc tgtcgtgtgc ctgctgaaca acttctaccc cagagaagcc 3840aaggtgcagt ggaaagtgga caatgccctg cagagcggca acagccaaga gagcgtgaca 3900gagcaggact ccaaggatag cacctatagc ctgagcagca ccctgacact gagcaaggcc 3960gactacgaga agcacaaagt gtacgcctgc gaagtgaccc accagggcct ttctagccct 4020gtgaccaaga gcttcaaccg gggcgaatgt taagagctcg ctgatcagcc tcgactgtgc 4080cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 4140gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 4200ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 4260acaatagcag gcatgctggg gatgcggtgg gctctatgga agcttgaatt cagctgacgt 4320gcctcggacc gctaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 4380tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc 4440tcagtgagcg agcgagcgcg cagctgcctg cagg 447436144DNAArtificial SequenceITR sequence 36cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct gcggccgcac gcgt 144371671DNAArtificial SequenceCBA sequence 37gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180ctttccattg acgtcaatgg gtggactatt tacggtaaac tgcccacttg gcagtacatc 240aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360tagtcatcgc tattaccatg ggtcgaggtg agccccacgt tctgcttcac tctccccatc 420tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg 480atgggggcgg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg 540cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc 600ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg 660agtcgctgcg ttgccttcgc cccgtgcccc gctccgcgcc gcctcgcgcc gcccgccccg 720gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg 780ctgtaattag cgcttggttt aatgacggct cgtttctttt ctgtggctgc gtgaaagcct 840taaagggctc cgggagggcc ctttgtgcgg gggggagcgg ctcggggggt gcgtgcgtgt 900gtgtgtgcgt ggggagcgcc gcgtgcggcc cgcgctgccc ggcggctgtg agcgctgcgg 960gcgcggcgcg gggctttgtg cgctccgcgt gtgcgcgagg ggagcgcggc cgggggcggt 1020gccccgcggt gcgggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg 1080gggggtgagc agggggtgtg ggcgcggcgg tcgggctgta acccccccct gcacccccct 1140ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtgcggg gcgtggcgcg 1200gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg 1260cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc cggcggctgt 1320cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga 1380cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg caccccctct 1440agcgggcgcg

gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg 1560gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc 1620ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca g 16713839DNAArtificial Sequencespacer 38ctcctgggca acgtgctggt tattgtgacc ggtgccacc 393960DNAArtificial SequenceIL-2 secretion signal sequence 39atgtaccgga tgcagctgct gagctgtatc gccctgtctc tggccctggt caccaattct 60401359DNAArtificial SequenceSequence encoding heavy chain of bevacizumab 40gaggtgcagc tggtggaatc tggcggcgga cttgttcaac ctggcggctc tctgagactg 60agctgtgccg cttctggcta caccttcacc aactacggca tgaactgggt ccgacaggcc 120cctggcaaag gccttgaatg ggtcggatgg atcaacacct acaccggcga gccaacatac 180gccgccgact tcaagcggag attcaccttc agcctggaca ccagcaagag caccgcctac 240ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc caagtatccc 300cactactacg gcagcagcca ctggtacttt gacgtgtggg gacagggcac actggtcaca 360gtgtctagcg cctctacaaa gggccccagc gttttcccac tggctcctag cagcaagtct 420accagcggag gaacagccgc tctgggctgt ctggtcaagg actactttcc cgagcctgtg 480accgtgtcct ggaattctgg cgctctgaca agcggcgtgc acacctttcc agctgtgctg 540caaagcagcg gcctgtactc tctgagcagc gtcgtgacag tgccaagcag ctctctgggc 600acccagacct acatctgcaa tgtgaaccac aagcctagca acaccaaggt ggacaagaag 660gtggaaccca agagctgcga caagacccac acctgtcctc catgtcctgc tccagaactg 720ctcggcggac cttccgtgtt cctgtttcct ccaaagccta aggacaccct gatgatcagc 780agaacccctg aagtgacctg cgtggtggtg gatgtgtccc acgaggatcc cgaagtgaag 840ttcaattggt acgtggacgg cgtggaagtg cacaacgcca agaccaagcc tagagaggaa 900cagtacaaca gcacctacag agtggtgtcc gtgctgaccg tgctgcacca ggattggctg 960aacggcaaag agtacaagtg caaggtgtcc aacaaggccc tgcctgctcc tatcgagaaa 1020accatcagca aggccaaggg ccagcctagg gaaccccagg tttacacact gcctccaagc 1080cgggaagaga tgaccaagaa ccaggtgtcc ctgacctgcc tcgtgaaggg cttctaccct 1140tccgatatcg ccgtggaatg ggagagcaat ggccagccag agaacaacta caagacaacc 1200cctcctgtgc tggacagcga cggctcattc ttcctgtaca gcaagctgac agtggacaag 1260tccagatggc agcagggcaa cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac 1320cactacaccc agaagtctct gagcctgtct cctggcaag 13594112DNAArtificial SequenceLinker sequence 41cggaagagaa ga 124263DNAArtificial SequenceT2A sequence 42ggctctggcg aaggcagagg cagcctgctt acatgtggcg acgtggaaga gaaccccgga 60cct 634360DNAArtificial SequenceIL-2 secretion signal sequence 43atgtatagaa tgcagctcct gtcctgcatt gccctgagcc tggctctcgt gaccaacagc 6044645DNAArtificial SequenceSequence encoding light chain of bevacizumab 44gacatccaga tgacacagag ccccagcagc ctgtctgcct ctgtgggaga cagagtgacc 60atcacctgta gcgccagcca ggacatctcc aactacctga actggtatca gcaaaagccc 120ggcaaggccc ctaaggtgct gatctacttc acaagcagcc tgcactccgg cgtgcccagc 180agattttctg gctctggcag cggcaccgac ttcaccctga ccatatctag cctgcagcct 240gaggacttcg ccacctacta ctgccagcag tacagcaccg tgccttggac atttggccag 300ggcacaaagg tggaaatcaa gcggactgtg gccgctccta gcgtgttcat ctttccacct 360agcgacgagc agctgaagtc tggcacagcc tctgtcgtgt gcctgctgaa caacttctac 420cccagagaag ccaaggtgca gtggaaagtg gacaatgccc tgcagagcgg caacagccaa 480gagagcgtga cagagcagga ctccaaggat agcacctata gcctgagcag caccctgaca 540ctgagcaagg ccgactacga gaagcacaaa gtgtacgcct gcgaagtgac ccaccagggc 600ctttctagcc ctgtgaccaa gagcttcaac cggggcgaat gttaa 6454521DNAArtificial SequenceLinker sequence 45gagctcgctg atcagcctcg a 2146225DNAArtificial SequenceBovine growth hormone polyA tail sequence 46ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgg 2254734DNAArtificial SequenceLinker sequence 47aagcttgaat tcagctgacg tgcctcggac cgct 3448141DNAArtificial SequenceITR 48aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120gagcgcgcag ctgcctgcag g 1414920PRTArtificial SequenceIL-2 signal sequence 49Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu1 5 10 15Val Thr Asn Ser 205021PRTArtificial SequenceT2A sequence 50Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu1 5 10 15Glu Asn Pro Gly Pro 20513814DNAArtificial SequencepITR.CBA.lucentis 51cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct gcggccgcac gcgtgacatt gattattgac tagttattaa tagtaatcaa 180ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 240atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 300ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt 360aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 420tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 480ctacttggca gtacatctac gtattagtca tcgctattac catgggtcga ggtgagcccc 540acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt 600attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggcgcg cgccaggcgg 660ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 720gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 780aaagcgaagc gcgcggcggg cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg 840cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg 900gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggctcgtttc 960ttttctgtgg ctgcgtgaaa gccttaaagg gctccgggag ggccctttgt gcggggggga 1020gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct 1080gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcgtgtgcgc 1140gaggggagcg cggccggggg cggtgccccg cggtgcgggg gggctgcgag gggaacaaag 1200gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc 1260tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg 1320gggctccgtg cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg 1380ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc 1440ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg 1500taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct 1560gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg 1620aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc 1680cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg 1740ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc 1800ttctttttcc tacagctcct gggcaacgtg ctggttattg tgaccggtgc caccatgtac 1860cggatgcagc tgctgagctg tatcgccctg tctctggccc tggtcaccaa ttctgaggtg 1920cagctggtgg aatctggcgg cggacttgtt caacctggcg gctctctgag actgagctgt 1980gccgcttctg gctacgactt cacccactac ggcatgaact gggtccgaca ggcccctggc 2040aaaggccttg aatgggtcgg atggatcaac acctacaccg gcgagccaac atacgccgcc 2100gacttcaagc ggagattcac cttcagcctg gacaccagca agagcaccgc ctacctgcag 2160atgaacagcc tgagagccga ggacaccgcc gtgtactact gcgccaagta tccctactac 2220tacggcacca gccactggta ctttgacgtg tggggacagg gcacactggt cacagtgtct 2280agcgcctcta caaagggccc cagcgttttc ccactggctc ctagcagcaa gtctaccagc 2340ggaggaacag ccgctctggg ctgtctggtc aaggactact ttcccgagcc tgtgaccgtg 2400tcctggaatt ctggcgctct gacaagcggc gtgcacacct ttccagctgt gctgcaaagc 2460agcggcctgt actctctgag cagcgtcgtg acagtgccaa gcagctctct gggcacccag 2520acctacatct gcaatgtgaa ccacaagcct agcaacacca aggtggacaa gaaggtggaa 2580cccaagagct gcgacaagac ccacaccggc aagcggaaga gaagaggctc tggcgaaggc 2640agaggcagcc tgcttacatg tggcgacgtg gaagagaacc ccggacctat gtatagaatg 2700cagctcctgt cctgcattgc cctgagcctg gctctcgtga ccaacagcga catccagctg 2760acacagagcc ccagcagcct gtctgcctct gtgggagaca gagtgaccat cacctgtagc 2820gccagccagg acatctccaa ctacctgaac tggtatcagc aaaagcccgg caaggcccct 2880aaggtgctga tctacttcac aagcagcctg cactccggcg tgcccagcag attttctggc 2940tctggcagcg gcaccgactt caccctgacc atatctagcc tgcagcctga ggacttcgcc 3000acctactact gccagcagta cagcaccgtg ccttggacat ttggccaggg cacaaaggtg 3060gaaatcaagc ggactgtggc cgctcctagc gtgttcatct ttccacctag cgacgagcag 3120ctgaagtctg gcacagcctc tgtcgtgtgc ctgctgaaca acttctaccc cagagaagcc 3180aaggtgcagt ggaaagtgga caatgccctg cagagcggca acagccaaga gagcgtgaca 3240gagcaggact ccaaggatag cacctatagc ctgagcagca ccctgacact gagcaaggcc 3300gactacgaga agcacaaagt gtacgcctgc gaagtgaccc accagggcct ttctagccct 3360gtgaccaaga gcttcaaccg gggcgaatgt taagagctcg ctgatcagcc tcgactgtgc 3420cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 3480gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 3540ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 3600acaatagcag gcatgctggg gatgcggtgg gctctatgga agcttgaatt cagctgacgt 3660gcctcggacc gctaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 3720tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc 3780tcagtgagcg agcgagcgcg cagctgcctg cagg 381452699DNAArtificial SequenceSequence encoding ranibizumab heavy chain 52gaggtgcagc tggtggaatc tggcggcgga cttgttcaac ctggcggctc tctgagactg 60agctgtgccg cttctggcta cgacttcacc cactacggca tgaactgggt ccgacaggcc 120cctggcaaag gccttgaatg ggtcggatgg atcaacacct acaccggcga gccaacatac 180gccgccgact tcaagcggag attcaccttc agcctggaca ccagcaagag caccgcctac 240ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc caagtatccc 300tactactacg gcaccagcca ctggtacttt gacgtgtggg gacagggcac actggtcaca 360gtgtctagcg cctctacaaa gggccccagc gttttcccac tggctcctag cagcaagtct 420accagcggag gaacagccgc tctgggctgt ctggtcaagg actactttcc cgagcctgtg 480accgtgtcct ggaattctgg cgctctgaca agcggcgtgc acacctttcc agctgtgctg 540caaagcagcg gcctgtactc tctgagcagc gtcgtgacag tgccaagcag ctctctgggc 600acccagacct acatctgcaa tgtgaaccac aagcctagca acaccaaggt ggacaagaag 660gtggaaccca agagctgcga caagacccac accggcaag 69953645DNAArtificial SequenceSequence encoding ranibizumab light chain 53gacatccagc tgacacagag ccccagcagc ctgtctgcct ctgtgggaga cagagtgacc 60atcacctgta gcgccagcca ggacatctcc aactacctga actggtatca gcaaaagccc 120ggcaaggccc ctaaggtgct gatctacttc acaagcagcc tgcactccgg cgtgcccagc 180agattttctg gctctggcag cggcaccgac ttcaccctga ccatatctag cctgcagcct 240gaggacttcg ccacctacta ctgccagcag tacagcaccg tgccttggac atttggccag 300ggcacaaagg tggaaatcaa gcggactgtg gccgctccta gcgtgttcat ctttccacct 360agcgacgagc agctgaagtc tggcacagcc tctgtcgtgt gcctgctgaa caacttctac 420cccagagaag ccaaggtgca gtggaaagtg gacaatgccc tgcagagcgg caacagccaa 480gagagcgtga cagagcagga ctccaaggat agcacctata gcctgagcag caccctgaca 540ctgagcaagg ccgactacga gaagcacaaa gtgtacgcct gcgaagtgac ccaccagggc 600ctttctagcc ctgtgaccaa gagcttcaac cggggcgaat gttaa 64554233PRTArtificial SequenceRanibizumab Heavy Chain 54Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp Lys Thr His Thr Gly Lys225 230554573DNAArtificial SequencepITR.CBA.Lucentis.tGFP 55cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct gcggccgcac gcgtgacatt gattattgac tagttattaa tagtaatcaa 180ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 240atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 300ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt 360aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 420tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 480ctacttggca gtacatctac gtattagtca tcgctattac catgggtcga ggtgagcccc 540acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt 600attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggcgcg cgccaggcgg 660ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 720gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 780aaagcgaagc gcgcggcggg cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg 840cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg 900gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggctcgtttc 960ttttctgtgg ctgcgtgaaa gccttaaagg gctccgggag ggccctttgt gcggggggga 1020gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct 1080gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcgtgtgcgc 1140gaggggagcg cggccggggg cggtgccccg cggtgcgggg gggctgcgag gggaacaaag 1200gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc 1260tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg 1320gggctccgtg cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg 1380ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc 1440ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg 1500taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct 1560gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg 1620aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc 1680cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg 1740ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc 1800ttctttttcc tacagctcct gggcaacgtg ctggttattg tgaccggtgc caccatgtac 1860cggatgcagc tgctgagctg tatcgccctg tctctggccc tggtcaccaa ttctgaggtg 1920cagctggtgg aatctggcgg cggacttgtt caacctggcg gctctctgag actgagctgt 1980gccgcttctg gctacgactt cacccactac ggcatgaact gggtccgaca ggcccctggc 2040aaaggccttg aatgggtcgg atggatcaac acctacaccg gcgagccaac atacgccgcc 2100gacttcaagc ggagattcac cttcagcctg gacaccagca agagcaccgc ctacctgcag 2160atgaacagcc tgagagccga ggacaccgcc gtgtactact gcgccaagta tccctactac 2220tacggcacca gccactggta ctttgacgtg tggggacagg gcacactggt cacagtgtct 2280agcgcctcta caaagggccc cagcgttttc ccactggctc ctagcagcaa gtctaccagc 2340ggaggaacag ccgctctggg ctgtctggtc aaggactact ttcccgagcc tgtgaccgtg 2400tcctggaatt ctggcgctct gacaagcggc gtgcacacct ttccagctgt gctgcaaagc 2460agcggcctgt actctctgag cagcgtcgtg acagtgccaa gcagctctct gggcacccag 2520acctacatct gcaatgtgaa ccacaagcct agcaacacca aggtggacaa gaaggtggaa 2580cccaagagct gcgacaagac ccacaccggc aagcggaaga gaagaggctc tggcgaaggc 2640agaggcagcc tgcttacatg tggcgacgtg gaagagaacc ccggacctat gtatagaatg 2700cagctcctgt cctgcattgc cctgagcctg gctctcgtga ccaacagcga catccagctg 2760acacagagcc ccagcagcct gtctgcctct gtgggagaca gagtgaccat cacctgtagc 2820gccagccagg acatctccaa ctacctgaac tggtatcagc aaaagcccgg caaggcccct 2880aaggtgctga tctacttcac aagcagcctg cactccggcg tgcccagcag attttctggc 2940tctggcagcg gcaccgactt caccctgacc atatctagcc tgcagcctga ggacttcgcc 3000acctactact gccagcagta cagcaccgtg ccttggacat ttggccaggg cacaaaggtg 3060gaaatcaagc ggactgtggc cgctcctagc gtgttcatct ttccacctag cgacgagcag 3120ctgaagtctg gcacagcctc tgtcgtgtgc ctgctgaaca acttctaccc cagagaagcc 3180aaggtgcagt ggaaagtgga caatgccctg cagagcggca acagccaaga gagcgtgaca 3240gagcaggact ccaaggatag cacctatagc ctgagcagca ccctgacact gagcaaggcc 3300gactacgaga agcacaaagt gtacgcctgc gaagtgaccc accagggcct ttctagccct 3360gtgaccaaga gcttcaaccg gggcgaatgt ggctccggag agggcagagg aagtctgcta 3420acatgcggtg acgtcgagga gaatcctggc ccaatggaga gcgacgagag cggcctgccc 3480gccatggaga tcgagtgccg catcaccggc accctgaacg gcgtggagtt cgagctggtg 3540ggcggcggag agggcacccc cgagcagggc cgcatgacca acaagatgaa gagcaccaaa 3600ggcgccctga ccttcagccc ctacctgctg agccacgtga tgggctacgg cttctaccac 3660ttcggcacct accccagcgg ctacgagaac cccttcctgc acgccatcaa caacggcggc 3720tacaccaaca cccgcatcga gaagtacgag gacggcggcg tgctgcacgt gagcttcagc 3780taccgctacg aggccggccg cgtgatcggc gacttcaagg tgatgggcac cggcttcccc 3840gaggacagcg tgatcttcac cgacaagatc atccgcagca acgccaccgt ggagcacctg 3900caccccatgg gcgataacga tctggatggc agcttcaccc gcaccttcag cctgcgcgac 3960ggcggctact acagctccgt ggtggacagc cacatgcact tcaagagcgc catccacccc 4020agcatcctgc agaacggggg ccccatgttc gccttccgcc gcgtggagga ggatcacagc 4080aacaccgagc tgggcatcgt ggagtaccag

cacgccttca agaccccgga tgcagatgcc 4140ggtgaagaat aagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 4200ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 4260cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 4320gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg 4380atgcggtggg ctctatggaa gcttgaattc agctgacgtg cctcggaccg ctaggaaccc 4440ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 4500ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 4560agctgcctgc agg 457356642DNAArtificial SequenceSequence encoding ranibizumab light chain 56gacatccagc tgacacagag ccccagcagc ctgtctgcct ctgtgggaga cagagtgacc 60atcacctgta gcgccagcca ggacatctcc aactacctga actggtatca gcaaaagccc 120ggcaaggccc ctaaggtgct gatctacttc acaagcagcc tgcactccgg cgtgcccagc 180agattttctg gctctggcag cggcaccgac ttcaccctga ccatatctag cctgcagcct 240gaggacttcg ccacctacta ctgccagcag tacagcaccg tgccttggac atttggccag 300ggcacaaagg tggaaatcaa gcggactgtg gccgctccta gcgtgttcat ctttccacct 360agcgacgagc agctgaagtc tggcacagcc tctgtcgtgt gcctgctgaa caacttctac 420cccagagaag ccaaggtgca gtggaaagtg gacaatgccc tgcagagcgg caacagccaa 480gagagcgtga cagagcagga ctccaaggat agcacctata gcctgagcag caccctgaca 540ctgagcaagg ccgactacga gaagcacaaa gtgtacgcct gcgaagtgac ccaccagggc 600ctttctagcc ctgtgaccaa gagcttcaac cggggcgaat gt 6425763DNAArtificial SequenceLinker sequence 57ggctccggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc 60cca 6358699DNAArtificial SequenceSequence encoding Turbo GFP 58atggagagcg acgagagcgg cctgcccgcc atggagatcg agtgccgcat caccggcacc 60ctgaacggcg tggagttcga gctggtgggc ggcggagagg gcacccccga gcagggccgc 120atgaccaaca agatgaagag caccaaaggc gccctgacct tcagccccta cctgctgagc 180cacgtgatgg gctacggctt ctaccacttc ggcacctacc ccagcggcta cgagaacccc 240ttcctgcacg ccatcaacaa cggcggctac accaacaccc gcatcgagaa gtacgaggac 300ggcggcgtgc tgcacgtgag cttcagctac cgctacgagg ccggccgcgt gatcggcgac 360ttcaaggtga tgggcaccgg cttccccgag gacagcgtga tcttcaccga caagatcatc 420cgcagcaacg ccaccgtgga gcacctgcac cccatgggcg ataacgatct ggatggcagc 480ttcacccgca ccttcagcct gcgcgacggc ggctactaca gctccgtggt ggacagccac 540atgcacttca agagcgccat ccaccccagc atcctgcaga acgggggccc catgttcgcc 600ttccgccgcg tggaggagga tcacagcaac accgagctgg gcatcgtgga gtaccagcac 660gccttcaaga ccccggatgc agatgccggt gaagaataa 69959232PRTArtificial SequenceTurbo GFP 59Met Glu Ser Asp Glu Ser Gly Leu Pro Ala Met Glu Ile Glu Cys Arg1 5 10 15Ile Thr Gly Thr Leu Asn Gly Val Glu Phe Glu Leu Val Gly Gly Gly 20 25 30Glu Gly Thr Pro Glu Gln Gly Arg Met Thr Asn Lys Met Lys Ser Thr 35 40 45Lys Gly Ala Leu Thr Phe Ser Pro Tyr Leu Leu Ser His Val Met Gly 50 55 60Tyr Gly Phe Tyr His Phe Gly Thr Tyr Pro Ser Gly Tyr Glu Asn Pro65 70 75 80Phe Leu His Ala Ile Asn Asn Gly Gly Tyr Thr Asn Thr Arg Ile Glu 85 90 95Lys Tyr Glu Asp Gly Gly Val Leu His Val Ser Phe Ser Tyr Arg Tyr 100 105 110Glu Ala Gly Arg Val Ile Gly Asp Phe Lys Val Met Gly Thr Gly Phe 115 120 125Pro Glu Asp Ser Val Ile Phe Thr Asp Lys Ile Ile Arg Ser Asn Ala 130 135 140Thr Val Glu His Leu His Pro Met Gly Asp Asn Asp Leu Asp Gly Ser145 150 155 160Phe Thr Arg Thr Phe Ser Leu Arg Asp Gly Gly Tyr Tyr Ser Ser Val 165 170 175Val Asp Ser His Met His Phe Lys Ser Ala Ile His Pro Ser Ile Leu 180 185 190Gln Asn Gly Gly Pro Met Phe Ala Phe Arg Arg Val Glu Glu Asp His 195 200 205Ser Asn Thr Glu Leu Gly Ile Val Glu Tyr Gln His Ala Phe Lys Thr 210 215 220Pro Asp Ala Asp Ala Gly Glu Glu225 230603631DNAArtificial SequencepITR.CBA.Eylea 60cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct gcggccgcac gcgtgacatt gattattgac tagttattaa tagtaatcaa 180ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 240atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 300ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt 360aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 420tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 480ctacttggca gtacatctac gtattagtca tcgctattac catgggtcga ggtgagcccc 540acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt 600attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggcgcg cgccaggcgg 660ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 720gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 780aaagcgaagc gcgcggcggg cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg 840cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg 900gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggctcgtttc 960ttttctgtgg ctgcgtgaaa gccttaaagg gctccgggag ggccctttgt gcggggggga 1020gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct 1080gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcgtgtgcgc 1140gaggggagcg cggccggggg cggtgccccg cggtgcgggg gggctgcgag gggaacaaag 1200gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc 1260tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg 1320gggctccgtg cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg 1380ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc 1440ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg 1500taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct 1560gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg 1620aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc 1680cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg 1740ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc 1800ttctttttcc tacagctcct gggcaacgtg ctggttattg tgaccggtgc caccatgtac 1860cggatgcagc tgctgagctg tatcgccctg tctctggccc tggtcaccaa ttctagcgat 1920accggcagac ccttcgtgga aatgtacagc gagatccccg agatcatcca catgaccgag 1980ggcagagagc tggtcatccc ctgcagagtg acaagcccca acatcaccgt gactctgaag 2040aagttccctc tggacacact gatccccgac ggcaagagaa tcatctggga cagccggaag 2100ggcttcatca tcagcaacgc cacctacaaa gagatcggcc tgctgacctg tgaagccacc 2160gtgaatggcc acctgtacaa gaccaactac ctgacacaca gacagaccaa caccatcatc 2220gacgtggtgc tgagccctag ccacggcatt gaactgtctg tgggcgagaa gctggtgctg 2280aactgtaccg ccagaaccga gctgaacgtg ggcatcgact tcaactggga gtaccccagc 2340agcaagcacc agcacaagaa actggtcaac cgggacctga aaacccagag cggcagcgag 2400atgaagaaat tcctgagcac cctgaccatc gacggcgtga ccagatctga ccagggcctg 2460tacacatgtg ccgccagctc tggcctgatg accaagaaaa acagcacctt cgtgcgggtg 2520cacgagaagg acaagaccca cacctgtcct ccatgtcctg ctccagaact gctcggcgga 2580ccttccgtgt tcctgtttcc tccaaagcct aaggacaccc tgatgatcag cagaacccct 2640gaagtgacct gcgtggtggt ggatgtgtcc cacgaggatc ccgaagtgaa gttcaattgg 2700tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ctagagagga acagtacaat 2760agcacctaca gagtggtgtc cgtgctgacc gtgctgcacc aggattggct gaacggcaaa 2820gagtacaagt gcaaggtgtc caacaaggcc ctgcctgctc ctatcgagaa aaccatctcc 2880aaggccaagg gccagcctag ggaaccccag gtttacacac tgcctccaag cagggacgag 2940ctgacaaaga accaggtgtc cctgacctgc ctggtcaagg gcttctaccc ttccgatatc 3000gccgtggaat gggagagcaa tggccagcct gagaacaact acaagacaac ccctcctgtg 3060ctggacagcg acggctcatt cttcctgtac agcaagctga cagtggacaa gagcagatgg 3120cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 3180cagaagtccc tgagcctgtc tcctggataa gagctcgctg atcagcctcg actgtgcctt 3240ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 3300ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 3360gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 3420atagcaggca tgctggggat gcggtgggct ctatggaagc ttgaattcag ctgacgtgcc 3480tcggaccgct aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 3540ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 3600gtgagcgagc gagcgcgcag ctgcctgcag g 3631611296DNAArtificial SequenceSequence encoding aflibercept 61agcgataccg gcagaccctt cgtggaaatg tacagcgaga tccccgagat catccacatg 60accgagggca gagagctggt catcccctgc agagtgacaa gccccaacat caccgtgact 120ctgaagaagt tccctctgga cacactgatc cccgacggca agagaatcat ctgggacagc 180cggaagggct tcatcatcag caacgccacc tacaaagaga tcggcctgct gacctgtgaa 240gccaccgtga atggccacct gtacaagacc aactacctga cacacagaca gaccaacacc 300atcatcgacg tggtgctgag ccctagccac ggcattgaac tgtctgtggg cgagaagctg 360gtgctgaact gtaccgccag aaccgagctg aacgtgggca tcgacttcaa ctgggagtac 420cccagcagca agcaccagca caagaaactg gtcaaccggg acctgaaaac ccagagcggc 480agcgagatga agaaattcct gagcaccctg accatcgacg gcgtgaccag atctgaccag 540ggcctgtaca catgtgccgc cagctctggc ctgatgacca agaaaaacag caccttcgtg 600cgggtgcacg agaaggacaa gacccacacc tgtcctccat gtcctgctcc agaactgctc 660ggcggacctt ccgtgttcct gtttcctcca aagcctaagg acaccctgat gatcagcaga 720acccctgaag tgacctgcgt ggtggtggat gtgtcccacg aggatcccga agtgaagttc 780aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcctag agaggaacag 840tacaatagca cctacagagt ggtgtccgtg ctgaccgtgc tgcaccagga ttggctgaac 900ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgctcctat cgagaaaacc 960atctccaagg ccaagggcca gcctagggaa ccccaggttt acacactgcc tccaagcagg 1020gacgagctga caaagaacca ggtgtccctg acctgcctgg tcaagggctt ctacccttcc 1080gatatcgccg tggaatggga gagcaatggc cagcctgaga acaactacaa gacaacccct 1140cctgtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 1200agatggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 1260tacacccaga agtccctgag cctgtctcct ggataa 1296

Claims

1. A method comprising introducing into an inner ear of a mammal a therapeutically effective amount of an adeno-associated virus (AAV) vector that comprises a nucleotide sequence encoding (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal peptide.

2. A method for increasing the level of an antibody or an antigen-binding antibody fragment in an inner ear of a mammal in need thereof, the method comprising: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide; wherein the introducing results in an increase in the level of the antibody or the antigen-binding antibody fragment in the inner ear of the mammal.

3. The method of claim 1 or 2, wherein the antibody or the antigen-binding antibody fragment binds specifically to vascular endothelial growth factor (VEGF).

4. The method of claim 3, wherein the antibody or antigen-binding antibody fragment decreases VEGF activity.

5. The method of any one of claims 1-4, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment.

6. The method of claim 5, wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.

7. The method of any one of claims 1-6, wherein the AAV vector further comprises a polyadenylation signal sequence.

8. The method of any one of claims 1-7, wherein the mammal is a human.

9. The method of any one of claims 1-8, wherein the mammal has been identified as having an inner ear disorder.

10. The method of any one of claims 1-8, wherein the mammal has been diagnosed as having an inner ear disorder.

11. The method of any one of claims 1-10, wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide.

12. The method of any one of claims 1-10, wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal.

13. A method for treating an inner ear disorder in a mammal in need thereof, the method comprising: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding: (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide; wherein the introducing results in the treatment of the inner ear disorder in the mammal.

14. The method of claim 13, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment.

15. The method of claim 14, wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.

16. The method of any one of claims 13-15, wherein the AAV vector further comprises a polyadenylation signal sequence.

17. The method of any one of claims 13-16, wherein the mammal is a human.

18. The method of any one of claims 13-17, wherein the mammal has been identified as having an inner ear disorder.

19. The method of any one of claims 13-17, wherein the mammal has been diagnosed as having an inner ear disorder.

20. The method of any one of claims 13-19, wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide.

21. The method of any one of claims 13-19, wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal.

22. A method of reducing VEGF activity in an inner ear of a mammal in need thereof, the method comprising: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide; wherein the polypeptide of (a) encodes an antibody that binds specifically to VEGF and reduces VEGF activity, the polypeptide of (b) encodes an antigen-binding antibody fragment that binds specifically to VEGF and reduces VEGF activity; wherein the introducing results in a reduction in VEGF activity in the inner ear of the mammal.

23. The method of claim 22, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment.

24. The method of claim 23, wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.

25. The method of any one of claims 22-24, wherein the AAV vector further comprises a polyadenylation signal sequence.

26. The method of any one of claims 22-25, wherein the mammal is a human.

27. The method of any one of claims 22-26, wherein the mammal has been identified or diagnosed as having an acoustic neuroma.

28. The method of any one of claims 22-26, wherein the mammal has been identified or diagnosed as having a vestibular schwannoma.

29. The method of any one of claims 22-26, wherein the mammal has been identified or diagnosed as having a neurofibromatosis type 2.

30. The method of any one of claims 22-29, wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide.

31. The method of any one of claims 22-29, wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal peptide.

32. A method of treating acoustic neuroma, vestibular schwannoma, or neurofibromatosis type 2 in an inner ear of a mammal, the method comprising: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide; or (b) a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide; wherein the polypeptide of (a) encodes an antibody that binds specifically to VEGF and reduces VEGF activity, the polypeptide of (b) encodes an antigen-binding antibody fragment that binds specifically to VEGF and reduces VEGF activity; wherein the introducing results in treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II, respectively, in the inner ear of the mammal.

33. The method of claim 32, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment.

34. The method of claim 33, wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.

35. The method of any one of claims 32-34, wherein the AAV vector further comprises a polyadenylation signal sequence.

36. The method of any one of claims 32-35, wherein the mammal is a human.

37. The method of any one of claims 32-36, wherein the mammal has been identified or diagnosed as having an acoustic neuroma.

38. The method of any one of claims 32-36, wherein the mammal has been identified or diagnosed as having a vestibular schwannoma.

39. The method of any one of claims 32-36, wherein the mammal has been identified or diagnosed as having neurofibromatosis type 2.

40. The method of any one of claims 32-39, wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide.

41. The method of any one of claims 32-40, wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal peptide.

42. The method of any one of claims 1-41, wherein the antibody comprises a Fc region that includes one or more amino acid substitutions that decreases the half-life of the antibody in a mammal as compared to a control antibody; or the antigen-binding antibody fragment thereof has a decreased in vivo half-life as compared to a control antigen-binding antibody fragment.

43. A method comprising introducing into an inner ear of a mammal a therapeutically effective amount of an adeno-associated virus (AAV) vector that comprises a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide.

44. A method for increasing the level of a soluble vascular endothelial growth factor (VEGF) receptor in an inner ear of a mammal in need thereof, the method comprising: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide; wherein the introducing results in an increase in the level of the soluble VEGF receptor in the inner ear of the mammal.

45. The method of claim 43 or 44, wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGF receptor-1 (VEGFR-1).

46. The method of claim 45, wherein the portion of the extracellular region of VEGFR-1 comprises a contiguous sequence from wildtype human VEGFR-1.

47. The method of claim 46, wherein the portion of the extracellular region of VEGFR-1 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1.

48. The method of claim 45, wherein the portion of the extracellular region of VEGFR-1 comprises a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-1.

49. The method of claim 43 or 44, wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGF receptor-2 (VEGFR-2).

50. The method of claim 49, wherein the portion of the extracellular region of VEGFR-2 comprises a contiguous sequence from wildtype human VEGFR-2.

51. The method of claim 50, wherein the portion of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2.

52. The method of claim 49, wherein the portion of the extracellular region of VEGFR-2 comprises a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-2.

53. The method of claim 43 or 44, wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGFR-1 and a portion of an extracellular region of VEGFR-2.

54. The method of claim 53, wherein: the portion of the extracellular region of VEGFR-1 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1; and the portion of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2.

55. The method of claim 54, wherein the soluble VEGF receptor is aflibercept.

56. The method of claim 43 or 44, wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGF receptor-3 (VEGFR-3).

57. The method of claim 56, wherein the portion of the extracellular region of VEGFR-3 comprises a contiguous sequence from wildtype human VEGFR-3.

58. The method of claim 57, wherein the portion of the extracellular region of VEGFR-3 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-3.

59. The method of claim 56, wherein the portion of the extracellular region of VEGFR-3 comprises a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-3.

60. The method of any one of claims 43-59, wherein the soluble VEGF receptor comprises a Fc domain.

61. The method of claim 60, wherein the Fc domain is an IgG1 Fc domain.

62. The method of claim 61, wherein the IgG1 Fc domain is a human wildtype IgG1 Fc domain.

63. The method of any one of claims 43-62, wherein the soluble VEGF receptor decreases the ability of a VEGF to bind to one or more of VEGFR-1, VEGFR-2, and VEGFR-3.

64. The method of any one of claims 43-63, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the soluble VEGF receptor.

65. The method of claim 64, wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.

66. The method of any one of claims 43-65, wherein the AAV vector further comprises a polyadenylation signal sequence.

67. The method of any one of claims 43-66, wherein the mammal is a human.

68. The method of any one of claims 43-67, wherein the mammal has been identified as having an inner ear disorder.

69. The method of any one of claims 43-67, wherein the mammal has been diagnosed as having an inner ear disorder.

70. A method for treating an inner ear disorder in a mammal in need thereof, the method comprising: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide; wherein the introducing results in the treatment of the inner ear disorder in the mammal.

71. The method of claim 70, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the soluble VEGF receptor.

72. The method of claim 71, wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.

73. The method of any one of claims 70-72, wherein the AAV vector further comprises a polyadenylation signal sequence.

74. The method of any one of claims 70-73, wherein the mammal is a human.

75. The method of any one of claims 70-74, wherein the mammal has been identified as having an inner ear disorder.

76. The method of any one of claims 70-74, wherein the mammal has been diagnosed as having an inner ear disorder.

77. A method of reducing a VEGF activity in an inner ear of a mammal in need thereof, the method comprising: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide; wherein the introducing results in a reduction in the VEGF activity in the inner ear of the mammal.

78. The method of claim 77, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the soluble VEGF receptor.

79. The method of claim 78, wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.

80. The method of any one of claims 77-79, wherein the AAV vector further comprises a polyadenylation signal sequence.

81. The method of any one of claims 77-80, wherein the mammal is a human.

82. The method of any one of claims 77-81, wherein the mammal has been identified or diagnosed as having an acoustic neuroma.

83. The method of any one of claims 77-81, wherein the mammal has been identified or diagnosed as having a vestibular schwannoma.

84. The method of any one of claims 77-81, wherein the mammal has been identified or diagnosed as having a neurofibromatosis type 2.

85. A method of treating acoustic neuroma, vestibular schwannoma, or neurofibromatosis type 2 in an inner ear of a mammal, the method comprising: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide; wherein the introducing results in treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II, respectively, in the inner ear of the mammal.

86. The method of claim 85, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the soluble VEGF receptor.

87. The method of claim 86, wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.

88. The method of any one of claims 85-87, wherein the AAV vector further comprises a polyadenylation signal sequence.

89. The method of any one of claims 85-88, wherein the mammal is a human.

90. The method of any one of claims 85-89, wherein the mammal has been identified or diagnosed as having an acoustic neuroma.

91. The method of any one of claims 85-89, wherein the mammal has been identified or diagnosed as having a vestibular schwannoma.

92. The method of any one of claims 85-89, wherein the mammal has been identified or diagnosed as having neurofibromatosis type 2.

93. The method of any one of claims 70-92, wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGF receptor-1 (VEGFR-1).

94. The method of claim 93, wherein the portion of the extracellular region of VEGFR-1 comprises a contiguous sequence from wildtype human VEGFR-1.

95. The method of claim 94, wherein the portion of the extracellular region of VEGFR-1 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1.

96. The method of claim 93, wherein the portion of the extracellular region of VEGFR-1 comprises a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-1.

97. The method of any one of claims 70-92, wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGF receptor-2 (VEGFR-2).

98. The method of claim 97, wherein the portion of the extracellular region of VEGFR-2 comprises a contiguous sequence from wildtype human VEGFR-2.

99. The method of claim 98, wherein the portion of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2.

100. The method of claim 97, wherein the portion of the extracellular region of VEGFR-2 comprises a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-2.

101. The method of any one of claims 70-92, wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGFR-1 and a portion of an extracellular region of VEGFR-2.

102. The method of claim 101, wherein: the portion of the extracellular region of VEGFR-1 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1; and the portion of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2.

103. The method of claim 102, wherein the soluble VEGF receptor is aflibercept.

104. The method of any one of claims 70-92, wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGF receptor-3 (VEGFR-3).

105. The method of claim 104, wherein the portion of the extracellular region of VEGFR-3 comprises a contiguous sequence from wildtype human VEGFR-3.

106. The method of claim 105, wherein the portion of the extracellular region of VEGFR-3 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-3.

107. The method of claim 104, wherein the portion of the extracellular region of VEGFR-3 comprises a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-3.

108. The method of any one of claims 70-107, wherein the soluble VEGF receptor comprises a Fc domain.

109. The method of claim 108, wherein the Fc domain is an IgG1 Fc domain.

110. The method of claim 109, wherein the IgG1 Fc domain is a human wildtype IgG1 Fc domain.

111. The method of any one of claims 70-110, wherein the soluble VEGF receptor decreases the ability of a VEGF to bind to one or more of VEGFR-1, VEGFR-2, and VEGFR-3.

112. The method of any one of claims 43-111, wherein the AAV vector further comprises a secretion sequence.

Images