Insulin-like growth factor-1 receptor (IGF1R) antibodies and related methods of treatment

Abstract

The present invention provides antibodies and antigen-binding fragments thereof configured to specifically bind to insulin-like growth factor-1 ("IGF1R"). Also provided are nucleic acid molecules encoding the antibodies and antigen-binding fragments thereof, pharmaceutical compositions, and therapeutic methods based on administration or expression of the anti-IGF1R antibodies and antigen-binding fragments described herein.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 671,695, filed July 15, 2024, and U.S. Provisional Application No. 63 / 585,198, filed September 25, 2023, the contents of which are incorporated herein by reference.

[0002] References to sequence lists The contents of the currently applied electronic sequence list (“037525-00589_SequenceListing.xml; size: 72,312 bytes; creation date: September 25, 2024”) are incorporated herein by reference in their entirety. Background Technology

[0003] Thyroid eye disease (TED), also known as thyroid-associated ophthalmopathy (TAO), Graves' ophthalmopathy or orbitopathy (GO), thyrotoxic exophthalmos, dysthyroid ophthalmopathy, and several other terms, is an orbital disorder associated with thyroid dysfunction. TAO is classified into two types. Active or acute TED typically lasts 1 to 3 years and is characterized by a persistent autoimmune / inflammatory response in the orbital soft tissues. Active TED causes expansion and remodeling of the ocular soft tissues. The autoimmune / inflammatory response of active TED eventually resolves spontaneously, and the condition transitions to inactive or chronic TED. Inactive TED is a term used to describe the long-term / permanent sequelae of active TED. In TED, inflammation and expansion of the orbital soft tissues (primarily the extraocular muscles and fat) force the eye forward (protrudes) out of its socket, a condition known as proptosis or exophthalmos. While most TED cases do not result in vision loss, the condition can lead to vision-threatening exposure keratopathy, troublesome diplopia (double vision), and compressive thyroid optic neuropathy.

[0004] The etiology of TED is currently unknown. However, antibodies against the associated activated insulin-like growth factor 1 receptor (“IGF1R”) have been detected in active TED. IGF1R is a widely expressed heterotetrameric protein involved in the regulation of proliferation and metabolic functions in many cell types. It is a two-subunit tyrosine kinase receptor. IGF1R-α contains a ligand-binding domain, while IGF1R-β is involved in signaling and contains a tyrosine phosphorylation site. Monoclonal antibodies against IGF1R have been developed and evaluated as a therapeutic strategy for several types of solid tumors and lymphomas.

[0005] Without being bound by any theoretical framework, it is believed that the thyroid-stimulating hormone (“TSH”) receptor (“TSHR”) and IGF1R form a physical and functional complex in orbital fibroblasts, and that blocking IGF1R appears to attenuate both IGF1-dependent and TSH-dependent signaling. It has been shown that blocking IGF1R with antibody antagonists reduces IGF-1-dependent and / or TSH-dependent signaling, thereby inhibiting the pathological activity of autoantibodies that act as agonists for either receptor.

[0006] The current standard of care for thyroid eye disease is teprotumumab (Tepezza). ® ), an IGF1R inhibitor indicated for the treatment of TED, is administered via intravenous (“IV”) infusion, with the first infusion at 10 mg / kg and subsequent infusions at 20 mg / kg every 3 weeks, for a total of 8 infusions over a 24-week period. These infusions are typically administered over 60 to 90 minutes and have caused Tepezza ® Systemic administration of IGF1R can lead to IGF1R inhibition in various parts of the body, which may result in adverse or unwanted effects (such as infusion reactions, exacerbation of irritable bowel disease, hyperglycemia, or hearing loss). Summary of the Invention

[0007] This article provides methods for treating or reducing the severity of TED using novel antibodies (or antigen-binding fragments thereof) that specifically bind to IGF1R and achieve specific therapeutic endpoints in TED treatment (e.g., reduction of exophthalmos, diplopia, TED clinical activity scores and subsets and individual measurements thereof) and improving the quality of life of TED patients. In some embodiments, these anti-IGF1R antibodies or antigen-binding fragments thereof are expressed by a virus or other vector administered to an individual in need of treatment. Such constructs may be designed, for example, to express single-chain variable fragments (“scFv”), i.e., recombinant antibody fragments containing light chain variable domains and heavy chain variable domains of immunoglobulins or portions thereof, in various configurations. In some embodiments, the virus or other vector locally (e.g., in one or more specific tissues or one or more specific cell types) expresses one or more anti-IGF1R antibodies or antigen-binding fragments. Thus, the novel gene therapies described herein allow for the localized expression of anti-IGF1R antibodies or antigen-binding fragments. In some embodiments, the anti-IGF1R antibodies or antigen-binding fragments described herein may be administered directly to at least one eye of an individual in need of treatment in the form of a pharmaceutical composition. In both cases where delivery is via a vector designed for local expression or direct administration (e.g., administration to the lacrimal gland), systemic administration, or at least clinically relevant systemic administration, can be avoided, thereby reducing or eliminating the need for systemic administration of Tepezza. ® (TED's current standard of care) associated with burdens and unwanted side effects.

[0008] In a first general aspect, this disclosure provides an antibody or antigen-binding fragment that specifically binds to the human IGF1R protein, comprising: a) a heavy chain variable domain comprising a CDR1 region having the polypeptide sequence of SEQ ID NO: 3 or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 3; a polypeptide sequence of SEQ ID NO: 4 or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 4; and / or a polypeptide sequence of SEQ ID NO: 5 or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 5; and / or b) a light chain variable domain comprising a polypeptide sequence of SEQ ID NO: 7 or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 3; 7. Having a CDR1 region with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity, having a polypeptide sequence of SEQ ID NO: 8 or a CDR2 region with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 8, and / or having a polypeptide sequence of SEQ ID NO: 9 or a CDR3 region with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 9.

[0009] In some embodiments, the heavy chain variable domain includes a CDR1 region having the polypeptide sequence of SEQ ID NO: 3, the heavy chain variable domain includes a CDR2 region having the polypeptide sequence of SEQ ID NO: 4, and / or the heavy chain variable domain includes a CDR3 region having the polypeptide sequence of SEQ ID NO: 5; and / or the light chain variable domain includes a CDR1 region having the polypeptide sequence of SEQ ID NO: 7, the light chain variable domain includes a CDR2 region having the polypeptide sequence of SEQ ID NO: 8, and / or the light chain variable domain includes a CDR3 region having the polypeptide sequence of SEQ ID NO: 9.

[0010] In some embodiments, the heavy chain variable domain includes a CDR1 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 3; the heavy chain variable domain includes a CDR2 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 4; and / or the heavy chain variable domain includes a CDR3 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 5; and / or the light chain variable domain includes a CDR1 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 7; the light chain variable domain includes a CDR2 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 7; the CDR2 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 3; and / or the heavy chain variable domain includes a CDR1 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 7; the heavy chain variable domain includes a CDR2 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 3 ...3 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: Compared to SEQ ID NO: 9, the polypeptide sequence has 1, 2, 3, 4 or 5 inserted, substituted or deleted polypeptide sequences; and / or the light chain variable domain includes a CDR3 region having 1, 2, 3, 4 or 5 inserted, substituted or deleted polypeptide sequences.

[0011] In some embodiments, the heavy chain variable domain comprises the polypeptide sequence of SEQ ID NO: 2; and / or the light chain variable domain comprises the polypeptide sequence of SEQ ID NO: 6. In some embodiments, the heavy chain variable domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 2; and / or the light chain variable domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 6. In some embodiments, the heavy chain variable domain comprises a polypeptide sequence having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 2; and / or the light chain variable domain comprises a polypeptide sequence having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 6.

[0012] In some embodiments, the antibody or antigen-binding fragment specifically binds to: a) an IGF1R protein having the polypeptide sequence of SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids; b) an α chain of an IGF1R protein having a polypeptide sequence comprising the sequence spanning positions 31-736 of SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids; and / or c) an β chain of an IGF1R protein having a polypeptide sequence comprising the sequence spanning positions 741-1367 of SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids.

[0013] In some embodiments, the antibody is a rabbit antibody or a mouse antibody.

[0014] In some embodiments, the antibody is a monoclonal antibody.

[0015] In some embodiments, the antibody comprises a monoclonal antibody, a transplanted antibody, a chimeric antibody, a human antibody, or a humanized antibody.

[0016] In some embodiments, the antigen-binding fragment includes Fab, Fab', F(ab')2, variable fragment (Fv), trifunctional antibody, tetrafunctional antibody, microantibody, bispecific F(ab')2, trispecific F(ab')2, bifunctional antibody, bispecific bifunctional antibody, single-chain variable fragment (scFv), scFv-Fc, Fab-Fc, VHH, or bispecific scFv.

[0017] In some embodiments, an antibody or antigen-binding fragment is operatively linked to a signal peptide. In some embodiments, the signal peptide is configured to induce the secretion of an antibody or antigen-binding fragment from human cells.

[0018] In a second general aspect, this disclosure provides a nucleic acid molecule encoding an antibody or antigen-binding fragment according to any embodiment described herein. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% congruence with a nucleotide sequence comprising SEQ ID NO: 21 or 22. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% congruence with SEQ ID NO: 21 and a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% congruence with SEQ ID NO: 22. In some embodiments, the nucleic acid molecule comprises SEQ ID NO: 21 and SEQ ID NO: 22. In some embodiments, the nucleic acid molecule comprises an optimized form of the codons of SEQ ID NO: 21 and / or SEQ ID NO: 22, or a fragment thereof.

[0019] In some embodiments, the nucleic acid molecule further includes a promoter or enhancer sequence operatively linked to a molecule encoding an antibody or antigen-binding fragment.

[0020] In some embodiments, the nucleic acid molecule further encodes a signal peptide sequence operatively linked to the encoded antibody or antigen-binding fragment.

[0021] In some embodiments, nucleic acid molecules may be contained within liposomes.

[0022] In a third general aspect, this disclosure provides a carrier that contains nucleic acid molecules according to any of the embodiments described herein.

[0023] In some embodiments, the vector comprises an adenovirus vector, an HIV vector, a lentiviral vector, an adeno-associated virus (“AAV”) vector, a plasmid vector, or a C-type vector. 3 DNA vector.

[0024] In some embodiments, the AAV carrier includes AAV2, AAV4, AAV8, or AAV9.

[0025] In some embodiments, the carrier may be contained within the liposome.

[0026] In a fourth general aspect, this disclosure provides a recombinant vector comprising an expression cartridge containing a polynucleotide encoding an antibody or antigen-binding fragment according to any embodiment described herein, the polynucleotide being operatively linked to a promoter. In some embodiments, the antibody or antigen-binding fragment is an scFv. In some embodiments, the scFv comprises: a heavy chain variable domain comprising a polypeptide sequence of SEQ ID NO: 2; and / or a light chain variable domain comprising a polypeptide sequence of SEQ ID NO: 6. In some embodiments, the scFv further comprises a linker sequence (e.g., a GS linker, such as SEQ ID NO: 42) between the heavy chain variable domain and the light chain variable domain. In other aspects, any other linker sequence may be used. In some embodiments, the heavy chain variable domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 2; and / or the light chain variable domain comprises a polypeptide sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 6. In some embodiments, the heavy chain variable domain comprises a polypeptide sequence having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 2; and / or the light chain variable domain comprises a polypeptide sequence having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 6. In some embodiments, the recombinant vector is a recombinant adeno-associated virus (“rAAV”) vector containing an AAV capsid.

[0027] In some embodiments, the scFv comprises: a) a heavy chain variable domain comprising a CDR1 region having a polypeptide sequence of SEQ ID NO: 3 or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 3; a polypeptide sequence of SEQ ID NO: 4 or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 4; and / or a polypeptide sequence of SEQ ID NO: 5 or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 5; and / or b) a light chain variable domain comprising a polypeptide sequence of SEQ ID NO: 7 or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 3; 7. Having a CDR1 region with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity, having a polypeptide sequence of SEQ ID NO: 8 or a CDR2 region with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 8, and / or having a polypeptide sequence of SEQ ID NO: 9 or a CDR3 region with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 9.

[0028] In some embodiments (e.g., when the recombinant vector is an rAAV vector), the expression cartridge may be side-attached by two inverted terminal repeat sequences (“ITRs”). In some embodiments, the ITR is an AAV2 ITR. In some embodiments, the AAV capsid contains a viral protein (“VP”) having at least 95%, 98%, or 100% identity with the AAV2 capsid protein (SEQ ID NO: 13), AAV5 capsid protein (SEQ ID NO: 15), AAV8 capsid protein (SEQ ID NO: 17), or AAV9 capsid protein (SEQ ID NO: 19). In some embodiments, the AAV capsid contains a VP having at least 95%, 98%, or 100% identity with the AAV9 capsid protein (SEQ ID NO: 19). In some embodiments, the AAV capsid contains a VP within a capsid for any other AAV serotype.

[0029] In a fifth general aspect, this disclosure provides a composition comprising any recombinant vector as described herein. In some embodiments of the composition, the recombinant vector is an rAAV vector, wherein the rAAV vector comprises: (a) an AAV2, AAV5, AAV8, and / or AAV9 shell, and (b) an expression cartridge, wherein the expression cartridge comprises a polynucleotide comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 100% homology with a nucleotide sequence comprising SEQ ID NO: 21 or 22, and wherein said polynucleotide is linked to a promoter.

[0030] In some embodiments, such as when the composition comprises an rAAV vector, the rAAV vector may comprise: (a) an AAV2, AAV5, AAV8, and / or AAV9 shell, and (b) an expression cartridge, wherein the expression cartridge contains a polynucleotide sequence having at least 80%, 85%, 90%, 95%, or 100% homology with SEQ ID NO: 21 or 22. In some embodiments, the AAV shell is AAV2, AAV5, AAV8, or AAV9.

[0031] In some embodiments, the composition according to this disclosure comprises an rAAV vector, wherein the rAAV vector comprises: (a) an AAV shell, and (b) an expression cartridge, wherein the expression cartridge comprises a polynucleotide encoding a polypeptide sequence having at least 80%, 85%, 90%, and 95% sequence identity with SEQ ID NO: 2 and / or 6, and wherein the polynucleotide is optionally linked to a promoter. In some embodiments, the polypeptide sequence comprises SEQ ID NO: 2 located at the N-terminus of SEQ ID NO: 6, while in other embodiments, SEQ ID NO: 6 is located at the N-terminus of SEQ ID NO: 2.

[0032] In a sixth general aspect, this disclosure provides pharmaceutical compositions comprising: any antibody or antigen-binding fragment described herein, any nucleic acid molecule described herein, any carrier described herein, or any composition described herein, and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises about 1 × 10 7 To approximately 1×10 14 One genome copy per milliliter of rAAV vector. In some embodiments, the composition comprises about 1 × 10⁻⁶ genome copies / mL rAAV vector. 12 To approximately 6.5 × 10 12 One genome copy per milliliter of rAAV vector.

[0033] In a seventh general aspect, this disclosure provides a kit comprising an antibody or antigen-binding fragment of any embodiment described herein, a carrier of any embodiment described herein, a composition of any embodiment described herein, or a pharmaceutical composition of any embodiment described herein, and a pharmaceutically acceptable carrier, along with instructions for use to treat an individual's ocular disease, condition, or symptom, the instructions including administration of an antibody, an antigen-binding fragment of an antibody, a carrier, a composition, or a pharmaceutical composition (e.g., topically or via injection) to the individual's eye. In some embodiments, the ocular disease, condition, or symptom is Graves' orbital lesion or TED.

[0034] In an eighth general aspect, this disclosure provides a method for treating an eye disease, condition, or symptom in an individual in need, the method comprising administering to the individual's eye an effective amount of: a) an antibody or antigen-binding fragment of any embodiment described herein, b) a carrier of any embodiment described herein, c) a composition of any embodiment described herein, or d) a pharmaceutical composition of any embodiment described herein. In some embodiments, administration to the individual's eye provides topical administration of an IGF1R antibody or antigen-binding fragment to the eye.

[0035] In some embodiments, the pharmaceutical composition is delivered to an accessory lacrimal gland. In some embodiments, the accessory lacrimal gland is a meibomian gland. In some embodiments, the pharmaceutical composition is delivered to the trabecular meshwork. In some embodiments, administration provides local application of an IGF1R antibody or antigen-binding fragment to the accessory lacrimal gland or the trabecular meshwork.

[0036] In some embodiments, application is performed by injection into the eye, accessory glands, or trabecular meshwork. In some embodiments, application is localized to the eye.

[0037] In some embodiments, the eye disease, condition, or symptom is TED. In some embodiments, the eye disease, condition, or symptom includes a) hypothyroidism-related visual impairment, b) decreased vision due to eyelid retraction, c) eyelid wiper epitheliopathy, d) dry eye, e) aqueous humor deficiency, f) diplopia, g) compressive optic neuropathy, h) eye pain, i) ptosis, j) and / or diplopia.

[0038] In some embodiments, an eye disease, condition, or symptom is caused by applying any of the carriers described herein to one or more tissues of an individual (e.g., at least one eye of the individual, or accessory lacrimal glands or trabecular meshwork). 8 Up to 10 14 Genome restoration therapy.

[0039] In some embodiments, the method causes: a) improvement in one or more symptoms of the ocular condition; and / or b) slowing of the progression of the ocular condition.

[0040] In some embodiments, the individual is a human individual.

[0041] In a ninth general aspect, this disclosure provides a recombinant cell transfected or transduced by a vector according to any of the embodiments described herein.

[0042] In a tenth general aspect, this disclosure provides a kit comprising a pharmaceutical composition of any embodiment described herein and instructions for use for treating a condition in a human individual, wherein the instructions for use include administering the pharmaceutical composition to the eye of the human individual. In some embodiments, this disclosure provides a pharmaceutical composition according to any embodiment described herein for manufacturing an agent for treating a condition in a human individual in need.

[0043] In an eleventh general aspect, this disclosure provides a method for treating a disease, condition, or symptom in an individual in need with a therapeutic antibody or antigen-binding fragment (e.g., scFv), wherein the method comprises administering to the individual an effective amount of a carrier containing a nucleic acid molecule encoding an antibody or antigen-binding fragment, and wherein the administration induces local expression of the antibody or antigen-binding fragment in a specific cell type or tissue of the individual. In some embodiments, the cell type or tissue is selected from: ocular cells and / or tissues, accessory lacrimal gland cells and / or tissues, trabecular meshwork cells and / or tissues. In some embodiments, the cell type or tissue is selected from: periorbital tissue, periorbital fat, scleral fibroblasts, conjunctival epithelial cells, and / or corneal stromal cells.

[0044] In some embodiments, administration of an effective amount of a carrier comprising a nucleic acid molecule encoding an antibody or antigen-binding fragment can be used to treat red eyes (e.g., caused by eyelid retraction) in individuals with TED. In some aspects, administration of an effective amount of a carrier comprising a nucleic acid molecule encoding an antibody or antigen-binding fragment can be used to treat compressive optic neuropathy (e.g., optic nerve compression due to external compressive forces from retrobulbar fat accumulation) in individuals with TED. Attached Figure Description

[0045] Figure 1 The vector map shows the rAAV plasmid vector (“pAAV-VH-VL-filled fragment-2”), which is configured to express and encapsulate scFv containing the heavy chain variable domain and light chain variable domain of one of the anti-IGF1R antibodies described herein.

[0046] Figures 2A to 2B This shows an example of delivering a viral vector to the lacrimal glands of a human individual.

[0047] Figure 3 Annotated photographs of the ELISA microplate show the results described in Example 2.

[0048] Figures 4 to 12 A graph containing binding data for the VL-VH and VH-VL scFv constructs and other analytes tested in the experiments described in Example 4.

[0049] Figures 13 to 14 Photographs of SDS-PAGE gels showing the purified VL-VH and VH-VL scFv constructs produced in the experiments described in Example 5.

[0050] Figures 15 to 23 A graph containing binding data for the purified VL-VH and VH-VL scFv constructs and other analytes tested in the experiments described in Example 5. Detailed Implementation

[0051] This disclosure provides novel antibodies and fragments thereof configured to specifically bind to IGF1R, as well as vectors, expression constructs, and formulations for use in gene therapy to treat GO and / or TED. Treatment may comprise transduction of human cells and / or tissues (e.g., exocrine glands, endocrine glands, adipose tissue, epithelial cells, endothelial cells, stromal cells, etc.) with an adenovirus-associated vector, adenovirus vector, lentiviral vector, and / or plasmid DNA encoding a transgenic gene of an scFv expressing an antibody against IGF1R, and appropriate promoters and / or enhancers, ITRs, and polyA tails to drive transgenic gene expression, thereby inducing protein translation. Additionally, a signal peptide (e.g., an IgK leader sequence) may be included in the transgenic construct to induce expression followed by secretion into cells. In some embodiments, the expression and secretion of the gene product (e.g., IGF1R-scFv) ultimately diffuses into the extraorbital fat pad, thereby blocking IGF-1 binding to IGF1R and inhibiting downstream IGF1R signaling. Specifically, IGF1R activation by IGF-1 induces AKT activation in orbital adipocytes and orbital fibroblasts. Activated AKT inhibits FOX01, subsequently activating the mTOR signaling cascade to induce PPARγ transcription factor activation, leading to hyaluronic acid (HA) expression and secretion, as well as adipogenesis. Increased HA and adipogenesis cause expansion of the posterior orbital fat pad, resulting in increased forces exerted on the posterior portion of the eyeball, causing the eyeball to move forward or "bulge." This embodiment will provide constitutive inhibition of IGF1R signaling and subsequent posterior orbital HA and fat expansion through, for example, the binding of the IGF1R-scFv gene product to the IGF1 receptor. Inhibition of HA production and adipogenesis will reduce the volume of posterior orbital adipose tissue, thereby causing the eyeball to retract into the orbit (i.e., reducing bulging).

[0052] definition

[0053] For ease of understanding of this disclosure, the following terms and abbreviations are defined as used herein:

[0054] As used herein, the term "antibody" encompasses all forms of antibodies, including but not limited to whole antibodies, monoclonal antibodies, antibody fragments, human antibodies, humanized antibodies, chimeric antibodies, and genetically engineered antibodies, as long as they retain characteristics such as specificity.

[0055] As used herein, the terms “antigen-binding fragment,” “antigen-binding antibody fragment,” “fragment,” and “antibody fragment” are used interchangeably to refer to any fragment comprising a portion of a full-length antibody and binding to IGF1R, generally at least the antigen-binding portion (e.g., a variable domain of the heavy or light chain) or its variable region (e.g., one or more CDRs) of a full-length antibody. Examples of antibody fragments include, but are not limited to, bifunctional antibodies, single-chain antibody molecules, multispecific antibodies, Fab, Fab', F(ab')2, Fv, or scFv. Furthermore, as used herein, the term “antibody” includes both the antibody and its antigen-binding fragment. Additionally, antibody fragments comprise single-chain polypeptides that possess the characteristics of a VH chain (i.e., capable of assembling with a VL chain to form a functional antigen-binding pocket) or a VL chain that binds to IGF1R (i.e., capable of assembling with a VH chain to form a functional antigen-binding pocket), thereby providing the property of inhibiting the binding of IGF-1 and / or IGF-2 to IGF1R.

[0056] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a formulation of an antibody molecule having a single amino acid composition. Therefore, the term "human monoclonal antibody" refers to an antibody exhibiting single-binding specificity, having variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is generated from a fusion tumor comprising B cells obtained from a self-transgenic non-human animal (e.g., a transgenic mouse) fused with an immortalized cell, having a genome containing human heavy chain transgenic genes and human light chain transgenic genes.

[0057] The term "expression cartridge" refers to a polynucleotide containing at least one polynucleotide sequence encoding a protein of interest (e.g., an anti-IGF1R antibody or its IGF1R binding fragment, such as the heavy chain, light chain, or scFv, or any other binding construct described herein). In some embodiments (e.g., in the case of an rAAV viral particle as the vector), the expression cartridge may further contain inverted terminal repeat (ITR) sequences flanking the at least one polynucleotide encoding the protein of interest.

[0058] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. As used herein, the term "humanized antibody" refers to an antibody whose framework or "complementarity-determining region" (CDR) has been modified to include a CDR of an immunoglobulin having a different specificity than that of its parent. In some embodiments, a murine CDR is grafted into the framework region of a human antibody to prepare a "humanized antibody".

[0059] As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from host cells, for example SP2-0, NSO, or CHO cells, or from animals transfected with human immunoglobulin genes (e.g., mice), or antibodies expressed using a recombinant expression vector transfected into host cells. These recombinant human antibodies possess variable and constant regions derived from human germline immunoglobulin sequences in rearranged form. The antigen-binding fragments and fusion proteins described herein can also be prepared recombinantly.

[0060] As used herein, the term "variable domain" (variable portions of the light chain (VL) and heavy chain (VH)) refers to each of the light and heavy chain pairs directly involved in antibody-antigen binding. The domains of the variable human light and heavy chains share the same general structure, and each domain contains four broadly conserved framework (FR) regions connected by three "hypervariable regions" (also known as complementarity-determining regions or "CDRs"). The framework regions adopt a β-lamellae configuration, and the CDRs can form loops connecting the β-lamellae structures. The CDRs in each chain maintain their three-dimensional structure through the framework regions and, together with CDRs from the other chain, form antigen-binding sites. The CDR3 regions of the antibody heavy and light chains play a crucial role in antibody binding specificity / affinity.

[0061] The terms “complementarity-determining region,” “CDR,” “hypervariant region,” or “antigen-binding portion of the antibody” are used interchangeably herein and refer to the amino acid residues in the antibody responsible for antigen binding. A hypervariant region contains amino acid residues derived from the complementarity-determining region or CDR. A “framework” or “FR” region refers to those variable domains other than the hypervariant residues as defined herein. Therefore, the light and heavy chains of the antibody contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and 1-R4 from the N-terminus to the C-terminus. In particular, CDR3 of the heavy chain is the region that contributes most significantly to antigen binding. CDR and FR regions can be determined, for example, according to the standard definition in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and / or those residues derived from the “hypervariant loop.”

[0062] As used herein, the term "fusion protein" refers to a protein or polypeptide comprising at least one first protein or polypeptide conjugated or linked to at least one second protein or polypeptide. In some aspects, at least one protein or polypeptide is conjugated or linked to at least one second protein or polypeptide via one or more linker sequences (e.g., intervening peptide sequences). Examples of fusion proteins are chimeric proteins. In some aspects, fusion proteins are scFv, bifunctional antibodies, trifunctional antibodies, or tetrafunctional antibodies. Fusion proteins can be produced using conventional techniques known in the art (e.g., recombinant DNA techniques) by conjugating or linking two or more genes that originally encode independent proteins. Thus, fusion proteins can comprise multimers of different or identical binding proteins expressed as a single linear polypeptide.

[0063] The terms "binding to IGF1R" or "specifically binding to IGF1R" are used interchangeably herein and refer to the binding of antibody to IGF1R in an in vitro assay, preferably in a binding assay where the antibody binds to a surface and the binding of IGF1R is measured using surface plasmon resonance (SPR). Binding refers to binding affinity (K... D ) is 10 -8 M or smaller, preferably 10 -13 Up to 10 -9 M. Binding to IGF1R can be studied using BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden). Binding affinity is determined by the terms ka (the antibody-antigen complex association rate constant), kd (dissociation constant), and K. D(kd / ka) definition. The antibodies used in the methods disclosed herein typically show approximately 10 -9 M or smaller K D .

[0064] The antibodies or antigen-binding fragments thereof used in the methods disclosed herein inhibit the binding of IGF-1 and / or IGF-2 to IGF1R. Inhibition is measured in the IC50 assay targeting the binding of IGF-1 / IGF-2 to IGF-IR on cells. 50 Measurements. Such measurements are known to those skilled in the art and described, for example, in U.S. Patent No. 7,579,157, the entire contents of which are incorporated herein by reference. The antibodies used in the methods disclosed herein target the IC50 of IGF-1 and IGF-2 binding to IGF1R. 50 Values ​​typically do not exceed 2 nM. IC 50 The value is measured as the average or median of at least three independent measurements. A single IC 50 The value may be excluded from the range.

[0065] As used herein, the term "inhibition of IGF-1 and / or IGF-2 binding to IGF1R" refers to the inhibition of IGF1R binding in vitro. 125 Labeled IGF-1 or IGF-2 binds to IGF-IR presented on the cell surface. Inhibition refers to an IC50 of 2 nM or lower. 50 value.

[0066] The phrase “therapeuticly effective” is intended to limit the amount of active ingredient used to treat a disease or condition or to achieve clinical indicators.

[0067] The term “therapeuticly acceptable” refers to a compound (or salt, prodrug, tautomer, zwitterionic form, etc.) that is suitable for contact with a patient’s tissues without adverse toxicity, irritation, or allergic reactions, is commensurate with a reasonable benefit / risk ratio, and is effective for its intended use.

[0068] As used herein, the intention to “treat” an individual or patient includes prevention, control, mitigation, improvement, and therapy. Treatment can also include disease prevention. Disease prevention can involve complete immunity from disease, such as in the prevention of pathogen infection, or it can involve preventing disease progression. For example, disease prevention may not mean completely eliminating any role related to the disease at any level, but can refer to preventing the symptoms of the disease at a clinically significant or detectable level. Disease prevention can also refer to preventing the progression of the disease to a later stage.

[0069] The terms “individual” and “patient” are used interchangeably herein to refer to all mammals, including humans. Instances of individuals include, but are not limited to, humans, monkeys, dogs, cats, horses, cattle, goats, sheep, pigs, and rabbits. In one embodiment, the individual or patient is a human.

[0070] The terms “affected by disease or symptom,” “suffering from disease or symptom,” or “having a disease or symptom” are used interchangeably herein and refer to an individual or patient suffering from any disease, symptom, syndrome, or condition. The use of one of these terms compared to the other does not imply an increase or decrease in the severity of the condition.

[0071] As used herein, the term “disease” is intended to be generally synonymous with and interchangeable with the terms “symptom,” “syndrome,” and “symptom” (as in medical symptom), because they all reflect an abnormal condition in which the normal function of a human or animal body or part thereof is impaired, usually manifested by prominent signs and symptoms, and resulting in a shortened lifespan or reduced quality of life for the human or animal.

[0072] When introducing elements of this disclosure or preferred embodiments thereof, the articles “a,” “an,” “the,” and “said” are intended to refer to the presence of one or more elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that other elements besides those listed may be present.

[0073] When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any or more of the listed items. For example, the expression "A and / or B" is intended to refer to either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" is intended to refer to A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0074] When a range of values ​​is disclosed, and the notation “n1…to n2” or “between n1…and n2” is used, where n1 and n2 are numbers, then unless otherwise specified, this notation is intended to include the numbers themselves and the range between them. This range may be integers or consecutive between the endpoints, and the endpoints are included. For example, the range “2 to 6 carbons” is intended to include two, three, four, five, and six carbons because carbon appears as an integer unit. For example, compared to the range “1 to 3 µM (micromoles)”, it is intended to include all values ​​between 1 µM, 3 µM, and accurate to any number of significant figures (e.g., 1.255 µM, 2.1 µM, 2.9999 µM, etc.).

[0075] The term "comprising" encompasses both "including" and "consisting of". For example, a composition "comprising" X may consist exclusively of X or may include something else, such as X+Y.

[0076] As used in this article, "thyroid ophthalmopathy" (TED), "thyroid-associated ophthalmopathy" (TAO), "thyroid-inflammatory ophthalmopathy (TIED)," "Greeves' ophthalmopathy" (GO), or "Greeves' orbital lesion" (GO) refer to the same condition or symptom and are used interchangeably. They all refer to inflammatory orbital lesions associated with some autoimmune thyroid diseases, most commonly Graves' disease (GD), but sometimes associated with other conditions such as Hashimoto's thyroiditis.

[0077] The terms “proptosis” and “exophthalmos” (also known as exophthalmus, exophthalmia, or exorbitism) refer to the forward protrusion, displacement, bulging, or extension of an organ. As used herein, the term refers to the forward protrusion, displacement, bulging, or extension of the eye socket. Some practitioners consider proptosis and exophthalmos to have the same meaning and are often used interchangeably, while others consider there to be subtle differences in their meaning. Some use exophthalmos to refer to severe proptosis or endocrine-related proptosis. Others use the term exophthalmos to describe proptosis associated with, for example, the eyes of individuals with TED (TAO or GO).

[0078] As used herein, the terms “pterygium” and “bulging eye” are used interchangeably and refer to the forward projection, displacement, protrusion, or extension of the eye from the orbit. Any increase in the soft tissue contents of the orbit, occurring laterally or posteriorly, due to the rigid bony structure of the orbit, which has only an anterior opening for expansion, will cause forward displacement of the eyeball. Pedrownium or bulging eye can result from several disease processes, including infection, inflammation, tumors, trauma, cancer metastasis, endocrine disorders, vascular diseases, and extraorbital lesions. TED (TAO or GO) is currently recognized as the most common cause of pterygium in adults. Bulging eye can be bilateral, as is common in TED (TAO or GO), or unilateral (as is common in orbital tumors).

[0079] The degree of eyeball protrusion can be measured using an exometry meter (an instrument used to measure the degree of forward displacement of the eye). This device allows for the measurement of the forward distance from the outer edge of the orbit to the anterior part of the cornea.

[0080] Computed tomography (CT) scans and magnetic resonance imaging (MRI) can also be used to assess the degree of eyeball protrusion or ptosis. CT scans are an excellent imaging diagnostic tool for diagnosing TED (TAO or GO). In addition to allowing observation of enlarged extraocular muscles, CT scans also provide surgeons or clinicians with a depiction of the orbital bony anatomy when orbital decompression is required. MRI provides excellent imaging of the orbital contents without the radiation exposure associated with CT scan studies due to its multiplanar and inherent contrast capabilities. MRI provides preferred imaging of the optic nerve, orbital fat, and extraocular muscles, but CT scans provide a preferred view of the orbital bony architecture.

[0081] Orbital ultrasound can also be used to diagnose and evaluate TED (TAO or GO) because it is rapid and highly reliable. It facilitates the assessment of extraocular muscle hyperreflectivity and enlargement, and continuous ultrasound examinations can also be used to assess the progression or stability of eye lesions.

[0082] Based on currently available or future available technologies, those skilled in the art will be able to determine the optimal diagnostic tools for diagnosing and assessing the degree of bulging eyes or eyeball protrusion.

[0083] While the generally accepted normal range for bulging eye is 12 to 21 mm, it is important to note that normal values ​​vary with age, sex, and race. For example, in normal adult white males, the average distance of eyeball protrusion is 16.5 mm, with an upper limit of 21.7 mm. In adult African Americans, the average is 18.2 mm, with an upper limit of 24.1 mm for males and 22.7 mm for females. In Mexican adults, the average is 15.2 mm for males and 14.8 mm for females; while in Iran, the average is 14.7 mm for the 20-70 age group. In Taiwanese adults, comparing normal individuals with those suffering from Graves' ophthalmopathy (TED), the average reading was 13.9 mm in the normal group and 18.3 mm in the TED group. Although the mean and upper limit of eyeball protrusion or bulging eye vary widely, it is generally accepted in the art that a difference greater than 2 mm between eyes is significant and abnormal.

[0084] Technicians in the field, such as ophthalmologists, surgeons, or other clinicians familiar with eye conditions and their treatment, will know the normal range for exophthalmos based on an individual's age, sex, and ethnicity, and will be able to diagnose or assess the presence of exophthalmos and track its progression.

[0085] Activity measurement or assessment

[0086] Several classification systems have been conceived to assess the clinical manifestations of TED (TAO or GO). In 1969, Werner reported the NOSPECS classification (no physical signs or symptoms, signs only, soft tissue involvement, exophthalmos, extraocular muscle signs, corneal involvement, and vision loss) (Werner, SC, American Journal of Ophthalmology, 1969, 68, Vol. 4, 646-648).

[0087] Werner also published an improved NOSPECS in 1977, which has since been widely used (Werner, SC, *American Journal of Ophthalmology*, 1977, 83, Vol. 5, 725-727). This classification grades clinical severity but does not provide a means of distinguishing between active TED (inflammatory progressive) and inactive TED (non-inflammatory stationary). Therefore, treatment indications were previously based solely on symptom severity, without considering whether the disease was active or inactive. In 1989, Mounts et al. described the Clinical Activity Score (CAS) (Mounts et al., *British Journal of Ophthalmology*, 1989, 73, Vol. 8, 639-644) as a way to assess the severity of active disease. This scoring system, based on typical signs of acute inflammation (pain, redness, swelling, and impaired function), was proposed as a clinical classification to easily distinguish between active and inactive diseases, and was revised in 1997 (Mounts et al., Clinical Endocrinology, 1997. 47, Vol. 1, 9-14). This scheme is further described below.

[0088] As used herein, the term CAS refers to the protocol for description and scoring as disclosed below. According to this protocol, one score is given for the presence of each parameter assessed in the following list. The sum of all scores defines clinical activity and provides the CAS. For patients on their first assessment, only items 1 through 7 are scored. A CAS ≥ 3 / 7 indicates active disease (TED). For patients on their second or subsequent assessments (usually 1 to 3 months later), items 8 through 10 are also scored; and a CAS ≥ 4 / 10 indicates active disease. A ten-item CAS scale also exists, but in clinical trials, a seven-item scale is generally used, as it is more suitable for longitudinal studies involving multiple assessments.

[0089] CAS consists of seven components:

[0090] 1. Spontaneous retroocular pain,

[0091] 2. Pain when attempting eye movements (upward, side-to-side, and downward gaze).

[0092] 3. Redness of the conjunctiva,

[0093] 4. Redness of the eyelids,

[0094] 5. Severe conjunctival swelling (conjunctival edema),

[0095] 6. Swelling of the lacrimal caruncle / folds, and

[0096] 7. Eyelid swelling.

[0097] Each component was scored as present (1 point) or absent (0 points). The score for each efficacy assessment was the sum of all present items; a range of 0 to 7 was obtained, where 0 or 1 was considered inactive disease and 7 was considered severe active ocular disease. A change of >2 points was considered clinically significant.

[0098] First, spontaneous orbital pain can be a feeling of pain or pressure on or behind the eyeball. This pain can be caused by increased intraocular pressure when the volume of orbital tissue increases due to excessive synthesis, fluid accumulation, and cell infiltration and proliferation of the extracellular matrix. Second, gaze-induced orbital pain can be pain in the eye when looking up, down, or sideways, or when attempting to look up, down, or sideways; that is, pain when moving the eyeball up, down, or sideways, or when attempting to gaze up, down, or sideways. This type of pain can be caused by stretching of inflamed muscles, especially when attempting to gaze upward. “Stretching pain” is not caused by pressing on the eyeball with a finger, while expected pressure pain is a characteristic expression of increased intraocular pressure. Both types of pain can be reduced after anti-inflammatory treatment. Therefore, these types of pain are considered to be directly related to autoimmune inflammation of the orbit and are therefore suitable for assessing TED activity.

[0099] Swelling in TED (TAO or GO) is considered as severe conjunctival swelling (conjunctival edema) and swelling of the caruncle and / or semilunar fold. Both are signs of active TED. Swelling of the eyelids may be caused by edema, fat prolapse through the orbital septum, or fibrotic degeneration. In addition to swelling, other symptoms indicating active TED include redness and / or pain of the conjunctiva, eyelids, caruncle, and / or semilunar fold.

[0100] Other grading systems have also been developed for evaluating TED (TAO or GO). VISA classification (visual acuity, inflammation, strabismus, and appearance) (Dolman, PJ and Rootman, J., Ophthalmic Plastic and Reconstructive Surgery, 2006, 22, Vol. 5, 319-324 and Dolman, PJ, Best Practice & Research Clinical Endocrinology & Metabolism, 2012, 26, Vol. 3, 229-248) and European Group of Graves' Orbitopathy (EUGOGO) classification (Bartalena, L. et al., European Journal of Endocrinology, 2008, 158, Vol. 3). 273-285) are two examples of this type. Both systems are based on the NO SPECS and CAS classifications and use indicators to assess the activity and severity of symptoms. More importantly, they allow clinicians to guide the treatment of GO patients. VISA is more commonly used in North America and Canada, while EUGOGO is more commonly used in Europe. Because the VISA and EUGOGO protocols are not interchangeable, only one should be used as a reference in a specific patient.

[0101] In some embodiments, improvements in one or more symptoms of TED can be determined using metrics and schemes known in the art. For example, improvements in any of the following symptoms may optionally be determined as follows:

[0102] (a) Protruding eye (sometimes called bulging eyeball): This can be determined or measured using an eyeball extrusion meter. Measurements are in millimeters and are consistent with eyelid retraction, Von Graefe's lid lag, and eyelid edema.

[0103] (b) Diplopia: Improvement can be determined or measured using eye trackers and / or MRI scans.

[0104] (c) Pain: Improvement can be determined or measured through patient assessment using a rating scale (e.g., the Eye Pain Assessment Survey, “OPAS”), the VRS Pain Scale, etc.

[0105] (d) Redness: Improvement can be determined or measured by clinical grading or color image analysis, for example using a 0-4 scale in 0.5 unit increments.

[0106] (e) Functional vision: Improvement can be determined or measured through visual function assessment via reading and / or simulators.

[0107] Quality of life (GO-QoL) in thyroid eye disease / Grevez's ophthalmopathy

[0108] In addition to bulging eyes (or protruding eyeballs) and CAS (conjunctivitis cerebral palsy), the Graves' ophthalmopathy quality of life (GO-QoL) questionnaire can be used to assess quality of life (QoL). This questionnaire is designed to determine improvements in quality of life following treatment. In some embodiments, the questionnaire can determine that, compared to glucocorticoid treatment, treatment with an antibody or antigen-binding fragment according to the methods disclosed herein results in fewer or no side effects.

[0109] The GO-QoL questionnaire has two self-assessment subscales. The first subscale assesses the impact of visual function on daily activities, while the second subscale assesses the impact on self-perceived appearance. Each subscale has eight questions that can be answered with: (i) Yes - very much; (ii) Yes - a little; or (iii) No - not at all. Each question is rated from 0 to 2, and the total raw score is then mathematically converted to a 0-100 scale, where 0 represents the greatest negative impact on quality of life and 100 represents no impact. A change of >8 points on the 0-100 scale is considered clinically significant. The pooled score is the raw score from both subscales converted back into a single 0-100 scale.

[0110] Severity measurement

[0111] For the eyelid opening, the distance between the eyelid margins (in mm) is measured when the patient is in a primary eye position, relaxed, and looking at a fixed distance.

[0112] For swelling of the eyelids, measure / assess as "absent / not obvious", "moderate" or "severe".

[0113] The assessment result for eyelid redness was either absent or present.

[0114] The assessment result for conjunctival redness was either absent or present.

[0115] The assessment result for conjunctival edema was either absent or present.

[0116] The assessment result for inflammation of the lacrimal caruncle or folds was either absent or present.

[0117] For individual patients, the same Hertel bulging meter and the same interocular distance were used to measure bulging of the eyeball, in millimeters.

[0118] Subjective diplopia is scored from 0 to 3 (0 = no diplopia; 1 = intermittent, i.e. diplopia occurs in the primary gaze position when tired or just waking up; 2 = non-continuous, i.e. diplopia occurs during excessive gaze; 3 = continuous, i.e. diplopia occurs continuously in the primary gaze position or reading gaze position).

[0119] For cases involving the eye muscles, turning is measured in degrees.

[0120] The assessment result for corneal involvement is: no / punctate or corneal lesions / ulcers.

[0121] For optic nerve involvement, i.e., optimal corrected visual acuity, color vision, optic disc, and relative pupillary afferent defects, the assessment result is either absent or present. Additionally, if optic nerve compression is suspected, visual field testing should be performed.

[0122] Severity classification

[0123] Vision-threatening thyroid eye disease: Patients with thyroid dysfunctional optic neuropathy (DON) and / or corneal rupture. This category requires immediate intervention.

[0124] Moderate to severe thyroid ophthalmopathy: Patients without vision-threatening conditions whose eye disease has an impact on their daily lives that justifies the risk of immunosuppression (if active) or surgical intervention (if inactive). Patients with moderate to severe thyroid ophthalmopathy typically present with one or more of the following: eyelid retraction ≥2 mm, moderate or severe soft tissue involvement, bulging of the eyeball exceeding the normal range for their race and sex by ≥3 mm, and non-persistent or persistent diplopia.

[0125] Mild thyroid eye disease: Thyroid eye disease characterized by only minor impacts on daily life, insufficient to justify immunosuppression or surgical treatment. These patients typically present with one or more of the following: mild eyelid retraction (<2 mm), mild soft tissue involvement, eyeball protrusion exceeding the normal range for their race and sex by <3 mm, transient diplopia or absence of diplopia, and corneal exposure responsive to lubricants.

[0126] Assessment of Gorman Grading of Diplopia

[0127] The Goleman assessment of subjective diplopia includes four categories: no diplopia (absence), diplopia occurring when the patient is tired or awake (intermittent), diplopia occurring during excessive gaze (non-persistent), and diplopia occurring continuously in the primary or reading position (persistent). Patients are scored according to the level of diplopia they experience. An improvement of ≥1 level is considered clinically significant.

[0128] Other tests that can be used to determine the efficacy of TED treatments can be found in U.S. Pre-Approval Publication No. 2019 / 0225696A1, which is incorporated herein by reference in its entirety.

[0129] IGF1R antibody and its antigen-binding fragment

[0130] The following provides sequences of the heavy and light chains of examples of antibodies that can be used in the methods disclosed herein, each antibody containing three CDRs on the heavy chain and three CDRs on the light chain. The sequences of the CDRs, heavy chain, light chain, and nucleic acid molecules encoding the CDRs, heavy chain, and light chain of the antibody are disclosed in the sequence listing. The CDRs of the antibody heavy chain may alternatively be referred to as CDRH1 (or HCDR1), CDRH2 (or HCDR2), and CDRH3 (or HCDR3), respectively. Similarly, the CDRs of the antibody light chain may alternatively be referred to as CDRL1 (or LCDR1), CDRL2 (or LCDR2), and CDRL3 (or LCDR3), respectively.

[0131] Mutant antibodies are also included within the scope of this disclosure. Therefore, variants of the sequences described in this application are also included within the scope of this disclosure. These variants include naturally occurring variants generated in vivo during an immune response or in vitro during the culture of immortal B cell clones due to somatic cell mutation. Alternatively, variants may arise due to the degeneracy of the genetic code or due to errors in transcription or translation.

[0132] Other variants of the antibody sequence with improved affinity and / or performance can be obtained using methods known in the art and are included within the scope of this disclosure. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can be used to improve translation efficiency in expression systems used to generate antibodies. Furthermore, polynucleotides comprising sequences optimized for antibody specificity or neutralizing activity by applying directed evolution methods to any nucleic acid sequence disclosed herein are also within the scope of this disclosure.

[0133] In one embodiment, the variant antibody sequence may have 70% or higher (i.e., 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher) amino acid sequence identity with the sequence described in this application. In some embodiments, the sequence identity is calculated relative to the full length of a reference sequence (i.e., the sequence described in this application). In some other embodiments, the identity percentage, as mentioned herein, is determined using BLAST version 2.1.3 with the default parameters [Blosum 62 matrix; gap opening penalty = 11 and gap expansion penalty = 1] specified by NCBI (National Center for Biotechnology Information; http: / / www.ncbi.nlmnih.gov / ).

[0134] The antibody or its antigen-binding fragment used with the methods disclosed herein may be any isotype (e.g., IgA, IgG, IgM; i.e., α, γ, or μ heavy chains). In one embodiment, the antibody is IgG. Within the IgG isotype, the antibody may be a subclass of IgG1, IgG2, IgG3, or IgG4. The antibody may have a κ or λ light chain.

[0135] Antibodies or antigen-binding antibody fragments used in conjunction with the methods disclosed herein may be administered by any means known to those skilled in the art.

[0136] In some embodiments, this disclosure provides an antibody or antigen-binding fragment that specifically binds to the human IGF1R protein, comprising a) a heavy chain variable domain, wherein the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence of SEQ ID NO: 3 or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 3, a polypeptide sequence of SEQ ID NO: 4 or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 4, and / or a polypeptide sequence of SEQ ID NO: 5 or having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 5.

[0137] In some embodiments, the antibody or antigen-binding antibody fragment comprises (alone or in addition to, for example, the heavy chain variable domain described above) a light chain variable domain comprising a polypeptide sequence having SEQ ID NO: 7 or a CDR1 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 7, a polypeptide sequence having SEQ ID NO: 8 or a CDR2 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 8, and / or a polypeptide sequence having SEQ ID NO: 9 or a CDR3 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 9.

[0138] In some aspects, the antibody or antigen-binding antibody fragment comprises: a heavy chain variable domain comprising a CDR1 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 3; a heavy chain variable domain comprising a CDR2 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 4; and / or a heavy chain variable domain comprising a CDR3 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 5; and / or a light chain variable domain comprising a CDR1 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 7; and a light chain variable domain comprising a CDR2 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 7. Compared to SEQ ID NO: 9, the polypeptide sequence has 1, 2, 3, 4 or 5 inserted, substituted or deleted polypeptide sequences; and / or includes a light chain variable domain containing a CDR3 region having 1, 2, 3, 4 or 5 inserted, substituted or deleted polypeptide sequences.

[0139] In some embodiments, the antigen-binding fragment may include Fab, Fab', F(ab')2, variable fragment (Fv), trifunctional antibody, tetrafunctional antibody, microantibody, bispecific F(ab')2, trispecific F(ab')2, bifunctional antibody, bispecific bifunctional antibody, single-chain variable fragment (scFv), scFv-Fc, Fab-Fc, nanobody (VHH), or bispecific scFv.

[0140] In some embodiments, an antibody or antigen-binding fragment according to any aspect described herein may comprise one or more putative CDR sequences shown in Table 1 (e.g., any selection or combination of these sequences). In some aspects, the antibody or antigen-binding fragment may comprise one or more CDR sequences, each having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with any sequence shown in Table 1. In some aspects, the antibody or antigen-binding fragment may comprise one or more CDR sequences having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions compared to any sequence shown in Table 1. For clarity, the antibody or antigen-binding fragment may comprise any selection or combination of the sequences shown in Table 1 (e.g., SEQ ID NO: 3; SEQ ID NO: 3, 36, and 39; or SEQ ID NO: 3, 5, 9, 36, 38, and 39), optionally having one or more substitutions, insertions, or deletions.

[0141]

[0142] Table 1 The putative anti-IGF1R CDR sequence can be incorporated into any antibody or its antigen-binding fragment described herein.

[0143] Nucleic acid molecules encoding IGF1R antibodies and their antigen-binding fragments

[0144] In some embodiments, the expression cartridge (e.g., an expression cartridge of any rAAV vector described herein) contains a polynucleotide encoding one or more anti-IGF1R antibodies or antigen-binding fragments thereof (e.g., capable of binding to human IGF1R protein) described herein. In some embodiments, the polynucleotide contains an ORF encoding any anti-IGF1R antibody or antigen-binding fragment described herein.

[0145] In some embodiments, the expression cartridge provides expression of one or more anti-IGF1R antibodies or antigen-binding fragments in at least one eye and / or lacrimal gland. In some embodiments, the expression cartridge induces expression of one or more anti-IGF1R antibodies or antigen-binding fragments in at least one meibomian gland. In some embodiments, the expression cartridge induces expression of one or more anti-IGF1R antibodies or antigen-binding fragments in the trabecular meshwork. In some embodiments, the expression cartridge induces expression of one or more anti-IGF1R antibodies or antigen-binding fragments in the retina. As used herein, “individual” means any mammal, including mice, rabbits, non-human primates (NHPs), and humans. In some embodiments, an individual is a human or an NHP. Furthermore, “individual” or “patient” may be used interchangeably with “subject”. In some embodiments, expression of one or more anti-IGF1R antibodies or antigen-binding fragments thereof in the treated eye is at any detectable level, while one or more anti-IGF1R antibodies or antigen-binding fragments thereof may not be expressed in an untreated individual or in the contralateral eye of a treated individual.

[0146] In some embodiments, the expression cartridge contains a polynucleotide encoding a protein capable of specifically binding to the human IGF1R protein. In some embodiments, the expression cartridge contains a polynucleotide that includes a nucleotide sequence encoding the amino acid sequence listed in Table 1. In some embodiments, the expression cartridge contains a polynucleotide that includes a nucleotide encoding the amino acid sequence identified in Table 1.

[0147] In some embodiments, the expression cartridge contains a polynucleotide that includes a nucleotide sequence encoding an antibody or antigen-binding fragment having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% congruence with any amino acid sequence shown in SEQ ID NO: 2-9 or 32-38. In some embodiments, the expression cartridge contains a polynucleotide that includes a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO: 2 or 6. In some embodiments, the expression cartridge contains a polynucleotide that includes a nucleotide sequence encoding a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% congruence with the amino acid sequence shown in SEQ ID NO: 2 or 6. In some embodiments, the expression cartridge contains a polynucleotide that includes a nucleotide sequence encoding an amino acid sequence shown in one or more of SEQ ID NO: 3-5, 7-9, or 34-40. In some embodiments, the expression cartridge contains a polynucleotide having a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21 or 22. In some embodiments, the expression cartridge contains a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 21. In some embodiments, the expression cartridge contains a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 22.

[0148] In some embodiments, the expression cartridge contains a polynucleotide containing a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment, wherein the anti-IGF1R antibody or antigen-binding fragment comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO:21 or 22.

[0149] The percentage of similarity can be determined using any suitable method known in the field. For example, a method for determining the percentage of similarity of a nucleic acid sequence is genome sequencing. Methods for determining the percentage of similarity with a nucleic acid sequence or amino acid sequence are known to those skilled in the art.

[0150] Codon Optimization

[0151] In some embodiments, the expression cartridge contains a polynucleotide encoding an anti-IGF1R antibody or antigen-binding fragment, wherein the polynucleotide comprises or consists of a nucleotide sequence codon-optimized for expression in target cells. In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cell is a human cell, a mouse cell, or a non-human primate (NHP) cell.

[0152] The codon optimization methods are known in the art and are applicable to achieving one or more desired results, such as increasing the expression of a synthetic gene in target cells. In some embodiments, the expression cartridge contains a nucleotide sequence that has been sequence-optimized relative to a reference sequence using sequence optimization methods. The sequence optimization methods are known in the art and include known sequence optimization tools, algorithms, and services. Non-limiting examples include services from GeneArt (LifeTechnologies), DNA2.0 (Menlo Park, CA), Geneious®, and GeneGPS® (Atum, Newark, CA).

[0153] In some embodiments, the expression cartridge of this disclosure comprises a polynucleotide encoding an anti-IGF1R antibody or antigen-binding fragment, which is sequence-optimized relative to a reference sequence using a sequence optimization method (e.g., GeneGPS®, Geneious®). In some embodiments, the reference sequence encoding an anti-IGF1R antibody or antigen-binding fragment is shown in SEQ ID NO: 21 or 22. In some embodiments, the expression cartridge comprises a polynucleotide encoding an anti-IGF1R antibody or antigen-binding fragment, which is sequence-optimized relative to a reference sequence selected from SEQ ID NO: 21 or 22. In some embodiments, the reference sequence encoding an anti-IGF1R antibody or antigen-binding fragment is shown in SEQ ID NO: 22. In some embodiments, the reference sequence encoding an anti-IGF1R antibody or antigen-binding fragment is shown in SEQ ID NO: 22.

[0154] In some embodiments, the sequence optimization method comprises the codon optimization algorithms described in US 7,561,972, US 7,561,973, US 8,126,653, and US 8,401,798, each of which is incorporated herein by reference in its entirety. In some embodiments, a polynucleotide sequence is sequence optimized relative to a reference sequence based on codon usage preferences in a host cell (e.g., mammalian cells, such as human cells, mouse cells, or non-human primate cells) using a sequence optimization method known in the art (e.g., GeneGPS®, Geneious®). In some embodiments, the polynucleotide sequence comprises or consists of a nucleotide sequence codon-optimized relative to a reference sequence using a sequence optimization method. In some embodiments, the polynucleotide sequence comprises or consists of a nucleotide sequence codon-optimized relative to a reference sequence for expression in human host cells.

[0155] Expression Card Box

[0156] The vector disclosed herein comprises an expression cartridge, which is a polynucleotide containing at least one polynucleotide sequence encoding a protein of interest (e.g., an anti-IGF1R antibody or an anti-IGF1R antibody-binding fragment, such as a heavy chain, light chain, or scFv, or any other binding construct described herein). In some embodiments, the expression cartridge encodes an anti-IGF1R antibody-binding fragment. In some embodiments, the expression cartridge encodes an anti-IGF1R antibody-binding fragment, which is scFv. In some embodiments, the expression cartridge contains other polynucleotide sequences, such as promoters, regulatory elements (e.g., one or more promoters), translation initiation sequences, coding sequences, and termination sequences.

[0157] In some embodiments, the expression cartridge of this disclosure comprises a polynucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment. In some embodiments, the expression cartridge provides expression of the anti-IGF1R antibody or antigen-binding fragment in at least one eye, lacrimal gland, and / or trabecular meshwork. In some embodiments, eyes, lacrimal glands, meibomian glands, and / or trabecular meshwork to which the vector described herein is applied may express IGF1R at a lower abundance compared to eyes, lacrimal glands, meibomian glands, and / or trabecular meshwork having only endogenous (i.e., natural) expression of IGF1R.

[0158] In some embodiments, the expression cartridges provided herein are bicistronic expression cartridges configured to allow the simultaneous expression of two anti-IGF1R antibodies and / or antigen-binding fragments (e.g., via incorporation into an internal ribosome entry site, “IRES”) using a single mRNA transcript. For example, a bicistronic expression cartridge may encode a protein comprising an anti-IGF1R antibody sequence selected from any sequence or embodiment described herein and an anti-IGF1R antigen-binding fragment sequence (e.g., scFv). In some embodiments, co-expression of two or more different anti-IGF1R antibodies or antigen-binding fragments in ocular cells may provide a synergistic therapeutic effect.

[0159] In some embodiments, the expression cartridge of this disclosure comprises a polynucleotide sequence encoding an anti-IGF1R scFv, said scFv comprising a light chain variable domain and a heavy chain variable domain of an immunoglobulin or a portion thereof. In some aspects, the light chain variable domain is located at the N-terminus of the heavy chain variable domain and optionally separated by a polynucleotide sequence encoding a linker sequence. In other aspects, the heavy chain variable domain is located at the N-terminus of the light chain variable domain and optionally separated by a polynucleotide sequence encoding a linker sequence. In some embodiments, the expression cartridge provides expression of scFv in at least one eye, lacrimal gland, and / or trabecular meshwork. In some embodiments, eyes, lacrimal glands, meibomian glands, and / or trabecular meshwork to which the vector described herein is applied may express IGF1R at a lower abundance compared to eyes, lacrimal glands, meibomian glands, and / or trabecular meshwork having only endogenous (i.e., natural) expression of IGF1R.

[0160] In some embodiments, the expression cartridges provided herein are bicistronic expression cartridges configured to allow simultaneous expression of two anti-IGF1R scFvs (e.g., via incorporation of IRES) using a single mRNA transcript, each scFv containing a light chain variable domain and / or a heavy chain variable domain of an immunoglobulin or a portion thereof. For example, the bicistronic expression cartridge may encode: a first protein containing an anti-IGF1R scFv, the scFv containing a heavy chain variable domain of an immunoglobulin located at the N-terminus of the light chain variable domain of the immunoglobulin, optionally separated by polynucleotide sequences encoding adapter sequences; and a second protein containing an anti-IGF1R scFv, the scFv containing a light chain variable domain of an immunoglobulin located at the N-terminus of the heavy chain variable domain of the immunoglobulin, optionally separated by polynucleotide sequences encoding adapter sequences. In some embodiments, co-expression of two or more different anti-IGF1R scFvs in ocular cells may provide a synergistic therapeutic effect.

[0161] In some embodiments, the expression cartridge of this disclosure includes a promoter. As used herein, the term "promoter" refers to a DNA sequence that guides the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., the minimal sequence sufficient to guide transcription. Promoter and corresponding protein or polypeptide expression may be ubiquitous, meaning they are highly active in a wide range of cells, tissues, and species, or cell-type specific, tissue-specific, or species-specific. A promoter may be "constitutive," meaning it has sustained activity; or "inducible," meaning it can be activated or deactivated by the presence or absence of biological or abiotic factors. The nucleic acid constructs or vectors of this disclosure also include enhancer sequences that may or may not be adjacent to the promoter sequence. The enhancer sequences influence promoter-dependent gene expression and may be located in the 5' or 3' region of the native gene.

[0162] Any suitable promoter region or promoter sequence therein can be used in the subject polynucleotide cartridge, provided that the promoter region promotes the expression of a polynucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment in at least one eye, lacrimal gland, at least one meibomian gland, and / or trabecular meshwork. In some embodiments, the promoter promotes the expression of the gene in a mammalian eye, lacrimal gland, meibomian gland, and / or trabecular meshwork. In some embodiments, the expression cartridge contains a cell-type-specific promoter. The promoter can specifically promote transcription in cells of the eye, lacrimal gland, meibomian gland, or trabecular meshwork. For example, in some embodiments, the promoter may include: a corneal stromal-specific promoter, such as a keratin-polysaccharide promoter (Carlson EC et al., "In Vivo Gene Delivery and Visualization of Corneal Stromal Cells Using an Adenoviral Vector and Keratocyte-Specific Promoter." *Investigative Ophthalmology & Visual Science*, July 2004, Vol. 45, 2194-2200); or a corneal epithelial / limbal stem cell-specific promoter, such as a keratin-12 or Pax-6 promoter (Wang, I. et al., "Cis-regulatory elements of the mouse Krt1.12 gene." *Molecular Vision*, 8, 94-101). (2002); Yoshihara, M. et al., “High-resolution promoter map of human limbal epithelial cells cultured with keratinocyte growth factor and rho kinase inhibitor”."Scientific Reports 7, 2845 (2017); trabecular meshwork cell-specific promoters, such as the chitinase 3-like 1 promoter (Liton PB et al., "Specific targeting of gene expression to a subset of human trabecular meshwork cells using the chitinase 3-like 1 promoter." Invest Ophthalmol Vis Sci. January 2005; 46(1):183-90); or iris cell-specific promoters, such as the phosphodiesterase 11A promoter (Janssen SF et al., "Gene expression and functional annotation of the human ciliary body epithelia." PLoS One.) 2012;7(9):e44973).

[0163] In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter comprises a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO: 10. In some embodiments, the promoter comprises SEQ ID NO: 10.

[0164] In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter comprises a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 25. In some embodiments, the promoter comprises or consists of SEQ ID NO: 25.

[0165] In some embodiments, the enhancer is a CMV enhancer. In some embodiments, the CMV enhancer comprises a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 26. In some embodiments, the enhancer comprises or consists of SEQ ID NO: 26.

[0166] In some embodiments, the expression cartridge contains a CMV enhancer and a promoter. In some embodiments, the CMV enhancer and promoter contain nucleotide sequences having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 27. In some embodiments, the enhancer and promoter comprise or consist of SEQ ID NO: 27.

[0167] Expression cartridges may contain poly(A) sequences. In some embodiments, the expression cartridges described herein contain a transcription termination signal. Elements that guide the efficient termination of heterologous nucleic acid transcripts and polyadenylation increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In some embodiments, the vector contains a polyadenylated sequence located at the 3' end of a polynucleotide encoding the polypeptide to be expressed. As used herein, the terms “polyA site” or “polyA sequence” refer to the DNA sequence that guides both termination and polyadenylation of nascent RNA transcripts via RNA polymerase II. Polyadenylated sequences can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, and thus promote increased translation efficiency. Cleavage and polyadenylation are guided by a poly(A) sequence in the RNA. The core poly(A) sequence of mammalian premRNA has two recognition elements flanking the cleavage-polyadenylation site. Typically, the nearly invariant AAUAAA hexamer is located 20–50 nucleotides upstream of a more variable element rich in U or GU residues. The cleavage of the nascent transcript occurs between these two elements and is associated with the addition of up to 250 adenosines to the 5' cleavage product. In some embodiments, the core poly(A) sequence is a desirable polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA). In some embodiments, the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGH polyA), a rabbit β-globulin polyA sequence (rβgpA), a variant thereof, or another suitable heterologous or endogenous polyA sequence known in the art. In some embodiments, the expression cartridge described herein contains a polyA sequence. In some embodiments, the polyA sequence is a BGH polyA sequence. In some embodiments, the BGH polyA sequence comprises or consists of a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 28. In some embodiments, the BGH polyA sequence comprises or consists of SEQ ID NO: 28.

[0168] In some embodiments, the rAAV vector of this disclosure comprises a marmot posttranscriptional regulatory element (WPRE). In some embodiments, the rAAV vector comprises a WPRE containing SEQ ID NO: 29.

[0169] Recombinant vector

[0170] In some embodiments, the expression cartridge is used to deliver an anti-IGF1R antibody or antigen-binding fragment to at least one eye and / or lacrimal gland of an individual, for example, to treat an eye condition. In some embodiments, the subject expression cartridge is used to deliver an anti-IGF1R antibody or antigen-binding fragment to at least one or more meibomian glands of an individual, for example, to treat an eye condition. Therefore, in some embodiments, the composition providing expression of an anti-IGF1R antibody or antigen-binding fragment in at least one eye (or any cell or tissue) of an individual is a gene delivery vector, wherein the gene delivery vector comprises the expression cartridge described herein.

[0171] In some embodiments, the gene delivery vector is an rAAV vector (e.g., an rAAV viral particle). In some embodiments, the expression cartridge has a functional AAV inverted terminal repeat (ITR) sequence attached to its 5' and 3' ends. A “functional AAV ITR sequence” means that the ITR sequence is intended to function for the recovery, replication, and encapsulation of the AAV vector. Therefore, the AAV ITR used in the gene delivery vector of this disclosure need not have a wild-type nucleotide sequence and can be altered by inserting, deleting, or substituting nucleotides, or the AAV ITR can be derived from any of several AAV serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In some embodiments, the AAV ITR is derived from AAV1. In some embodiments, the AAV ITR is derived from AAV2. In some embodiments, the AAV ITR is derived from AAV3. In some embodiments, the AAV ITR is derived from AAV4. In some embodiments, the AAV ITR is derived from AAV5. In some embodiments, the AAV ITR is derived from AAV6. In some embodiments, the AAV ITR is derived from AAV7. In some embodiments, the AAV ITR is derived from AAV8. In some embodiments, the AAV ITR is derived from AAV9. In some embodiments, the AAV ITR is derived from AAV10. In some embodiments, the rAAV vector has a completely or partially deleted wild-type rep and / or cap gene. In some embodiments, the rAAV vector retains a functional side-linked ITR sequence. In some embodiments, the AAV ITR is... Table 2 One of the ones identified in the text.

[0172]

[0173] Table 2 Exemplary ITR sequence

[0174] In some embodiments, the rAAV vector comprises an AAV capsid derived from any adeno-associated virus serotype known or anticipated to be found in the art, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc. For example, the AAV capsid may be a wild-type (or “natural”) capsid. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV1. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV2. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV3. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV4. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV5. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV6. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV7. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV8. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV9. In some embodiments, the rAAV vector comprises an AAV capsid derived from AAV10. Commonly recognized AAV capsids include AAV2, AAV5, AAV8, and AAV9 (see Table 3). However, like ITRs, the polynucleotide encoding the capsid does not necessarily have a wild-type nucleotide sequence, but can be altered by inserting, deleting, or substituting nucleotides in the VP1, VP2, or VP3 sequences, as long as the polynucleotide encoding the capsid can transduce cells of the eye and / or lacrimal gland. In other words, the AAV capsid can be a variant AAV capsid. In some embodiments, the rAAV vector is a “pseudotyped” AAV vector generated using a capsid gene of one AAV serotype and a rep gene from a different AAV serotype, for example, a pseudotyped AAV2 generated using a rep from AAV2 and a cap from AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. For example, the rAAV vector may be rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8, rAAV2 / 9, etc., wherein the "X / Y" tag at the end of each of the aforementioned rAAVs indicates the serotype (X) of the rep protein used to generate the respective rAAV and the serotype (Y) of the capsid. In some embodiments, rAAV is rAAV2 / 1. In some embodiments, rAAV is rAAV2 / 3. In some embodiments, rAAV is rAAV2 / 4. In some embodiments, rAAV is rAAV2 / 5. In some embodiments, rAAV is rAAV2 / 6. In some embodiments, rAAV is rAAV2 / 7.In some embodiments, rAAV is rAAV2 / 8. In some embodiments, rAAV is rAAV2 / 9. In some aspects, the ITR may also be selected from a specific serotype (e.g., an ITR from AAV2, AAV5, or any other AAV may be incorporated into any plasmid or other vector described herein).

[0175] In some embodiments, rAAV is replication-deficient because the rAAV vector cannot independently replicate further and encapsulate its genome. For example, in such replication-deficient embodiments, cells from the eye and / or lacrimal gland of an individual are transduced with a replication-deficient rAAV vector configured to express anti-IGF1R antibody or IGF1R antigen-binding fragment transfection genes in the transduced tissue; however, because the rAAV vector lacks the AAV rep and cap genes, as well as accessory function genes, the rAAV cannot replicate.

[0176] In some embodiments, the rAAV vector of this disclosure encapsulating an expression cartridge as described herein can be manufactured using helper-free production. rAAV is typically manufactured by encapsulating an infectious rAAV vector in a host cell using components from a live helper virus (e.g., adenovirus). The rAAV helper-free production system allows the manufacture of infectious rAAV vectors without the use of a live helper virus. In the helper-free system, the host encapsulation cell line is co-transfected with three plasmids. The first plasmid contains adenovirus genes (i.e., E2A, E4, and VA RNA genes) encoding the products required for encapsulating the rAAV vector. The second plasmid contains the desired AAV genes (i.e., the rep and cap genes). The third plasmid contains an expression cartridge, such as a multinucleotide sequence encoding an anti-IGF1R antibody or an IGF1R antigen-binding fragment, and a promoter, side-attached by an ITR. The host encapsulation cell line may be, for example, AAV-293 host cells. Suitable host cells contain additional components required for encapsulating the infectious rAAV vector, not supplied by the plasmids. In some embodiments, the cap gene may encapsulate any AAV capsid protein, such as that described herein. In some embodiments, the promoter is a promoter sequence as described herein. In some embodiments, the promoter sequence is a CAG sequence. In some embodiments, the polynucleotide encodes an anti-IGF1R antibody or IGF1R antigen binding fragment having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology with SEQ ID NO: 2 or 6. In some embodiments, the polynucleotide encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology with one or more of the CDRs identified as SEQ ID NO: 3-5, 7-9, and / or 31-37.

[0177] AAV serotypes that indicate infection of the eyes and / or lacrimal glands include AAV2, AAV5, AAV5w8, and AAV9 (Rocha et al., above). Exemplary amino acid and nucleotide sequences of several AAV capsid proteins are identified in Table 3.

[0178] In some embodiments, the AAV shell protein shares at least 95%, 98%, or 100% identity with the AAV2 VP1 protein (SEQ ID NO: 11). In some embodiments, the polynucleotide sequence encoding the AAV2 VP1 protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 12. In some embodiments, the AAV shell protein shares at least 95%, 98%, or 100% identity with the AAV2 VP3 protein (SEQ ID NO: 13). In some embodiments, the polynucleotide sequence encoding the AAV2 VP3 protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 14. In some embodiments, the AAV shell protein shares at least 95%, 98%, or 100% identity with the AAV5 shell protein (SEQ ID NO: 15). In some embodiments, the polynucleotide sequence encoding the AAV5 shell protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 16. In some embodiments, the AAV capsid protein shares at least 95%, 98%, or 100% identity with the AAV8 capsid protein (SEQ ID NO: 17). In some embodiments, the polynucleotide sequence encoding the AAV8 capsid protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 18. In some embodiments, the AAV capsid protein shares at least 95%, 98%, or 100% identity with the AAV9 capsid protein (SEQ ID NO: 19). In some embodiments, the polynucleotide sequence encoding the AAV9 capsid protein shares at least 95%, 98%, or 100% identity with SEQ ID NO: 20.

[0179]

[0180]

[0181] Table 3 AAV shell sequence

[0182] Exemplary rAAV vector

[0183] In some embodiments, the rAAV vector comprises an AAV shell. In some embodiments, the rAAV vector described herein comprises an expression cartridge containing a polynucleotide containing a nucleotide sequence encoding an anti-IGF1R antibody or an antigen-binding fragment. In some embodiments, the rAAV vector described herein comprises an expression cartridge containing a polynucleotide containing a nucleotide sequence encoding an anti-IGF1R scFv (e.g., containing SEQ ID NO: 2 and / or 6). In some embodiments, the rAAV vector described herein comprises a bicistronic expression cartridge containing a polynucleotide containing a nucleotide sequence encoding two anti-IGF1R antibodies, two anti-IGF1R antigen-binding fragments, or one anti-IGF1R antibody and one antigen-binding fragment. In some embodiments, the polynucleotide is operatively linked to a promoter. In some embodiments, the rAAV vector comprises an AAV shell and an expression cartridge, wherein the expression cartridge contains a polynucleotide operatively linked to a promoter, wherein the polynucleotide contains a nucleotide sequence encoding an anti-IGF1R antibody or an antigen-binding fragment. In some embodiments, the promoter is any promoter described herein or known in the art. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the expression cartridge further comprises a 5' ITR and / or a 3' ITR. In some embodiments, the 5' ITR is an AAV2 5' ITR. In some embodiments, the 3' ITR is an AAV2 3' ITR. In some embodiments, the expression cartridge comprises the following nucleotide sequences from 5' to 3': a 5' ITR (e.g., AAV2 5' ITR), a promoter (e.g., a CMV promoter), a 5' untranslated region (5' UTR), a polynucleotide sequence comprising a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment, a polyA sequence, and a 3' ITR (e.g., AAV2 3' ITR). In some embodiments, the expression cartridge comprises the following nucleotide sequences from 5' to 3': a 5' ITR (e.g., AAV2 5' ITR), an enhancer (e.g., a CMV enhancer), a promoter (e.g., a CMV promoter), a 5' UTR, a polynucleotide sequence comprising a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment, a WPRE sequence, a polyA sequence, and a 3' ITR (e.g., AAV2 3' ITR). In some embodiments, the 5' UTR includes a Kozak sequence located immediately upstream and adjacent to the start codon in the nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment.

[0184] In some embodiments, the rAAV vector comprises an AAV shell and an expression cartridge, the expression cartridge containing a polynucleotide operatively linked to a promoter, wherein the polynucleotide contains a nucleotide sequence encoding a humanized anti-IGF1R antibody or antigen-binding fragment as described herein. In some embodiments, the rAAV vector comprises an AAV shell and an expression cartridge, the expression cartridge containing a polynucleotide operatively linked to a CAG promoter or a CMV promoter, wherein the polynucleotide contains a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment as described herein. In some embodiments, the polynucleotide from 5' to 3' comprises (i) a nucleotide sequence encoding a signal peptide, and (ii) a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment as described herein.

[0185] In some embodiments, the rAAV vector comprises an AAV shell and an expression cartridge containing a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide, from 5' to 3', comprises (i) a nucleotide sequence encoding a signal peptide and (ii) a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment, wherein the anti-IGF1R antibody or antigen-binding fragment comprises or is composed of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO: 2 and / or 6. In some embodiments, the anti-IGF1R antibody or antigen-binding fragment comprises or is composed of the amino acid sequence shown in SEQ ID NO: 2 or 6.

[0186] In some embodiments, the rAAV vector comprises an AAV2 shell and an expression cartridge, the expression cartridge containing a polynucleotide operatively linked to a promoter, wherein the polynucleotide contains a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment as described herein. In some embodiments, the rAAV vector comprises an AAV2 shell and an expression cartridge, the expression cartridge containing a polynucleotide operatively linked to a CAG promoter or a CMV promoter, wherein the polynucleotide contains, from 5' to 3', (i) a nucleotide sequence encoding a signal peptide and (ii) a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment as described herein.

[0187] In some embodiments, the rAAV vector comprises an AAV2 shell and an expression cartridge containing a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide, from 5' to 3', comprises (i) a nucleotide sequence encoding a signal peptide and (ii) a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment, wherein the anti-IGF1R antibody or antigen-binding fragment comprises or is composed of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO: 2 or 6. In some embodiments, the anti-IGF1R antibody or antigen-binding fragment comprises or is composed of the amino acid sequences shown in or SEQ ID NO: 3-5, 7-9, or 34-40. In some embodiments, the rAAV vector comprises an AAV2 shell and an expression cartridge containing a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide from 5' to 3' comprises (i) a nucleotide sequence encoding a signal peptide and (ii) a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment, wherein the anti-IGF1R antibody or antigen-binding fragment comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO: 2 or 6.

[0188] In some embodiments, the rAAV vector comprises an AAV9 shell and an expression cartridge containing a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide, from 5' to 3', comprises (i) a nucleotide sequence encoding a signal peptide and (ii) a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment, wherein the anti-IGF1R antibody or antigen-binding fragment comprises or is composed of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO: 2 or 6. In some embodiments, the anti-IGF1R antibody or antigen-binding fragment comprises or is composed of the amino acid sequences shown in or SEQ ID NO: 3-5, 7-9, or 34-40. In some embodiments, the rAAV vector comprises an AAV9 shell and an expression cartridge containing a polynucleotide operatively linked to a promoter (e.g., a CAG promoter or a CMV promoter), wherein the polynucleotide from 5' to 3' comprises (i) a nucleotide sequence encoding a signal peptide and (ii) a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment, wherein the anti-IGF1R antibody or antigen-binding fragment comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO: 2 and / or 6.

[0189] In some embodiments, the rAAV vector may comprise a nucleotide sequence encoding anti-IGF1R scFv, wherein the anti-IGF1R scFv comprises a heavy chain variable domain and a light chain variable domain of an immunoglobulin. In some embodiments, the heavy chain variable domain comprises or consists of an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2; and / or the light chain variable domain comprises or consists of an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the heavy chain variable domain is located at the 5' of the nucleotide sequence encoding the light chain variable domain; in other embodiments, this orientation may be reversed. In some embodiments, the anti-IGF1R scFv may further comprise a nucleotide sequence encoding a signal peptide located at the 5' of the nucleotide sequence encoding either the heavy chain variable domain or the light chain variable domain. In some embodiments, the rAAV vector comprises an AAV2, AAV9 shell, or a shell derived from any other AAV serotype.

[0190] In some embodiments, the promoter is any promoter described herein or known in the art. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the expression cartridge further comprises a 5' ITR and / or a 3' ITR. In some embodiments, the 5' ITR is an AAV2 5' ITR. In some embodiments, the 3' ITR is an AAV2 3' ITR. In some embodiments, the expression cartridge comprises, from 5' to 3', the following nucleotide sequences: a 5' ITR (e.g., AAV2 5' ITR), a promoter (e.g., a CMV promoter), a 5' untranslated region (5' UTR), a polynucleotide sequence comprising a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment, a polyA sequence, and a 3' ITR (e.g., AAV2 3' ITR). In some embodiments, the expression cartridge comprises the following nucleotide sequences from 5' to 3': a 5' ITR (e.g., AAV2 5' ITR), an enhancer (e.g., a CMV enhancer), a promoter (e.g., a CMV promoter), a 5' UTR, a polynucleotide sequence comprising a nucleotide sequence encoding an anti-IGF1R antibody or antigen-binding fragment, a WPRE sequence, a polyA sequence, and a 3' ITR (e.g., AAV2 3' ITR). In some embodiments, the 5' UTR comprises a Kozak sequence located immediately upstream and adjacent to the start codon in the nucleotide sequence encoding the anti-IGF1R antibody or antigen-binding fragment.

[0191] How to use

[0192] The methods and compositions described herein can be used to treat ocular conditions and reduce associated symptoms of ocular conditions. The terms “treatment”, “treating,” etc., are generally used herein to refer to achieving the desired pharmacological and / or physiological effects. These effects may be preventative in terms of complete or partial prevention of the disease or its symptoms, such as reducing the likelihood of an individual developing the disease or its symptoms, delaying the onset or progression of the disease or its symptoms in an individual; and / or therapeutic in terms of partial or complete cure of the disease and / or adverse effects attributable to the disease. As used herein, “treatment” encompasses any treatment of a disease in mammals, including but not limited to: (a) inhibiting the progression of the disease; (b) alleviating, reducing, or decreasing one or more symptoms of the disease or their increase; (c) alleviating, reducing, or decreasing one or more signs of the disease or their increase; and (d) inducing disease remission. Therapeutic agents may be administered before, during, or after the onset of the disease or injury. Treatment of ongoing diseases is of particular interest, where treatment stabilizes or reduces unwanted clinical symptoms in a patient. This treatment is ideally administered before complete loss of function of the affected tissue. Therapeutic therapy is ideally administered during the symptomatic phase of the disease and, in some cases, after the symptomatic phase.

[0193] As used herein, the terms “administer,” “administering,” and “administration” refer to the delivery of a substance (e.g., a carrier configured to express an anti-IGF1R antibody or antigen-binding fragment; or an anti-IGF1R antibody or antigen-binding fragment) to an individual in a pharmacologically useful manner (e.g., for the treatment of an individual’s disease, condition, or symptom).

[0194] Therefore, in some embodiments, this document provides a method for treating an individual's ocular condition, the method comprising administering to the individual an rAAV carrier or a pharmaceutical composition provided herein. As used herein, the terms "individual" and "patient" are used interchangeably to refer to mammals, including but not limited to humans and non-human primates, including apes and humans; mammalian locomotion animals (e.g., horses); mammalian livestock (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). An individual may suffer from any of the symptoms or ocular conditions described herein.

[0195] In some embodiments, the individual is an adult, such as an adult over 40 years of age or an adult between 45 and 55 years of age. In some embodiments, the individual is an adult over 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years of age.

[0196] In some embodiments, the ocular condition treated according to the methods described herein is TED. In some embodiments, the ocular disease, condition, or symptom includes a) hypothyroidism-related visual impairment, b) decreased vision due to eyelid retraction, c) eyelid brush epithelial lesions, d) dry eye, e) aqueous humor deficiency, f) diplopia, g) compressive optic neuropathy, h) eye pain, i) ptosis, and / or j) diplopia. In some embodiments, the ocular disease, condition, or symptom is treated by applying 10% of any of the carriers described herein to one or more tissues of the individual (e.g., at least one eye of the individual, or accessory lacrimal glands or trabecular meshwork). 8 Up to 10 14 A vector genome for treatment.

[0197] In some embodiments, the treatment methods described herein include further administration of one or more additional therapeutic agents to an individual. In some embodiments, the additional therapeutic agent is a drug that increases tear production. In some embodiments, the additional therapeutic agent that increases tear production is a cholinergic agonist. In some embodiments, the additional therapeutic agent is another AAV-based gene therapy construct.

[0198] In some embodiments, treatment to increase tear production comprises administering an effective amount of a nicotinic acetylcholine receptor (nAChR) agonist or a pharmaceutically acceptable salt thereof. In some embodiments, an nAChR agonist is one described herein. nAChRs are a class of pentamer-gated ion channels that have high affinity and selectivity for both nicotinic acid and acetylcholine (which is similar to the protonated form of nicotinic acid) and comprise a combination of α and β subunits. Examples of nAChR subtypes include, but are not limited to, α3β4, α4β2, α3α5β4, and α4α6β2. An nAChR agonist can be characterized as a complete or partial agonist, determined by the ability of the nAChR agonist to activate the receptor to produce the response to acetylcholine (ACh) compared to the response of a given receptor. Generally, an nAChR agonist is a complete agonist if the response induced upon binding to a given receptor is equal to or greater than the response produced by ACh. If an nAChR agonist induces a smaller response after binding to the receptor than the response produced by ACh, then it is a partial agonist.

[0199] For example, appropriate cell-based assays can be used to generate nAChR agonist responses that can be used to determine receptor activation. Cells engineered to express a specific nAChR receptor subtype and to produce an electrical response upon binding to and activation by an nAChR agonist can be used to characterize the agonist profile of a compound and thereby determine the level of receptor activation.

[0200] In some embodiments, an nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to at least one of the nAChR isoforms selected from α3β4, α3α5β4, α4β2, and α4α6β2. As used herein, “selectively binds” or “is selective for” means that the compound has a higher affinity for the nAChR isoform and / or a lower half-maximum effective concentration (EC50) for said nAChr isoform relative to at least one reference nAChR isoform. Selectivity may be associated with at least a 5-fold difference in affinity, at least a 10-fold difference in affinity, at least a 20-fold difference in affinity, or at least a 50-fold difference in affinity. In some embodiments, an nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to nAChR isoform α3β4. In some embodiments, an nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to nAChR isoform α3α5β4. In some embodiments, an nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to nAChR isotype α4β2. In some embodiments, an nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to nAChR isotype α4α6β2. In some embodiments, an nAChR agonist or a pharmaceutically acceptable salt thereof selectively binds to nAChR isotype α7. In some embodiments, an nAChR agonist or a pharmaceutically acceptable salt thereof does not selectively bind to nAChR isotype α7.

[0201] The nAChR agonists considered in this disclosure include varenicline, pharmaceutically acceptable salts thereof, and compound 1 or pharmaceutically acceptable salts thereof. In some embodiments, the nAChR agonist is not varenicline.

[0202] Varencrine is characterized as a full agonist of the nAChR subtype α7 and partial agonists of subtypes α3β4, α4β2, α6β2, α3α5β4, and α4α6β2. In some embodiments, the nAChR agonist is varencrine or a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts of varencrine include varencrine tartrate. Further relevant information about varencrine can be found, for example, in U.S. Patents 6,951,938, 6,890,927, 7,265,119, 9,504,644, 9,504,645, 9,532,944, 9,597,284, 10,456,386, 11,224,598, and U.S. Application Publication 2022 / 0233528.

[0203] As described in this article, compound 1 refers to the following structure:

[0204]

[0205] The alternative structural representation of compound 1 is shown here:

[0206]

[0207] Compound 1 can also be referred to by its chemical name. For example, compound 1 is also known as (R)-5-((E)-2-pyrrolidine-3-ylvinyl)pyrimidine, or variants thereof, including simpanicline 5-{(E)-2-[(3R)-pyrrolidine-3-yl]vinyl}pyrimidine and (R,E)-5-((2-pyrrolidine-3-yl)vinyl)pyrimidine.

[0208] Compound 1 is a complete agonist for the nAChR isoforms α4β2, α3β4, α3α5β4, and α4α6β2. Compound 1 is a complete agonist for the nAChR isoforms α4β2 and α3β4.

[0209] Compound 1 is a partial agonist of the α3β2 subtype.

[0210] Compound 1 is a weak partial agonist of the α7 isoform. In one instance, a concentration of 300 μmol of compound 1 citrate induced only 25% of the maximum ACh-induced current.

[0211] In some embodiments, the nAChR agonist may be compound 1 or a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts of compound 1 include galactose disodium (e.g., hemi-galactose disodium dihydrate) and citrate (e.g., monocitrate). Patent-related information regarding compound 1 can be found, for example, in the following: U.S. Patents 7,098,331, 7,714,001, 8,063,068, 8,067,443, 8,604,191, 9,145,396, 9,981,949, 8,633,222, 8,153,821, 8,633,227, 10,709,707, U.S. Patent Application Publication 2020-0345734, and PCT Publication WO 2017 / 177024.

[0212] In some embodiments, the nAChR activator is (R)-5-((E)-2-pyrrolidine-3-ylvinyl)pyrimidine or a pharmaceutically acceptable salt thereof. In some embodiments, the nAChR activator is (R)-5-((E)-2-pyrrolidine-3-ylvinyl)pyrimidine hemi-galactose dihydrate. In some embodiments, the nAChR activator is (R)-5-((E)-2-pyrrolidine-3-ylvinyl)pyrimidine monocitrate.

[0213] In some embodiments, nAChR agonists increase tear production in individuals with impaired tear secretion.

[0214] Treatment

[0215] In some embodiments, this disclosure provides a method of treating an individual with an eye disease or condition, comprising administering to the individual a vector (e.g., rAAV) containing nucleotides encoding an anti-IGF1R antibody or antigen-binding fragment as described herein.

[0216] In some embodiments, this disclosure provides an rAAV vector in a method for treating ocular symptoms in an individual in need, the method comprising administering a recombinant adeno-associated virus (rAAV) vector or an anti-IGF1R antibody or antigen-binding fragment to the tear film or ocular tissue (in the context of this disclosure, "ocular tissue" includes tissue associated with the anatomy or function of the eye, such as orbital tissue, such as the extraorbital fat pad, and / or tissue of ocular glands) of at least one eye of the individual, the rAAV vector comprising an AAV shell and an expression cartridge containing a polynucleotide operably linked to a promoter encoding an anti-IGF1R antibody or antigen-binding fragment. For example, administration to at least one ocular tissue may include administration to at least one lacrimal gland (e.g., a primary or accessory lacrimal gland) or at least one meibomian gland and / or the trabecular meshwork of the individual. In some embodiments, the rAAV vector is administered to multiple lacrimal glands of the individual. In some embodiments, the rAAV vector comprises an expression cartridge encoding an anti-IGF1R antibody or an anti-IGF1R antigen-binding fragment.

[0217] Administration to at least one eye may be achieved via topical application, injection, or any other mode of administration of a pharmaceutical agent to the eye or any individual tissue or associated gland as described herein or known in the art. For example, a pharmaceutical composition comprising any rAAV vector described herein, any anti-IGF1R antibody or antigen-binding fragment described herein, or a plasmid (or other) vector configured to express any anti-IGF1R antibody or antigen-binding fragment described herein may be applied topically as a liquid formulation to the eye or at least one tissue or associated gland thereof by an individual or a medical professional. In other embodiments, a pharmaceutical composition comprising any rAAV vector described herein, any plasmid (or other) vector configured to express any anti-IGF1R antibody or antigen-binding fragment described herein, or any anti-IGF1R antibody or antigen-binding fragment described herein may be injected into the primary or accessory lacrimal gland, meibomian gland, or trabecular meshwork of at least one eye of an individual. This article describes various other administration modalities, such as administration of the vector and anti-IGF1R antibody or fragment described herein, which result in the direct or indirect diffusion of the anti-IGF1R antibody or fragment into the extraorbital fat pad of an individual's eye, thereby blocking the binding of IGF-1 to IGF1R and inhibiting downstream IGF1R signaling.

[0218] In some embodiments, this disclosure provides an rAAV vector for use or adaptable for treating an individual with an eye disease, condition, or symptom. In some embodiments, the rAAV vector for use or adaptable for use comprises an AAV shell and an expression cartridge containing a polynucleotide encoding an anti-IGF1R antibody or antigen-binding fragment, wherein the polynucleotide is operatively linked to a promoter.

[0219] In some embodiments, this disclosure provides an rAAV vector in a method for treating ocular symptoms in an individual in need, the method comprising administering the recombinant adeno-associated virus (rAAV) vector described herein to at least one eye of the individual or to at least one lacrimal gland of the individual's eye.

[0220] In some embodiments, this disclosure provides an rAAV vector in a method for treating ocular symptoms in an individual in need, the method comprising administering a recombinant adeno-associated virus (rAAV) vector to at least one eye of the individual or to at least one lacrimal gland of the individual's eye, the vector comprising an expression cartridge containing a nucleic acid sequence encoding SEQ ID NO: 2 or 6.

[0221] In some embodiments, the expression of anti-IGF1R antibody or antigen-binding fragment is maintained for approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year.

[0222] As used herein, “about” or “approximately” when applied to one or more values ​​of interest means a value similar to the stated reference value. In some embodiments, unless otherwise stated or apparent from the context, “about” means a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value in either direction (except where such numbers would exceed 100% of the possible value).

[0223] In some embodiments, this disclosure provides a vector (e.g., an rAAV vector) in a method for treating ocular symptoms in an individual in need. For example, the method may include administering a recombinant adeno-associated virus (rAAV) vector to at least one eye of an individual or to at least one lacrimal gland of an individual's eye. The rAAV vector comprises an AAV capsid and an expression cartridge containing a polynucleotide operatively linked to a promoter encoding an anti-IGF1R antibody or an antigen-binding fragment. In some embodiments, the rAAV vector for use is administered to the lacrimal gland of an individual. In some embodiments, the rAAV vector for use comprises an expression cartridge encoding an anti-IGF1R antibody or an anti-IGF1R antigen-binding antibody fragment.

[0224] In some embodiments, this disclosure provides an rAAV vector for use or adaptable for treating an individual with an eye disease, condition, or symptom. In some embodiments, the rAAV vector for use or adaptable for use comprises an AAV shell and an expression cartridge containing a polynucleotide encoding an anti-IGF1R antibody or antigen-binding fragment, wherein the polynucleotide is operatively linked to a promoter.

[0225] In some embodiments, the method includes administering a dose of rAAV to an individual suffering from TED-related symptoms, wherein the symptoms are characterized by one or more of the symptoms described herein. In some embodiments, the dose is administered before the onset of one or more symptoms. In some embodiments, the administration prevents the onset of one or more symptoms. In some embodiments, the administration reduces the severity of one or more symptoms. For example, in some embodiments, the method includes administering a dose of rAAV to an individual experiencing one or more symptoms of TED, such as a) hypothyroidism-related visual disturbances, b) decreased vision due to eyelid retraction, c) eyelid brush epithelial lesions, d) dry eye, e) aqueous humor deficiency, f) diplopia, g) compressive optic neuropathy, h) eye pain, i) exophthalmos, and / or j) diplopia, wherein the dose of rAAV is administered and prevents one or more symptoms or reduces their severity. In some embodiments, the dose is administered after the onset of one or more symptoms, wherein the administration reduces the severity of one or more symptoms.

[0226] In some embodiments, the method includes a dosing regimen of administering rAAV to an individual suffering from TED-related symptoms, wherein the symptoms are characterized by one or more of the symptoms described herein, and the dosing regimen includes a first dose of rAAV and at least one additional dose, wherein the first dose of rAAV is administered to the individual before or after the onset of one or more symptoms, and wherein at least one additional dose is administered to the individual after the first dose, thereby preventing one or more symptoms or reducing their severity. In some embodiments, the dosing regimen includes a dosing frequency and / or dose selected based on pharmacokinetic parameters of rAAV. In some embodiments, a clinician administers rAAV at a frequency and / or dose that achieves or maintains one or more desired effects (e.g., a clinically measurable reduction or improvement in any symptom of any symptom of any symptom described herein). In some embodiments, a clinically measurable reduction or improvement in any symptom is achieved at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after administration of rAAV to the individual, or within this timeframe. In some embodiments, a clinically measurable reduction or improvement of any symptom is achieved at least 1, 2, 3, 4, 5, or 6 weeks after administration of rAAV to an individual, or within this timeframe. In some aspects, one or more desired effects comprise prevention of one or more symptoms associated with an individual's condition. In some embodiments, one or more desired effects are a reduction in the severity of one or more symptoms associated with an individual's condition. In some embodiments, one or more desired effects comprise one or more clinically measurable changes achieved 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after administration of a first dose of rAAV, or within this timeframe, or 1, 2, 3, 4, 5, or 6 weeks after administration of a first dose of rAAV, or within this timeframe. In some embodiments, one or more desired effects occur at any point in time after administration of the first dose of rAAV. In some embodiments, one or more desired effects are achieved after administration of at least one additional dose of rAAV. In some embodiments, one or more desired effects are achieved at any point in time during the dosing regimen. In some embodiments, one or more desired effects are achieved after administration of a first dose of rAAV, and at least one additional dose of rAAV is administered to the individual to prevent reversal of one or more desired effects. In some embodiments, the method includes administering a first dose of rAAV to the individual before, immediately after, or during the onset of one or more symptoms, and administering an additional dose after approximately 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, or 2 years following the first dose.

[0227] In some embodiments, administration of the rAAV described herein results in the measurable expression, or measurable expression by, an antibody or fragment encoded by the rAAV in one or more cells, tissues, or organs of an individual within 1, 2, 3, 4, 5, 6, or 7 days after administration of the rAAV. In some embodiments, the expression of an antibody or fragment encoded by the rAAV is detectable in one or more cells, tissues, or organs of an individual within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks after administration.

[0228] In some embodiments, treatment of the condition described herein or any of its symptoms is achieved by administering the anti-IGF1R antibody or fragment described herein (e.g., in protein form in a liquid carrier suitable for topical application). For example, any antibody or fragment described herein may be administered topically to the cells, tissues, or organs of a human individual (e.g., in a liquid form administered to the eye). Administration of the antibody or fragment described herein may provide a therapeutic effect (e.g., a measurable reduction or improvement of one or more symptoms of any condition described herein).

[0229] In some embodiments, methods are provided for treating an eye disease, condition, or symptom, said eye disease, condition, or symptom comprising a) hypothyroidism-related visual impairment, b) decreased vision due to eyelid retraction, c) eyelid brush epithelial lesions, d) dry eye syndrome, e) aqueous humor deficiency, f) diplopia, g) compressive optic neuropathy, h) eye pain, i) exophthalmos, and / or j) diplopia. In some embodiments, the method comprises administering to an individual a dose of an anti-IGF1R antibody or an IGF1R binding fragment thereof, wherein the administration reverses or reduces one or more symptoms of the condition described herein. In some embodiments, the dose is administered before the onset of one or more symptoms. In some embodiments, the administration prevents the onset of at least one of one or more symptoms. In some embodiments, the administration reduces the severity of at least one of one or more symptoms. For example, in some embodiments, the method comprises administering to an individual with TED characterized by one or more symptoms (e.g., exophthalmos or diplopia) a dose of rAAV, wherein the administration of said dose of rAAV prevents or reduces the severity of at least one or more symptoms. In some embodiments, the dosage is administered after the onset of one or more symptoms, wherein the administration reduces the severity of at least one of the one or more symptoms.

[0230] In some embodiments, the method comprises a dosing regimen of administering rAAV to an individual suffering from TED-related symptoms, wherein the symptoms are characterized by one or more of the symptoms described herein, wherein the dosing regimen comprises a first dose of rAAV and at least one additional dose, wherein the first dose of rAAV is administered to the individual before or after the onset of one or more symptoms, and wherein at least one additional dose is administered to the individual after the first dose, thereby preventing at least one of the one or more symptoms or reducing their severity. In some embodiments, the dosing regimen comprises a dosing frequency and / or dose selected based on pharmacokinetic parameters of a carrier (e.g., any rAAV carrier described herein) or an anti-IGF1R antibody or antigen-binding fragment (if administered directly). In some embodiments, a clinician administers the carrier or anti-IGF1R antibody or antigen-binding fragment at a frequency and / or dose that achieves or maintains one or more desired effects. In some embodiments, one or more desired effects are the prevention of one or more symptoms associated with the individual's condition. In some embodiments, one or more desired effects are the reduction of the severity of one or more symptoms associated with the individual's condition. In some embodiments, the severity of one or more symptoms is measured using methods described herein or known in the art for assessing TED-related lesions. In some embodiments, one or more desired effects are achieved immediately following administration of a first dose of the carrier. In some embodiments, one or more desired effects occur at any point in time after administration of a first dose of the carrier. In some embodiments, one or more desired effects are achieved after administration of at least one additional dose of the carrier (e.g., an rAAV carrier). In some embodiments, one or more desired effects are achieved at any point in time during the dosing regimen. In some embodiments, one or more desired effects are achieved after administration of a first dose of the carrier, and at least one additional dose of the carrier is administered to the individual to prevent reversal of one or more desired effects. In some embodiments, the method includes administering a first dose of the carrier to the individual before, immediately after, or during the onset of one or more symptoms, and administering an additional dose after approximately 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, or 2 years following the first dose.

[0231] In some embodiments, the method includes administering a dose of a carrier to an individual suffering from symptoms associated with loss of accommodation, wherein the symptoms are characterized by one or more of the symptoms described herein. In some embodiments, the dose is administered before the onset of one or more symptoms. In some embodiments, the administration prevents the onset of at least one of the one or more symptoms. In some embodiments, the administration reduces the severity of at least one of the one or more symptoms. For example, in some embodiments, the method includes administering a dose of a carrier to an individual suffering from loss of accommodation characterized by one or more symptoms, wherein the administration of the dose of the carrier prevents at least one of the one or more symptoms or reduces their severity. In some embodiments, the dose is administered after the onset of one or more symptoms, wherein the administration reduces the severity of at least one of the one or more symptoms.

[0232] In some embodiments, the method comprises a dosing regimen of administering a carrier (e.g., an rAAV carrier) to an individual suffering from a symptom associated with loss of accommodation, wherein the symptom is characterized by one or more symptoms described herein, and the dosing regimen comprises a first dose of the carrier and at least one additional dose, wherein the first dose of the carrier is administered to the individual before or after the onset of one or more symptoms, and wherein at least one additional dose is administered to the individual after the first dose, thereby preventing at least one of the one or more symptoms or reducing their severity. In some embodiments, the dosing regimen comprises a dosing frequency and / or dose selected based on pharmacokinetic parameters of the carrier. In some embodiments, a clinician administers the carrier at a frequency and / or dose to achieve or maintain one or more desired effects. In some embodiments, one or more desired effects are prevention of one or more symptoms associated with the individual's symptom. In some embodiments, one or more desired effects are reduction of the severity of one or more symptoms associated with the individual's symptom. In some embodiments, the severity of one or more symptoms is measured using methods described herein or known in the art for assessing lesions associated with loss of accommodation. In some embodiments, one or more desired effects are achieved immediately following the administration of the first dose of the carrier. In some embodiments, one or more desired effects occur at any point in time following administration of the first dose of the carrier. In some embodiments, one or more desired effects are achieved after administration of at least one additional dose of the carrier. In some embodiments, one or more desired effects are achieved at any point in time during the dosing regimen. In some embodiments, one or more desired effects are achieved after administration of the first dose of the carrier, and at least one additional dose of the carrier is administered to the individual to prevent reversal of one or more desired effects. In some embodiments, the method includes administering the first dose of the carrier to the individual before, immediately after, or during the onset of one or more symptoms, and administering an additional dose after approximately 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, or 2 years following the first dose.

[0233] In some embodiments, the method includes administering a dose of a carrier to an individual suffering from TED-related symptoms, wherein the symptoms are characterized by one or more of the symptoms described herein. In some embodiments, the dose is administered before the onset of one or more symptoms. In some embodiments, the administration prevents the onset of at least one of the one or more symptoms. In some embodiments, the administration reduces the severity of at least one of the one or more symptoms. For example, in some embodiments, the method includes administering a dose of a carrier to an individual suffering from compressive optic neuropathy characterized by one or more symptoms, wherein the administration of the dose of the carrier prevents at least one of the one or more symptoms or reduces their severity. In some embodiments, the dose is administered after the onset of one or more symptoms, wherein the administration reduces the severity of at least one of the one or more symptoms.

[0234] In some embodiments, the method includes administering a dose of a carrier (e.g., an rAAV carrier) to an individual suffering from symptoms associated with meibomian gland dysfunction (MDI), wherein the symptoms are characterized by one or more of the symptoms described herein. In some embodiments, the dose is administered before the onset of one or more symptoms. In some embodiments, the administration prevents the onset of one or more symptoms. In some embodiments, the administration reduces the severity of one or more symptoms. For example, in some embodiments, the method includes administering a dose of a carrier to an individual suffering from MDI characterized by one or more symptoms (e.g., dry eyes, burning, itching, redness, discharge, blurred vision), wherein the administration of the dose of the carrier prevents at least one of the one or more symptoms or reduces their severity. In some embodiments, the dose is administered after the onset of one or more symptoms, wherein the administration reduces the severity of at least one of the one or more symptoms.

[0235] In some embodiments, the method includes a dosing regimen of administering a carrier to an individual suffering from TED-related symptoms, wherein the symptoms are characterized by one or more symptoms described herein, wherein the dosing regimen includes a first dose of the carrier and at least one additional dose, wherein the first dose of the carrier is administered to the individual before or after the onset of one or more symptoms, and wherein the at least one additional dose is administered to the individual after the first dose, thereby preventing at least one of the one or more symptoms or reducing their severity. In some embodiments, the dosing regimen includes a dosing frequency and / or dose selected based on pharmacokinetic parameters of the carrier. In some embodiments, a clinician administers the carrier at a frequency and / or dose to achieve or maintain one or more desired effects. In some embodiments, one or more desired effects are prevention of one or more symptoms associated with the individual's condition. In some embodiments, one or more desired effects are reduction of the severity of one or more symptoms associated with the individual's condition. In some embodiments, the severity of one or more symptoms is measured using methods described herein or known in the art for assessing TED-related lesions. In some embodiments, one or more desired effects are achieved immediately following administration of the first dose of the carrier. In some embodiments, one or more desired effects occur at any point in time after administration of the first dose of the carrier. In some embodiments, one or more desired effects are achieved after administration of at least one additional dose of the carrier. In some embodiments, one or more desired effects are achieved at any point during the dosing regimen. In some embodiments, one or more desired effects are achieved after administration of a first dose of the carrier, and at least one additional dose of the carrier is administered to the individual to prevent reversal of at least one of the one or more desired effects. In some embodiments, the method comprises administering a first dose of the carrier to the individual before, immediately after, or during the onset of one or more symptoms, and administering an additional dose after approximately 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, or 2 years following the first dose.

[0236] In some embodiments, this disclosure provides a method for treating the following conditions in an individual in need: a) hypothyroidism-related visual impairment, b) decreased vision due to eyelid retraction, c) eyelid brush epithelial lesions, d) dry eye, e) aqueous humor deficiency, f) diplopia, g) compressive optic neuropathy, h) eye pain, i) bulging eye, and / or j) diplopia, said method comprising administering one or more therapeutically effective doses of the carrier described herein to the individual.

[0237] In some embodiments, this disclosure provides a vector (e.g., an rAAV vector) described herein for use in manufacturing a medicament for treating the following conditions in individuals in need: a) hypothyroidism-related visual impairment, b) decreased vision due to eyelid retraction, c) eyelid brush epithelial lesions, d) dry eye syndrome, e) aqueous humor deficiency, f) diplopia, g) compressive optic neuropathy, h) eye pain, i) bulging eye, and / or j) diplopia.

[0238] In some embodiments, this disclosure provides a carrier (e.g., an rAAV carrier) described herein for use in a method of treating an individual in need of the following conditions: a) hypothyroidism-related visual impairment, b) decreased vision due to eyelid retraction, c) eyelid brush epithelial lesions, d) dry eye, e) aqueous humor deficiency, f) diplopia, g) compressive optic neuropathy, and / or h) eye pain, the method comprising administering an effective amount of the carrier to the individual.

[0239] In some embodiments, this disclosure provides a carrier as described herein for manufacturing a medicament for treating TED in individuals in need.

[0240] In some embodiments, this disclosure provides a vector described herein for use in a method of treating an individual in need of TED, the method comprising administering to the individual an effective amount of any vector described herein or an effective amount of any anti-IGF1R antibody or antigen-binding fragment described herein.

[0241] Application mode

[0242] In some embodiments, this disclosure provides a method comprising administering a carrier or an anti-IGF1R antibody or antigen-binding fragment to an individual's eye, the carrier being, for example, an rAAV carrier comprising an AAV shell and an expression cartridge, the expression cartridge comprising a polynucleotide encoding an anti-IGF1R antibody or antigen-binding fragment operably linked to a promoter. Administering may be to the individual's tear film or ocular tissue (e.g., to the individual's primary or accessory lacrimal glands, to the individual's meibomian glands, or to the trabecular meshwork of the individual's eye).

[0243] The lacrimal gland functional unit consists of the main lacrimal gland and accessory lacrimal glands, the ocular surface, and interconnected nerve innervation. In each eye, the main lacrimal gland is located in the lacrimal fossa of the frontal bone in the superior temporal orbit. Accessory glands, called Wolfring's gland and Krause's gland, are located in the eyelids. Approximately 2 to 5 Wolfring's glands and approximately 40 Krause's glands are present in the upper eyelid. Approximately 6 to 8 Krause's glands are present in the lower eyelid. The specific location and anatomy of the lacrimal gland functional unit are well known (Conrady et al., *Journal of Ophthalmology*; 2016: 7542929 (2016)). Together, the lacrimal glands secrete the tear film onto the ocular surface via the lacrimal ducts. The lacrimal glands also express and secrete proteins and products necessary for corneal regeneration and increased transparency into the tear film, such as transforming growth factor-β and retinol (Conrady et al., *Journal of Ophthalmology*; 2016: 7542929 (2016); Pan et al., *Optometry and Vision Science*; 95:27-31 (2018)). In addition to secreting tears into the eyes, the lacrimal ducts drain tears into the nasal cavity.

[0244] Administration of a carrier (e.g., an rAAV carrier) to the lacrimal gland can be achieved through topical application to the ocular surface, direct injection into the lacrimal gland, and / or topical application to the lacrimal gland. The lacrimal gland can be accessed surgically or by manipulating the eyelids. Manipulating the eyelids provides access to the tissue for topical application (e.g., by irrigating the tissue with a pharmaceutical composition containing the carrier). Direct injection into the lacrimal gland can be achieved by penetrating the skin over the lacrimal gland (…). Figure 2A Or by manipulating the eyelids to access the lacrimal gland ( Figure 2B The administration of the carrier to the lacrimal gland can be achieved via transconjunctival injection. In some embodiments, the carrier is administered to the lacrimal gland via direct injection, such as... Figure 2A As depicted in the figure. In some embodiments, the carrier is administered to the lacrimal gland by manipulating the eyelid, as... Figure 2B As depicted in the text.

[0245] In some embodiments, cells within the eye, lacrimal gland, and / or nasolacrimal duct are transduced or transfected via a vector. These cells include, but are not limited to, acinar cells, ductal cells, and / or myoepithelial cells, as well as cells of the iris and ciliary body (“ICB”), lens epithelial cells, meibomian glands, and trabecular meshwork cells. In some embodiments, transduced or transfected cells within the eye, lacrimal gland, and / or nasolacrimal duct express a therapeutically effective amount of anti-IGF1R antibody or antigen-binding fragment. In some embodiments, transduced or transfected cells in the eye, lacrimal gland, and / or nasolacrimal duct secrete a therapeutically effective amount of anti-IGF1R antibody or antigen-binding fragment into the tear film. In some embodiments, cells within the meibomian gland are transduced or transfected via a vector. In some embodiments, cells within the trabecular meshwork are transduced or transfected via a vector. In some embodiments, a therapeutically effective amount of anti-IGF1R antibody or antigen-binding fragment is secreted into the individual's nasal cavity. In some embodiments, a therapeutically effective amount of anti-IGF1R antibody or antigen-binding fragment is secreted onto the individual's ocular surface.

[0246] In vivo delivery of rAAV vectors to the eyes and / or lacrimal glands to express transgenic genes in the tear film has been demonstrated. In mice, the primary lacrimal glands were directly injected with rAAV vectors encoding luciferase transgenic genes, with serotypes AAV2, AAV4, AAV5, AAV 5w8, AAV x5, AAV9, AAV12, and bovine AAV (BAAV). AAV9, AAV 5w8, AAV5, and AAV2 can each transduce lacrimal duct cells and acinar cells of the lacrimal gland (Rocha et al., above).

[0247] In some embodiments, a carrier is applied to an individual's lacrimal gland. In some embodiments, the lacrimal gland is the primary lacrimal gland. In some embodiments, the lacrimal gland is either an individual's Valsalva gland or a Gosling gland.

[0248] The compositions and carriers according to this disclosure can be administered to an individual's lacrimal glands by any suitable method. For example, the subject composition can be administered directly to the primary or accessory lacrimal glands via injection.

[0249] Access to the lacrimal gland in a human individual can be achieved, for example, by manually lifting the upper eyelid to expose the palpebral lobe of the lacrimal gland and delivering the therapeutic agent using a syringe (e.g., with a 30G needle).

[0250] In some embodiments, a carrier is applied to the trabecular mesh structure of an individual's eye.

[0251] In some embodiments, the application of the carrier or any antigen-binding fragment described herein is performed by injection using a liquid carrier (e.g., one or more lipid nanoparticles or liposomes) or an erodible insert (e.g., a polymeric gel containing the carrier to be applied).

[0252] In some embodiments, the carrier or antigen-binding fragment described herein is applied to the endothelial cells of an individual's cornea. This can be achieved by injection into adjacent or suitable tissues (e.g., intracorneal injection). In some embodiments, the carrier is applied to other cells and / or tissues of the individual. For example, application can be performed via conjunctival injection, injection into the corneal stroma, suprachoroidal injection, subconjunctival injection, intravitreal injection, intra-anterior chamber injection, injection into the meibomian glands, or subcutaneous eyelid injection. For some tissues, carrier application can be performed via injection using a liquid carrier (e.g., one or more lipid nanoparticles) or using an erosive insert (e.g., a polymeric gel containing the carrier to be applied).

[0253] The carriers disclosed herein are generally delivered to an individual in the form of a pharmaceutical composition. The pharmaceutical composition comprises a pharmaceutically acceptable solvent (e.g., water) and one or more excipients. In some embodiments, the pharmaceutical composition comprises a buffer solution at approximately neutral pH (pH 5, 6, 7, 8, or 9). In some embodiments, the pharmaceutical composition comprises phosphate-buffered saline (e.g., PBS at approximately pH 7). The pharmaceutical composition may contain a pharmaceutically acceptable salt. The salt concentration may be selected to ensure that the pharmaceutical composition is isotonic or nearly isotonic with the target tissue.

[0254] In various embodiments, the pharmaceutical composition of this disclosure comprises about 1 × 10 8 One genome copy / mL (GC / mL), approximately 5 × 10 8 GC / mL, approximately 1×10 9 GC / mL, approximately 5 × 10 9 GC / mL, approximately 1×10 10 GC / mL, approximately 5 × 10 10 GC / mL, approximately 1×10 11 GC / mL, approximately 5 × 10 11 GC / mL, approximately 1×10 12 GC / mL, approximately 5 × 10 12 GC / mL, approximately 5 × 10 13 GC / mL or approximately 1×10 14 A carrier with a GC / mL concentration. In various embodiments, the pharmaceutical compositions of this disclosure contain about 1 × 10⁻⁶. 8 One genome copy / mL (GC / mL), approximately 5 × 10 8 GC / mL to approximately 1×10 9 GC / mL, approximately 1×10 9 GC / mL to approximately 5 × 10 9 GC / mL, approximately 5 × 10 9 GC / mL to approximately 1×10 10 GC / mL, approximately 1×10 10GC / mL to approximately 5 × 10 10 GC / mL, approximately 5 × 10 10 GC / mL to approximately 1×10 11 GC / mL, approximately 1×10 11 GC / mL to approximately 5 × 10 11 GC / mL, approximately 5 × 10 11 GC / mL to approximately 1×10 12 GC / mL, approximately 1×10 12 GC / mL to approximately 5 × 10 12 GC / mL, approximately 5 × 10 12 GC / mL to approximately 5 × 10 13 GC / mL or approximately 5 × 10 13 GC / mL to approximately 1×10 14 A carrier with a GC / mL concentration. In various other embodiments, the pharmaceutical compositions of this disclosure contain about 1 × 10⁻⁶. 8 One genome copy / mL (GC / mL), approximately 5 × 10 8 GC / mL to approximately 5 × 10 9 GC / mL, approximately 5 × 10 9 GC / mL to approximately 5 × 10 10 GC / mL, approximately 5 × 10 10 GC / mL to approximately 5 × 10 11 GC / mL, approximately 5 × 10 11 GC / mL to approximately 5 × 10 12 GC / mL or approximately 5 × 10 12 GC / mL to approximately 1×10 14 A carrier with a GC / mL concentration. In some embodiments, the pharmaceutical compositions disclosed herein contain about 5 × 10⁻⁶ GC / mL. 8 GC / mL to approximately 5 × 10 10 GC / mL, approximately 5 × 10 10 GC / mL to approximately 5 × 10 12 GC / mL or approximately 5 × 10 12 GC / mL to approximately 1×10 14 GC / mL vector.

[0255] In some embodiments, the pharmaceutical composition disclosed herein contains about 1 × 10 12 GC / mL to approximately 6.2 × 10⁻⁶ 12 A carrier with a GC / mL concentration. In some embodiments, the pharmaceutical compositions disclosed herein contain about 1 × 10⁻⁶ GC / mL. 12 GC / mL or approximately 6.2 × 10⁻⁶ 12 GC / mL vector.

[0256] In some embodiments, the pharmaceutical compositions of this disclosure are administered in total volumes of about 10 µL, about 20 µL, about 30 µL, about 40 µL, about 50 µL, about 60 µL, about 70 µL, about 80 µL, about 90 µL, about 100 µL, 110 µL, about 120 µL, about 130 µL, about 140 µL, about 150 µL, about 160 µL, about 170 µL, about 180 µL, about 190 µL, or about 200 µL. In some embodiments, the pharmaceutical composition of this disclosure is administered in the following total volumes: about 10 µL to about 20 µL, about 20 µL to about 30 µL, about 30 µL to about 40 µL, about 40 µL to about 50 µL, about 50 µL to about 60 µL, about 60 µL to about 70 µL, about 70 µL to about 80 µL, about 80 µL to about 90 µL, about 90 µL to about 100 µL, about 100 µL to 110 µL, about 110 µL to about 120 µL, about 120 µL to about 130 µL, about 130 µL to about 140 µL, about 140 µL to about 150 µL, about 150 µL to about 160 µL, about 160 µL to about 170 µL, about 170 µL to about 180 µL, about 180 µL to about 190 µL, or about 190 µL to about 200 µL.

[0257] The number of genome copies per milliliter can be determined using a quantitative polymerase change reaction (qPCR) with a standard curve generated from a reference sample containing a known concentration of the polynucleotide genome of a vector (or virus, in the case of a viral vector). For AAV, the reference sample is typically the transfer plasmid used to generate the rAAV vector, but other reference samples may be used.

[0258] Alternatively, the concentration of the viral vector can be determined by measuring the valence of the vector to the cell line. Viral valence is typically expressed as viral particles per unit volume (vp) (e.g., vp / mL). In various embodiments, the pharmaceutical compositions of this disclosure contain about 1 × 10⁻⁶ 8 1 viral particle / mL (vp / mL), approximately 5 × 10 8 vp / mL, approximately 1×10 9 vp / mL, approximately 5 × 10 9 vp / mL, approximately 1×10 10 vp / mL, approximately 5 × 10 10 vp / mL, approximately 1×10 11 vp / mL, approximately 5 × 10 11 vp / mL, approximately 1×10 12 vp / mL, approximately 5 × 10 12vp / mL, approximately 5 × 10 13 vp / mL or approximately 1×10 14 The rAAV carrier has a volume of vp / mL. In various other embodiments, the pharmaceutical compositions of this disclosure contain about 1 × 10⁻⁶. 8 1 viral particle / mL (vp / mL) to approximately 5 × 10 8 vp / mL, approximately 5 × 10 8 vp / mL to approximately 1×10 9 vp / mL, approximately 1×10 9 vp / mL to approximately 5 × 10 9 vp / mL, approximately 5 × 10 9 vp / mL to approximately 1×10 10 vp / mL, approximately 1×10 10 vp / mL to approximately 5 × 10 10 vp / mL, approximately 5 × 10 10 vp / mL to approximately 1×10 11 vp / mL, approximately 1×10 11 vp / mL to approximately 5 × 10 11 vp / mL, approximately 5 × 10 11 vp / mL to approximately 1×10 12 vp / mL, approximately 1×10 12 vp / mL to approximately 5 × 10 12 vp / mL, approximately 5 × 10 12 vp / mL to approximately 5 × 10 13 vp / mL or approximately 5 × 10 13 vp / mL to approximately 1×10 14 rAAV vector with vp / mL.

[0259] Efficacy evaluation

[0260] Multiple tests can be used to assess an individual's ocular condition before, during, and after treatment with any of the methods or compositions disclosed herein. In some embodiments disclosed herein, effective treatment for an individual may be indicated by one or more of the following tests: a) a dry eye score test based on a visual analog scale, b) Schirmer's test, c) corneal fluorescein staining test, and d) ocular surface disease index test. In some embodiments, effective treatment for an individual may be indicated by one or more of the following tests: Symptom Assessment in Dry Eye Questionnaire (SANDE), meibomian gland atrophy / loss, gland expression, meibomian gland imaging / meibomian gland measurement, tear film lipid layer thickness, corneal fluorescein staining, tear break-up time (TBUT), distance-corrected near visual activity (DCNVA), intraocular pressure measurement (e.g., barometric profilometry), visual acuity, slit-lamp examination, or ophthalmoscopy. Tests used to assess signs and symptoms of ocular conditions can be administered under standardized or reproducible conditions to obtain an individual test score. Conditions include exposing the individual to artificially created environments that adversely stimulate the individual, or environments (temperature, humidity, airflow) that are monitored and carefully controlled.

[0261] For example, in some embodiments, improvements in one or more symptoms of TED can be determined using metrics and schemes known in the art. For example, improvements in any of the following symptoms may optionally be determined as follows:

[0262] (a) Protruding eye (sometimes called bulging eyeball): This can be determined or measured and improved using an eyeball protrusion meter. Measurements are in millimeters and are consistent with eyelid retraction, von Greif's eyelid lag, and eyelid edema.

[0263] (b) Diplopia: Improvement can be determined or measured using eye trackers and / or MRI scans.

[0264] (c) Pain: Improvement can be determined or measured through patient assessment using a rating scale (e.g., the Eye Pain Assessment Survey, “OPAS”), the VRS Pain Scale, etc.

[0265] (d) Redness: Improvement can be determined or measured by clinical grading or color image analysis, for example using a 0-4 scale in 0.5 unit increments.

[0266] (e) Functional vision: Improvement can be determined or measured through visual function assessment via reading and / or simulator.

[0267] The effectiveness of the methods described in this paper can be evaluated using any suitable method known in the field.

[0268] In some embodiments, the methods described herein reduce one or more symptoms of an ocular condition compared to the symptoms of the ocular condition before application of the carrier. As used herein, “symptom” includes any of the diagnostic criteria or symptoms associated with a given ocular condition, including those described herein. Non-limiting examples of symptoms that can be relieved by treatment according to the methods described herein include, for example, deterioration of vision, and the requirement for corrective lenses (e.g., eyeglasses or contact lenses) and / or surgery.

[0269] In some embodiments, the treatment methods described herein cause a delay in the onset of ocular symptoms. In some embodiments, the onset of ocular symptoms is delayed relative to the onset of ocular symptoms in a control individual. In some embodiments, the onset of ocular symptoms is delayed relative to the onset of ocular symptoms in the contralateral eye. In some embodiments, the treatment methods described herein cause a delay in the progression of ocular symptoms. In some embodiments, the progression of ocular symptoms is delayed relative to the progression of ocular symptoms in a control individual. In some embodiments, the progression of ocular symptoms is delayed relative to the progression of ocular symptoms in the contralateral eye. A “control individual” may be, for example, an untreated control individual. In some embodiments, a control individual is an age-matched individual who has not been treated with a vector comprising an expression cartridge containing a polynucleotide encoding an anti-IGF1R antibody or an antigen-binding fragment. In some embodiments, a control individual is an age-matched untreated individual. A “contralateral eye” refers to the eye of an individual opposite the eye that has been treated with the composition according to this disclosure. The contralateral eye can be used as a treatment control, provided that the individual has bilateral disease, or in the case of a model animal, has been subjected to an experimental protocol that induces treatment in both eyes.

[0270] The treatment methods described herein can cause a slowing of the progression of ocular symptoms. In some embodiments, compared to a control individual, the progression of symptoms is slowed by about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, or more than about 95%.

[0271] In some embodiments, the treatment methods described herein cause a delay in the onset of symptoms in individuals of approximately 6 to 9 months, approximately 9 to 12 months, approximately 12 to 15 months, approximately 15 to 18 months, approximately 18 to 21 months, approximately 21 to 24 months, approximately 2 to 3 years, approximately 3 to 4 years, approximately 4 to 5 years, approximately 5 to 6 years, approximately 6 to 7 years, approximately 7 to 8 years, approximately 8 to 9 years, approximately 9 to 10 years, approximately 10 to 15 years, approximately 15 to 20 years, or more than 20 years, compared to control individuals.

[0272] In some embodiments, the treatments described herein result in an increase in visual acuity in an individual. In some embodiments, such as relative to the contralateral eye or relative to a control individual, the treatments described herein result in a delayed decrease in visual acuity in an individual. Visual acuity can be measured using a visual acuity chart that allows an individual to identify letters from a distance, including, for example, the Snellen acuity chart and the Early Treatment Research for Diabetic Retinopathy (ETDRS) visual acuity chart (Bailey and Kitchin, Vision Research 90 (2013) 2-9; Bennet et al., Semin Pediatr Neurol. 2019 Oct; 31:30-40). Visual acuity can also be assessed using binocular distance-corrected near vision (DCNVA).

[0273] In some embodiments, after administration of the rAAV carrier, the vision of an individual treated according to the methods described herein remains unchanged for at least about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years.

[0274] In some embodiments, after administration of the rAAV carrier, the visual acuity of an individual treated according to the methods described herein does not decrease by more than 5% for at least about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years. In some embodiments, after administration of the rAAV carrier, the visual acuity of an individual treated according to the methods described herein does not decrease by more than 10% for at least about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 15 months, about 15 months to about 18 months, about 18 months to about 21 months, about 21 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years.

[0275] Another indicator of vision may be the requirement for corrective lenses (e.g., contact lenses or eyeglasses), the intensity of which typically increases as the condition progresses. In some embodiments, individuals treated according to the methods described herein require corrective lenses prior to the application of the rAAV carrier, and the methods result in a consistent requirement for the intensity of corrective lenses for at least about 6 to about 9 months, about 9 to about 12 months, about 12 to about 15 months, about 15 to about 18 months, about 18 to about 21 months, about 21 to about 24 months, about 2 years to about 3 years, about 3 to about 4 years, about 4 years to about 5 years, about 5 years to about 6 years, about 6 years to about 7 years, about 7 years to about 8 years, about 8 years to about 9 years, about 9 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, or more than 20 years after the application of the rAAV carrier.

[0276] In some embodiments, effective treatment for an individual is indicated by the Dry Eye Symptom Assessment Questionnaire (SANDE). In some embodiments, effective treatment for an individual is indicated by meibomian gland atrophy / loss. In some embodiments, effective treatment for an individual is indicated by gland expression. In some embodiments, effective treatment for an individual is indicated by meibomian gland imaging / meibomian gland measurement. In some embodiments, effective treatment for an individual is indicated by tear film lipid layer thickness. In some embodiments, effective treatment for an individual is indicated by corneal fluorescein staining; in some embodiments, effective treatment for an individual is indicated by tear film breakup time (TBUT). In some embodiments, effective treatment for an individual is indicated by distance-corrected near visual acuity (DCNVA). In some embodiments, effective treatment for an individual is indicated by intraocular pressure measurement. In some embodiments, effective treatment for an individual is indicated by visual acuity. In some embodiments, effective treatment for an individual is indicated by slit-lamp examination. In some embodiments, effective treatment for an individual is indicated by ophthalmoscopy. The methods described herein for assessing effective treatment for an individual are known to those skilled in the art.

[0277] Maintaining effective treatment over time

[0278] The efficacy of the treatment methods described herein can be assessed at any suitable time point after application, such as approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 12 months, approximately 18 months, approximately 24 months, approximately 3 years, approximately 4 years, or approximately 5 years after application of the rAAV carrier described herein. In some embodiments, efficacy is measured at two or more time points after application of the rAAV carrier described herein, such as every 3 months, every 6 months, or every 12 months after application of the carrier.

[0279] This disclosure provides effective treatment for a period of time, during which efficacy measures (e.g., visual acuity) are maintained. As used in this disclosure and in relation to maintaining efficacy measures (e.g., visual acuity) in an individual's score, the term "maintain" means a statistically significant improvement that does not decrease to below a certain threshold over time. Efficacy measures maintained after treatment according to the disclosed method can be maintained without additional drug administration or after one or more subsequent doses.

[0280] Dosage sequence and administration method

[0281] The timing of dose administration to an individual depends on various considerations, including the duration of effect of each dose, the transduction (or transfection) efficiency of the rAAV vector, and the effect of the dose on the body. For example, if a patient's symptoms do not improve, the dosage may be adjusted or repeated, as described herein, at the discretion of the healthcare provider, to improve or otherwise control or limit the individual's ocular symptoms. For example, the time interval between the administration of one or more doses may be extended, or the time interval between the days on which one or more doses are administered to the individual may be extended. As a non-limiting example, the administration of one or more doses may be modified to administer one or more doses after measuring the symptoms of the ocular symptom.

[0282] As used herein, the term “dosage” may refer to a dose of the pharmaceutical composition disclosed herein, or a dose for the treatment of reducing symptoms of an eye condition.

[0283] In some embodiments described herein, the dose of the vector is the dose of the rAAV vector carrying the expression cartridge. In such cases, delivery of an appropriate dose (e.g., an effective amount) of the gene product is achieved by administering an appropriate amount / valent of the vector to the target site, which allows for the expression of an effective amount of the gene product over a period of time. As used herein, "effective amount" means the amount or dose of the vector, treatment, or composition described herein that is sufficient to reduce the symptoms and / or signs of the ocular condition described herein. As used herein, the term "amount" means an absolute amount (e.g., the absolute amount of protein or non-viral vector or rAAV vector particles) or a concentration (e.g., the concentration of protein in solution), and whether the amount referred to in a given example means an absolute amount, a concentration, or both, will be understood by those skilled in the art based on the context provided herein.

[0284] In some embodiments, the vector is an rAAV vector (e.g., rAAV viral particles). In some embodiments, the rAAV vector is applied to the lacrimal gland. In some embodiments, the rAAV vector is applied to the lacrimal gland by topical application. In some embodiments, the rAAV vector is applied to the lacrimal gland by direct injection. In some embodiments, the rAAV vector is applied to the trabecular meshwork. In some embodiments, the dosage of the rAAV vector is such that the gene product is stably produced for a period of time (e.g., about 1 day, about 2 days, about 4 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 12 months, or longer). In some embodiments, the dosage of the rAAV vector is such that the gene product is stably produced for about 1 week. In some embodiments, the dosage of the rAAV vector is such that the gene product is stably produced for about 2 weeks. In some embodiments, the dosage of the rAAV vector is such that the gene product is stably produced for about 3 weeks. In some embodiments, the dosage of the rAAV vector is such that the gene product is stably produced for about 4 weeks. In some embodiments, the dosage of the rAAV vector is such that the gene product is stably produced for about 1 month. In some embodiments, the dosage of the rAAV vector results in stable production of the gene product for approximately 2 months. In some embodiments, the dosage of the rAAV vector results in stable production of the gene product for approximately 3 months. In some embodiments, the dosage of the rAAV vector results in stable production of the gene product for approximately 4 months. In some embodiments, the dosage of the rAAV vector results in stable production of the gene product for approximately 5 months. In some embodiments, the dosage of the rAAV vector results in stable production of the gene product for approximately 6 months. In some embodiments, the dosage of the rAAV vector results in stable production of the gene product for approximately 9 months. In some embodiments, the dosage of the rAAV vector results in stable production of the gene product for approximately 12 months.

[0285] In some embodiments, the method described herein comprises administering to an individual an effective amount of the rAAV vector described herein, wherein the rAAV vector comprises a polynucleotide encoding at least one gene product (e.g., an anti-IGF1R antibody or an antigen-binding fragment). In some embodiments, the method comprises delivering a first dose and one or more subsequent doses of the rAAV vector. The one or more subsequent doses are administered some time after the first dose. In some embodiments, this time period between the first dose and the next subsequent dose is at least 1 day, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 9 months, at least 12 months, or longer. In some embodiments, this time period between the first dose and the next subsequent dose is between 1 and 7 days, between 1 and 4 weeks, between 2 and 6 weeks, between 4 and 8 weeks, between 1 and 3 months, between 2 and 4 months, between 3 and 6 months, between 4 and 12 months, or between 6 and 24 months. In some embodiments, the time interval between one or more subsequent doses is at least 1 day, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 9 months, at least 12 months, or longer. In some embodiments, the time interval between one or more subsequent doses is between 1 and 7 days, between 1 and 4 weeks, between 2 and 6 weeks, between 4 and 8 weeks, between 1 and 3 months, between 2 and 4 months, between 3 and 6 months, between 4 and 12 months, or between 6 and 24 months.

[0286] In some embodiments, the method comprises a first dose and one or more subsequent doses of the rAAV carrier. In some embodiments, one or more subsequent doses are administered a period of time after the first dose. This period of time between the first dose and the next subsequent dose is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours. The period of time between the first dose and the next subsequent dose is between 1 and 3 hours, 2 and 4 hours, 3 and 6 hours, or 4 and 8 hours. The period of time between one or more subsequent doses is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours. The period of time between one or more subsequent doses is between 1 and 3 hours, 2 and 4 hours, 3 and 6 hours, or 4 and 8 hours.

[0287] Pharmaceutical Compositions and Kits

[0288] In some embodiments, this disclosure provides a pharmaceutical composition comprising a carrier (e.g., an rAAV carrier) or an anti-IGF1R antibody or antigen-binding fragment as described herein. In some embodiments, the pharmaceutical composition comprises a carrier or anti-IGF1R antibody or antigen-binding fragment as described herein and a pharmaceutically acceptable carrier, delivery agent, or excipient.

[0289] In some embodiments, this disclosure provides the use of the carrier or pharmaceutical composition described herein for the manufacture of an agent for treating an eye disease, condition, or symptom. In some embodiments, this disclosure provides the use of a carrier or anti-IGF1R antibody or antigen-binding fragment or a pharmaceutical composition comprising any of these components for the treatment of an eye disease, condition, or symptom.

[0290] In some embodiments, a pharmaceutically acceptable carrier comprises phosphate-buffered saline. In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intralacrimal). In some embodiments, the pharmaceutical composition is formulated for administration into the lacrimal gland. In some embodiments, the pharmaceutical composition is formulated for administration onto the ocular surface.

[0291] In some embodiments, this disclosure provides a pharmaceutical composition for treating an individual's ocular condition, wherein the pharmaceutical composition comprises a carrier (e.g., an rAAV carrier) encoding an anti-IGF1R antibody or antigen-binding fragment according to any aspect described herein and at least one pharmaceutically acceptable carrier. Carriers of any concentration suitable for effective transduction or transfection of cells of the eye, lacrimal gland, meibomian gland, and / or trabecular meshwork can be prepared for in vitro or in vivo contact with cells of the eye, lacrimal gland, meibomian gland, and / or trabecular meshwork. For example, a concentration of 10... 8 One vector genome / mL or higher, such as 5 × 10⁸ vectors. 8 One vector genome / mL, 10 9 One vector genome / mL, 5×10 9 One vector genome / mL, 10 10 One vector genome / mL, 5×10 10 One vector genome / mL, 10 11 One vector genome / mL, 5×10 11 One vector genome / mL, 10 12 One vector genome / mL, 5×10 12 One vector genome / mL, 10 13 One vector genome / mL, 1.5 × 10⁻⁶ 13 One vector genome / mL, 3×10 13 One vector genome / mL, 5×10 13 One vector genome / mL, 7.5 × 10⁻⁶13 One vector genome / mL, 9×10 13 One vector genome / mL, 1×10 14 One vector genome / mL, 5×10 14 One vector genome / mL or higher, but usually not exceeding 1×10⁻⁶. 15 One vector genome / mL. Similarly, any total number of vectors (e.g., rAAV vectors) suitable for providing appropriate transduction or transfection to cells and / or trabecular meshwork structures of the eye, lacrimal gland, meibomian gland, or other cellular structures to confer the desired effect or treat a disease can be administered to the eyes of mammals or primates. In various embodiments, at least 10 genotypes per eye are injected. 5 2.5×10 5 5×10 5 7.5×10 5 10 6 2.5×10 6 5×10 6 7.5×10 6 10 7 2.5×10 7 5×10 7 7.5×10 7 10 8 2.5×10 8 5×10 8 7.5×10 8 10 9 2.5×10 9 5×10 9 7.5×10 9 10 10 2.5×10 10 5×10 10 7.5×10 10 10 11 2.5×10 11 5×10 11 7.5×10 11 10 12 2.5×10 12 5×10 12 7.5×10 12 10 13 2.5×10 13 5×10 13 7.5×10 13 10 14 2.5×10 14 5×10 14 7.5×10 14 10 152.5×10 15 5×10 15 Or 7.5×10 15 10 16 2.5×10 16 5×10 16 Or 7.5×10 16 One or more carriers, but typically not exceeding 1×10⁻⁶ 15 One carrier. For example, in some embodiments, approximately 1 × 10⁻⁶ is applied to each eye. 9 To approximately 1×10 10 Approximately 1×10 10 To approximately 1×10 11 Approximately 1×10 11 To approximately 1×10 12 Approximately 1×10 12 To approximately 1×10 13 Or approximately 1×10 13 To approximately 1×10 15 A genomic copy of the vector. In some aspects, the total number of vectors administered to the eyes of the treated human individual or animal may include concentrations within the range defined by any pair of concentrations described in this paragraph. Any suitable number of vector administrations may be performed to the eyes of mammals or primates. In some embodiments, the method comprises a single administration; in other embodiments, multiple administrations may be performed over time as deemed appropriate by the attending clinician.

[0292] In some embodiments, a suitable amount or concentration of the carrier (or any other expression construct configured to express an anti-IGF1R antibody or antigen-binding fragment) in the therapeutic formulation may be a concentration that effectively expresses an anti-IGF1R antibody or antigen-binding fragment of 100 pg / mL to 50 µg / mL in the individual's tear film after administration of the composition to the individual. For example, the amount or concentration of the carrier may be the amount or concentration that produces the following results: expression of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 µg / mL of anti-IGF1R antibody or antigen-binding fragment (or concentrations within the range defined by any of the foregoing values) in the tear film of an individual. Expression can be measured at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hours after application or after a longer duration, such as 1, 2, 3, 4 or 5 days.

[0293] The carrier can be formulated into any suitable unit dose, including but not limited to 1×10⁻⁶. 8 One vector genome or more, for example 1×10 9 1×10 10 1×10 11 1×10 12 Or 1×10 13 One vector genome or more, in some cases 1×10 14 One vector genome, but usually no more than 4 × 10⁻⁶. 15 One vector genome. In some embodiments, the viral vector is formulated to any suitable unit dose, including but not limited to 1 × 10⁻⁶. 7 1×10 8 1×10 9 1×10 10 1×10 11 1×10 12 Or 1×10 13 One vector genome or more. In some embodiments, the unit dose is up to about 5 × 10⁻⁶. 15 One vector genome, for example 1×10 14 One vector genome or fewer, such as 1×10 13 1×10 12 1×10 11 1×1010 Or 1×10 9 One vector genome or fewer, in some cases 1×10 8 One vector genome or fewer, and usually no less than 1 × 10⁻⁶ 8 One vector genome. In some embodiments, the unit dose is 1 × 10⁻⁶. 10 Up to 1×10 11 One vector genome. In some cases, the unit dose is 1×102 10 Up to 3×10 12 One vector genome. In some embodiments, the unit dose is 1 × 10⁻⁶. 9 Up to 3×10 13 One vector genome. In some embodiments, the unit dose is 1 × 10⁻⁶. 8 Up to 3×10 14 A vector genome. In some embodiments, the vector is an rAAV vector comprising an AAV shell and an expression cartridge configured to express an anti-IGF1R antibody or antigen-binding fragment, and the unit dose is at least, at most, exactly, or about 1 × 10⁻⁶. 5 1×10 6 1×10 7 1×10 8 1×10 9 1×10 10 1×10 11 1×10 12 1×10 13 1×10 14 1×10 15 Or 1×10 16 The concentration of an rAAV vector, or the concentration within the range defined by any pair of concentrations described in this paragraph.

[0294] In some embodiments, this disclosure provides a vector comprising a plasmid configured to express an anti-IGF1R antibody or an antigen-binding fragment, wherein the unit dose is at least, at most, exactly, or about 1 × 10⁻⁶. 7 5×10 7 1×10 8 5×10 8 1×10 9 5×10 9 1×10 10 5×10 10 1×10 11 5×10 11 1×10 12 5×10 12 1×10 13 5×10 13 1×1014 5×10 14 Or 1×10 15 The concentration of each plasmid (or the concentration within any of the aforementioned concentration ranges) is adjusted.

[0295] In some embodiments, the unit dose of the pharmaceutical composition may be measured using the multiplicity of infection (MOI). MOI refers to the ratio or fold of the viral genome of the vector or rAAV vector provided herein to the number of cells into which nucleic acids can be delivered. In some embodiments, the MOI may be 1 × 10⁻⁶. 6 In some embodiments, the MOI can be 1×10. 5 Up to 1×10 7 In some cases, the MOI can be 1×10. 4 Up to 1×10 8 In some embodiments, the recombinant virus of this disclosure is at least about 1 × 10⁻⁶. 1 1×10 2 1×10 3 1×10 4 1×10 5 1×10 6 1×10 7 1×10 8 1×10 9 1×10 10 1×10 11 1×10 12 1×10 13 1×10 14 1×10 15 1×10 16 1×10 17 and 1×10 18 MOI. In some embodiments, the recombinant virus of this disclosure is 1×10 8 Up to 3×10 14 MOI. In some embodiments, the recombinant virus of this disclosure is at most about 1×10⁻⁶. 1 1×10 2 1×10 3 1×10 4 1×10 5 1×10 6 1×10 7 1×10 8 1×10 9 1×10 10 1×10 11 1×10 12 1×10 13 1×1014 1×10 15 1×10 16 1×10 17 and 1×10 18 MOI.

[0296] In some embodiments, the amount of the pharmaceutical composition comprises about 1 × 10⁻⁶. 8 To approximately 1×10 15 One rAAV carrier, approximately 1×10 9 To approximately 1×10 14 One rAAV carrier, approximately 1×10 10 To approximately 1×10 13 One rAAV carrier or approximately 1×10 11 Approximately 3×10 12 One rAAV carrier.

[0297] In preparing the subject rAAV composition, any host cell used to generate the rAAV vector can be used, including, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell can also be an encapsulation cell in which the AAV rep and cap genes are stably maintained; or a production cell in which the rAAV vector genome is stably maintained and encapsulated. Exemplary encapsulation and production cells are derived from SF-9, 293, A549, or HeLa cells. The rAAV vector is purified and formulated using standard techniques known in the art.

[0298] In some embodiments, this disclosure provides the use of the rAAV carriers described herein in the manufacture of pharmaceutical agents. In some embodiments, this disclosure provides the use of the rAAV carriers described herein for the manufacture of pharmaceutical agents used in the methods described herein.

[0299] In some embodiments, this disclosure provides a kit comprising the rAAV described herein and instructions for use. In some embodiments, the kit comprises the rAAV described herein and a pharmaceutical instruction manual containing instructions for use of the kit. In some embodiments, the kit comprises the rAAV described herein and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising rAAV, and instructions for use to treat or delay the progression of the disease, condition, or symptom described herein in an individual in need.

[0300] Example

[0301] The specific examples below should be understood as illustrative only and in no way limit the remainder of this disclosure.

[0302] Example 1: Preparation of rAAV vector for expressing anti-IGF1R constructs

[0303] Prepare rAAV vector nucleotides containing expression cartridges encoding antigen-binding fragments of anti-IGF1R antibodies. Figure 1 The image depicts a vector map of the plasmid containing this rAAV vector and provides the nucleotide sequence of this vector (SEQ ID NO: 23). In this case, the antigen-binding fragment of the anti-IGF1R antibody is an scFv construct containing a heavy chain variable domain (SEQ ID NO: 2) linked to a light chain variable domain (SEQ ID NO: 6) via a GS adapter sequence (SEQ ID NO: 42), as shown below. Figure 1 As shown in Table 1, the putative CDR regions in the two domains were predicted using the Chothia, North, and IMGT CDR definitions. The “Chothia,” “North,” and “IMGT” columns in Table 1 refer to the alternative numbering system. See Dondelinger, M, et al., “Understanding the significance and implications of antibody numbering and antigen-binding surface / residue definition.” Frontiers in Immunology 9 (2018):2278. Figure 1 As shown, this plasmid of the rAAV vector further illustrates the use of additional components described in this application, such as the CMV enhancer and promoter (SEQ ID NO: 27), the WPRE sequence (SEQ ID NO: 29), the BGH polyA sequence (SEQ ID NO: 28), and the 5' and 3' ITRs (SEQ ID NO: 30 and 31, respectively).

[0304] This plasmid, similar plasmids, or other vectors described herein can be used to transfect or transduce one or more cells, tissues, or organs (e.g., the eye or accessory lacrimal gland) of a human individual to produce expression of an anti-IGF1R antibody or its antigen-binding fragment (e.g., the exemplary scFv used in this example). Furthermore, this plasmid and other similar plasmids described herein can be used to create rAAV vectors that can be used to transduce cells, tissues, or organs of a human individual.

[0305] Example 2: IGF1R ELISA assay

[0306] Exemplary scFv constructs based on the heavy chain variable domain and light chain variable domain sequences described herein were expressed by plasmid constructs to evaluate the binding ability and other properties of such constructs.

[0307] First, plasmid DNA from the VH-VL and VL-VH scFv constructs was transfected into HeLa cells. The VH-VL construct contains SEQ ID NO: 44 and the VL-VH construct contains SEQ ID NO: 43. Both constructs further include a 6×His tag sequence at the C-terminus for purification. Cells were cultured and conditioned medium was collected for a human IGF1R ELISA (Abcam: ab275102). The transgenic product from the conditioned medium was used as a sample in a human IGF1R ELISA machine to assess the ability of the transgenic product to bind to the intended target IGF1R.

[0308] The results of this IGF1R ELISA experiment are shown in Figure 3 And is defined in Table 4 below. Figure 3 In the diagram, wells associated with the two test constructs are labeled (blue rectangles); wells A to H in the left column represent the standard curve. These results indicate that both conformations of the scFv transgenic gene (VH-VL and VL-VH) can bind to IGF1R. However, this analysis did not indicate the binding level or binding affinity.

[0309]

[0310] Table 4 . Figure 3 An overview of the ELISA results shown.

[0311] Example 3: Combining affinity / inhibition and kinetic studies

[0312] Conditioned culture media containing VH-VL and VL-VH scFv constructs were sent to Gifford Bioscience Limited (Birmingham, United Kingdom) for Biacore® assays to determine the kinetic parameters of binding between the transgenic product and IGF1R (R&D Systems 305-GR-050). A method for determining the association rate (“k-coupling rate”) of binding to the following substances was developed using a Biacore T200® instrument. on ") and affinity ("k DThe method used included: natural IGF1 ligand (R&DSystems® 291-G1-01M), VH-VL and VL-VH scFv transgene products, and the commercially available human IGF1R monoclonal antibody tetumumab (Proteogenix® PX-TA1242-100). Two off-target receptors, insulin receptor A (“IR-A”) and insulin receptor B (“IR-B”), were used as experimental controls. Additionally, the ability of these reagents to block the binding of the IGF1 ligand to its endogenous receptor IGF1R was evaluated. In short, the ligand (IGF1R, 20 μg / mL at 10 mM sodium acetate pH) was desorbed, pre-cleaned, and immobilized on the CM5 S series sensor (Cytiva® 100530). 4.5 (in part), prepare the Biacore T200 surface plasmon resonance (“SPR”) sensor. The IGF1R protein is immobilized on the sensor surface via amine coupling: the CM5 sensor surface is activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (“EDC”)-N-hydroxy-succinimide (“NHS”), followed by coupling the IGF1R protein to the surface, and then inactivating all unreacted EDC-NHS groups with ethanolamine.

[0313] For binding and reproducibility studies, prepare IGF1 and tetumumab at single concentrations in operating buffer (HBS-T or PBS-T) (supply / reconstitute in PBS). Inject samples in quadruplicates, then reconstitute to baseline before starting the next binding cycle. Include buffer blank injections before and after each analyte quadruplicate set to subtract blank cycles.

[0314] For kinetic affinity studies, IGF1 and ticutometab were prepared in PBS-T at 10 concentrations using a 2-fold serial dilution. Samples were injected in ascending concentration order and then regenerated back to baseline. Buffer blank injections were included before and after each analyte concentration series to subtract blank cycles, thereby correcting for any systematic sensor drift.

[0315] For binding inhibition studies, tetrumumab was prepared in PBS-T at a single concentration and IGF1 at two concentrations. For each binding cycle, as a “capture” step, tetrumumab was first injected to allow the mAb to bind to the immobilized IGF1R surface. With the mAb still bound (i.e., no regeneration step), IGF1 was subsequently injected during a second “binding” step to bind to any remaining IGF1R that had not bound to the mAb. The surface was then regenerated back to baseline. A buffer blank injection was used instead of tetrumumab during the capture step to measure the maximum IGF1 binding response (100% binding or 0% inhibition) in the absence of tetrumumab. With tetrumumab injected and captured during the first “capture” step, a buffer blank injection was used during the second “binding” step to provide a reference cycle without IGF1 binding, subtracting the mAb dissociation rate. All sample combinations were repeated in duplicate.

[0316] For kinetic affinity calculations: Reference channels, namely Fc2-1, Fc3-1, and Fc4-1, were subtracted from the binding reactions of IGF1 and ticutoxumab kinetic affinity concentration series with IGF1R (Fc2), IR-A (Fc3), and IR-B (Fc4) to remove any bulk sample effect. Buffer blank cycles were then subtracted from the data to remove any sensor drift. IGF1 binding data were fitted to a two-state binding model, with the refractive index (“RI”) correction factor set to a local constant of “0”. Similarly, reference channels were subtracted from the ticutoxumab binding data, followed by subtraction of blank buffer cycles. The data were then fitted to a unit point binding model, with the RI correction factor set to a local constant of “0”.

[0317] For the calculation of binding inhibition: IGF1 binding responses in the absence of tetumumab (cycles 7 to 12) were zeroed on the Y-axis to the baseline report point, averaged within 5 seconds prior to the start of IGF1 injection. The reference channel was subtracted from the data, followed by the buffer + buffer blank, with cycle 7 used as the blank. The data were then exported as a .csv file.

[0318] In the presence of tetrumumab (cycles 13 to 18), the IGF1 binding reaction was also zeroed to the baseline reporter point, followed by the subtraction of the reference channel, and then the subtraction of tetrumumab + buffer, with cycle 13 used as a blank (to correct for the slow tetrumumab dissociation rate). The data were then exported as a .csv file.

[0319] Two exported datasets of IGF1 binding in the absence and presence of tetrumumab (using different blank cycles to correct for different baseline drifts) were imported into Excel for further analysis. Data on the time intervals between IGF1 injection and dissociation (400 to 1200 seconds) were plotted. The maximum binding response amplitude of two duplicate IGF1 assays at 100 nM and 10 nM in the absence or presence of 50 nM tetrumumab was averaged during the last 5 seconds of the association phase. The inhibition of IGF1 binding in the presence of tetrumumab was calculated as the percentage reduction in binding response.

[0320] result The target receptor (IGF1R) and the two associated off-target receptors (IR-A and IR-B) are immobilized on Fc2-4 at very similar levels, ensuring equal receptor density on the sensor surface. Reproducibility studies in HBS-T and PBS-T showed that using PBS-T as the operating buffer resulted in more consistent binding responses between IGF1 and tetamumab. Furthermore, eliminating the mismatch between the HBS-T operating buffer and the PBS sample buffer resulted in a smaller bulk effect. Extending the stabilization period to 180 seconds also improved recovery from baseline and thus improved binding response consistency. The kinetic binding affinities for IGF1 binding to IGF1-R, IR-A, and IR-B yielded KDs of 5.1 nM, 89.6 nM, and 272 nM, respectively. The binding response data best fit the two-state model. Without being bound by theory, this suggests a negative cooperativity of IGF1 binding to all three receptors, similar to the known negative cooperativity of insulin binding to IR-A and IR-B.

[0321] The kinetic affinity of ticutlimumab was determined to be best-fitted by a unit-point model, with a KD of 96 pM (0.096 nM). Binding of ticutlimumab to IR-A and IR-B was negligible. In binding competition studies, blocking the IGF1-R surface with 50 nM ticutlimumab reduced IGF1 binding by 59% at 10 nM and by 54% at 100 nM, compared to IGF1 binding at the same concentration in the absence of ticutlimumab. However, even at sufficiently high concentrations, the blocking effect of 50 nM ticutlimumab on the IGF1R surface did not reach the equilibrium plateau (50 nM = 500 × KD, equivalent to >99% binding).

[0322] The results of this study are provided Figures 4 to 10 and Table 5 (Overview) Figure 10 (as shown in the results).

[0323] The data demonstrate that the binding of IGF1 and IGF1R in PBS-T exhibits remarkably similar and reproducible continuous responses, and these responses stabilize to the original baseline after regeneration. Figures 6 to 7 In PBS-T, the binding of tetrumumab to IGF1R showed a slightly more variable sequential response. Figure 7 The reaction stabilized rapidly to the original baseline after regeneration. Figures 8 to 9 Display IGF-1 ( Figure 8 ) and tetromumab® Figure 9 The kinetic activity results, and Figure 10 The data show the kinetic activity of IGF-1 binding to IGF-1R in the presence and absence of tetumumab. The addition of tetumumab resulted in 59% inhibition of 10 nM IGF-1 and 54% inhibition of 100 nM IGF-1, as summarized in Table 5 below.

[0324]

[0325] Table 5 Tiltromab inhibits IGF1 binding.

[0326] Example 4: Binding determination using VL-VH and VH-VL scFv constructs

[0327] The following set of studies was conducted to test scFv transgenic products (VL-VH and VH-VL) from unpurified conditioned medium. Plasmid DNA encoding the respective VH-VL and VL-VH constructs was transfected into HeLa cells and cultured for 72 hours. The conditioned medium was collected, frozen, and transported on dry ice for sample analysis. The conditioned medium was not purified or enriched. The experimental samples (VH-VL and VL-VH) were serially diluted with Advanced DMEM, 5% fetal bovine serum (FBS), 1× penicillin-streptomycin, and 1× Glutamax. Analytes were tested as described in Table 6 below.

[0328]

[0329] Table 6 The concentration parameters of the analytes used in this experiment.

[0330] The target receptor (IGF1R) and the two associated off-target receptors (IR-A and IR-B) were immobilized on Fc2-4 at levels similar to those described in Example 3, ensuring equal receptor density on the sensor surface. For this phase of the study, the receptors were immobilized at a lower density (<4,000 RU) than in Phase 1 (approximately 10,000 RU) to minimize sample rebinding during the dissociation phase of the binding cycle. This provides more reliable measurements of sample dissociation rates and subsequent KD value calculations.

[0331] The recombinant small protein VL-VH scFv (“VL-VH”) is selective for IGF1R. No binding response was observed at the relevant receptors IR-A or IR-B. The concentration of the His-tagged protein of interest in VL-VH and VH-VL conditioned medium samples was estimated to be 4 nM (based on ELISA data from conditioned medium samples). The VL-VH binding response was fitted to a single-site binding model, yielding a KD value of 7.53 nM.

[0332] The unpurified second small protein, VH-VL scFv (“VH-VL”), does not bind to any of the three receptors.

[0333] The kinetic affinity values ​​for binding between the endogenous ligand IGF1 and the mAb tetumumab were also determined under conditions of lower receptor protein levels immobilized on the surface compared to Phase 1. The response amplitude of IGF1 to IGF1R was smaller than that observed in Phase 1, but the KD value of 8.74 nM was similar to the previously determined KD value (5.1 nM). Similarly, the tetumumab binding response at IGF1R was smaller compared to Phase 1 (KD value of 0.096 nM), but had a similarly high affinity KD value (0.42 nM). These data indicate that the unpurified transgenic product present in the conditioned medium of the VL-VH scFv construct exhibited a binding affinity of 8.74 nM for IGF-1R; while the alternative scFv conformation (VH-VL) did not exhibit binding affinity. Furthermore, the association constant of the transgenic product present in the conditioned medium exhibited an association constant similar to that of the purified commercial product tetumumab. This data is described in Table 7 below.

[0334]

[0335] Table 7 Binding data for the IGF1R target receptor of unpurified samples of VL-VH and VH-VL scFv constructs compared to IGF1 ligands and tetumumab.

[0336] Other results of this study show that Figures 11 to 12 In short, IGF1R (Fc2) was used as a ligand (flow channel) to test unpurified samples of VL-VH and VH-VL constructs at concentrations from 0.25 to 4 nM, and the largest observed binding reaction was 107 RU ( ) for the VL-VH construct at 4 nM. Figure 11 No binding reaction of the VH-VL construct was detected. Figure 12 The kinetic affinity data for the VL-VH and VH-VL constructs are summarized in Table 8 below.

[0337]

[0338] Table 8 Kinetic affinity fitting of unpurified samples of VL-VH and VH-VL scFv constructs.

[0339] Example 5: Analysis of purified VH-VL and VL-VH scFv constructs

[0340] Purified VH-VL and VL-VH scFv constructs were subsequently generated for further investigation. In short, 296-6E human embryonic kidney cells were transfected with plasmid DNA encoding VL-VH and VH-VL, including a 6×His tag. Cells were cultured in Freestyle F17 (ThermoFisher® A1383501) supplemented with 0.1% Pluronic F-68 (Gibco® 24040-032), 4 nM GlutaMax (Gibco 35050-061), and 25 μg / mL Geneticin (Gibco® 10131-027). Cell density was maintained at 0.3–3.0 × 10⁶ cells / mL in shake flasks. 6 Cells / mL, the shake flasks were incubated at 37°C, 5% CO2, and shaken at 135 rpm. PolyPlus PEIPro was used to transfect 296-6E cells with the VL-VH plasmid at a 1:1 w / v DNA:PEI ratio, in a 600 mL scale and the VH-VL plasmid at a 200 mL scale. Due to previous results using unpurified proteins, and given the previously excellent binding data, additional studies with this transfected gene product were required; therefore, a larger quantity of VL-VH was prepared. Cell cultures were harvested by centrifugation 4–6 days post-transfection. The conditioned medium supernatant was clarified by centrifugation at 9,300 × g for 30 min.

[0341] To purify these transgenic products via 6×His tagging, Ni-NTA His-Bind (EMD Millipore) resin was loaded onto a 1 cm diameter Bio-Rad® Econo-column, as recommended by the resin manufacturer. Supernatants from each conditioned medium were adjusted to 20 mM imidazole and top-loaded into the Ni-NTA resin column by gravity flow for 3–4 hours at 4°C. The resin was washed with 20 column volumes of Buffer A (1×PBS (2.7 mM KCl, 1.7 mM KH₂PO₄, 136 mM NaCl, 10.1 mM Na₂HPO₄) + 20 mM imidazole, pH 7.4), followed by elution of the His-tagged proteins with Buffer B (Buffer A + 280 mM imidazole, pH 7.4). The eluates were collected and absorbance measured at 280 nM on a spectrophotometer, then pooled. Selected eluates were analyzed by SDS-PAGE and stained with Instant Blue. Figure 13 The SDS-PAGE results of the affinity chromatography eluent of the VL-VH construct are shown, and Figure 14 The SDS-PAGE results of the affinity chromatography eluents of the VH-VL construct are shown. An overview of the purification results is also provided in Tables 9 and 10. Figures 13 to 14 As depicted in lane 4 of the SDS-PAGE gel shown, the eluted portion (IMAC pool) exhibits a single protein band at approximately 25 kDa. The eluted protein sample was subsequently dialyzed in 1×PBS (2.7 mM KCl, 1.7 mM KH2PO4, 136 mM NaCl, 10.1 mM Na2HPO4) at pH 7.4 to remove imidazole.

[0342]

[0343] Extinction coefficient A 280 2.1 = 1 mg / mL

[0344] Table 9. Analysis of the purified VL-VH scFv product generated in this experiment.

[0345]

[0346] Extinction coefficient A 280 2.1 = 1 mg / mL

[0347] Table 10 The assay for the purified VH-VL scFv product produced in this experiment was changed to a determination.

[0348] Protein samples were rapidly frozen in liquid nitrogen and stored at -80°C, then transported on dry ice for further Biacore analysis. Purified VL-VH and VH-VL samples were subsequently subjected to Biacore analysis again to assess binding affinity and the ability to block the binding of IGF1 ligands to their natural receptor IGF1R. Results showed higher levels of binding to IFG1-R and inhibition of IGF-1 binding to IFG1-R, with both the VL-VH and VH-VL constructs exhibiting improved performance compared to commercially available tetumumab. These results provide... Figures 15 to 23 And Tables 11 to 13. These results indicate that the VL-VH construct exhibits significantly higher levels of IGF1R binding and inhibition of IGF1 binding, exceeding the levels of the commercially available and marketable tetamumab control. Although both VH-VL and VL-VH bind to IGF1R and inhibit IGF1 binding, there are significant differences between them, highlighting the importance of cloning sequence and transgenic product conformation.

[0349] Figures 15-16 Draw VL-VH respectively Figure 15 ) and VH-VL ( Figure 16 The kinetic affinity of the construct for IGF1R. For Figures 15-16 The measurements shown were performed using IGF1R (Fc4) as the immobilized ligand, with VL-VH and VH-VL constructs tested at concentrations ranging from 0.1 to 50 nM. The maximum observed binding response was 430 RU for the VL-VH construct at 50 nM and 359 RU for the VH-VL construct at 50 nM. Kinetic affinity fittings are described in Table 11 below.

[0350]

[0351] Table 11 Kinetic affinity fitting of purified VL-VH and VH-VL scFv constructs.

[0352] Figures 17 to 20 Instructions are provided for purifying VL-VH ( Figures 17-18 ) and VH-VL ( Figures 19 to 20 Binding response data for the VL-VH construct inhibiting IGF1-IGF1R binding. Tables 12 and 13 summarize the results of these studies. These data demonstrate that the commercially available human monoclonal antibody tetumumab exhibits 59% inhibition of IGF1-IGF1R binding; while the VL-VH construct exhibits 78.9% inhibition. Figure 18 Furthermore, the VH-VL construct exhibited 54.6% inhibition ( Figure 19 ).

[0353] Inhibition of IGF1 binding by VL-VH

[0354]

[0355] Table 12 Inhibition of IGF1 and IGF1R binding by VL-VH at different test concentrations.

[0356] Inhibition of IGF1 binding by VH-VL

[0357]

[0358] Table 13 Inhibition of IGF1 and IGF1R binding by VH-VL at different test concentrations.

[0359] As illustrated in the studies discussed above, purified samples of the VH-VL and VL-VH scFv constructs expressed better than their unpurified equivalents. Without being bound by theory, potential competition from crude proteins and non-specific proteins could interfere with the interaction between VH-VL and the VL-VH scFv constructs in the crude sample; therefore, the results of the study described above (Example 4) are not inconsistent with the results of the study based on purified samples of the VH-VL and VL-VH scFv constructs (Example 5).

[0360] Example 6: Comparison of purified VH-VL and VL-VH scFv constructs with tetumumab

[0361] The purified VH-VL and VL-VH constructs generated in Example 5 were evaluated relative to the commercially available and marketable product tetumumab, which served as a baseline. This study demonstrated that the purified VL-VH scFv transgene product had a significantly lower IC50 in terms of blocking IGF1 binding to IGF1R and the percentage of protein binding to IGF1R. 50 and nominally lower EC 50 .like Figures 21 to 23 As demonstrated, VL-VH IC effectively blocks IGF1 binding. 50 11.49 nM, VH-VL IC 50 The concentration was 22.26 nM, and the tetumumab IC50 was... 50 The concentration was 31.85 nM. Therefore, compared with commercially available tetumumab, both the VL-VH and VH-HL constructs showed enhanced inhibition of IGF1 binding to IGF1R. Furthermore, the ECG of the VL-VH construct binding to IGF1R was significantly lower. 50 The EC50 of tetumumab was 21.74 nM, compared to which the EC50 of tetumumab was 21.74 nM. 50 It is 24.09 nM.

[0362] Overall, these results indicate that the VL-VH scFv construct is compatible with tetoduramab (a human monoclonal antibody, which is Tepezza) ® Compared to the active biological therapeutic agent (which is FDA-approved for the treatment of thyroid eye disease), it showed superior results, while the alternative VH-VL scFv construct also showed better results than tetumumab, but not as good as VL-VH. Furthermore, VL-VH showed superior results compared to VH-VL, indicating that the conformation of the variable domain is important.

[0363]

[0364] Finally, it should be understood that although aspects of this specification are highlighted by reference to specific embodiments, those skilled in the art will readily recognize that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that, unless explicitly stated otherwise, the subject matter described herein is by no means limited to the specific compounds, compositions, objects, apparatuses, methods, schemes, and / or reagents described herein. Furthermore, those skilled in the art will recognize that certain changes, modifications, substitutions, alterations, additions, subtractions, and sub-combinations thereof can be made based on the teachings herein without departing from the spirit of this specification.

[0365] The use of the terms "may" or "may" in reference to embodiments or aspects of embodiments also carries the alternative meaning of "may not" or "cannot". Therefore, if an embodiment or aspect of an embodiment disclosed in this specification may or may be included as part of the subject matter of the invention, then a negative limitation or exclusionary condition also expressly means that the embodiment or aspect of an embodiment may not or cannot be included as part of the subject matter of the invention. Similarly, the use of the term "optionally" in reference to embodiments or aspects of embodiments means that the embodiment or aspect of an embodiment may be included as part of the subject matter of the invention or may not be included as part of the subject matter of the invention. Whether such negative limitations or exclusionary conditions apply will depend on whether the negative limitation or exclusionary condition is set forth in the claimed subject matter.

[0366] Although the numerical ranges and values ​​used to illustrate the broad scope of the invention are approximations, the numerical ranges and values ​​described in specific examples are reported as precisely as possible. However, any numerical range or value inherently contains some error that is necessarily caused by the standard deviation found in its individual test measurements. The description of numerical ranges of values ​​herein is intended only as a way of individually referring to the individual values ​​belonging to the ranges. Unless otherwise indicated herein, the individual values ​​of the numerical ranges are incorporated into this specification as if individually described herein (e.g., any disclosure of a range with integer endpoints should be interpreted as also describing a subrange defined by any pair of integers within a wider range).

[0367] Unless otherwise indicated herein or clearly contradicted by the context, the terms “a,” “an,” “the,” and similar reference terms used in the context of describing the invention (particularly in the context of the following claims) should be interpreted to cover both the singular and the plural. Furthermore, sequence indicators for identified elements (e.g., “first,” “second,” or “third,” etc.) are used to distinguish between elements and do not indicate or imply a desired or limited number of such elements, nor do they indicate a particular position or order of such elements unless otherwise specifically stated. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all example or exemplary language provided herein (e.g., “for example”) is intended only to better illustrate the invention and does not limit the scope of the invention as otherwise claimed. The language in the specification should not be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0368] When used in claims, whether as applied or added according to amendments, the open transitional term “comprising” (and its equivalent open transitional phrases, such as including, containing, and having) covers all expressly stated elements, limitations, steps, and / or features, alone or in combination with unstated subject matter; named elements, limitations, and / or features are necessary, but other unstated elements, limitations, and / or features may be added and still form an construct within the scope of the claims. Specific embodiments disclosed herein may be further limited in claims by using the closed transitional phrases “consisting of” or “substantially consisting of” instead of “comprising” or as modifications of “comprising”. When used in claims, whether as applied or added according to amendments, the closed transitional phrase “consisting of” excludes any elements, limitations, steps, or features not expressly stated in the claims. The closed transitional phrase “substantially consisting of” limits the scope of the claims to expressly stated elements, limitations, steps, and / or features and any other elements, limitations, steps, and / or features that do not substantially affect the essential and novel features of the claimed subject matter. Therefore, the meaning of the open transition phrase "comprising" is as defined to cover all elements, limitations, steps, and / or features of a particular statement, as well as any optional additional unspecified elements, limitations, steps, and / or features. The meaning of the closed transition phrase "consisting of" is as defined to include only those elements, limitations, steps, and / or features of a particular statement in the claims, while the meaning of the closed transition phrase "substantially constitutes" is as defined to include only those elements, limitations, steps, and / or features of a particular statement in the claims and those elements, limitations, steps, and / or features that do not substantially affect the essential and novel features of the claimed subject matter. Therefore, the open transition phrase "comprising" (and its equivalent open transition phrases) includes, as a limitation, the claimed subject matter specified by the closed transition phrases "consisting of" or "substantially constitutes". Therefore, the embodiments described herein or claimed as in conjunction with the phrase "comprising" expressly or inherently describe, implement, and support the phrases "substantially constitutes" and "consisting of".

[0369] All patents, patent publications, and other disclosures referenced and identified in this specification are individually and expressly incorporated herein by full reference for the purpose of describing and disclosing, for example, the compositions and methods described in these disclosures that may be used in conjunction with the present invention. These disclosures provide only those prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventor has no prior right to such disclosures by virtue of a prior invention or for any other reason. All statements regarding dates or representations regarding the content of these documents are based on information available to the applicant and do not constitute any admission of the accuracy of the dates or content of these documents.

[0370] Finally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims. Therefore, the invention is not limited to what is accurately shown and described.

[0371] sequence list

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

Claims

1. An antibody or antigen-binding fragment that specifically binds to the human insulin-like growth factor 1 receptor ("IGF1R") protein, comprising: a) Heavy-chain variable fields, which contain A polypeptide sequence having SEQ ID NO: 3 or a CDR1 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO:

3. A polypeptide sequence having SEQ ID NO: 4 or a CDR2 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 4, and / or A polypeptide sequence having SEQ ID NO: 5 or a CDR3 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 5; and / or b) Light chain variable fields, which contain A polypeptide sequence having SEQ ID NO: 7 or a CDR1 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO:

7. A polypeptide sequence having SEQ ID NO: 8 or a CDR2 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 8, and / or The polypeptide sequence having SEQ ID NO: 9 or the CDR3 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO:

9.

2. The antibody or antigen-binding fragment according to claim 1, wherein... The heavy chain variable domain comprises a CDR1 region having the polypeptide sequence of SEQ ID NO:

3. The heavy chain variable domain comprises a CDR2 region having a polypeptide sequence of SEQ ID NO: 4, and / or The heavy chain variable domain comprises a CDR3 region having the polypeptide sequence of SEQ ID NO: 5; and / or The light chain variable domain includes a CDR1 region having the polypeptide sequence of SEQ ID NO:

7. The light chain variable domain includes a CDR2 region having the polypeptide sequence of SEQ ID NO: 8, and / or The light chain variable domain includes a CDR3 region having the polypeptide sequence of SEQ ID NO:

9.

3. The antibody or antigen-binding fragment according to claim 1, wherein... The heavy chain variable domain includes a CDR1 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO:

3. The heavy chain variable domain includes a CDR2 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 4, and / or The heavy chain variable domain includes a CDR3 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 5; and / or The light chain variable domain includes a CDR1 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO:

7. The light chain variable domain includes a CDR2 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO: 8, and / or The light chain variable domain includes a CDR3 region having 1, 2, 3, 4, or 5 inserted, substituted, or deleted polypeptide sequences compared to SEQ ID NO:

9.

4. The antibody or antigen-binding fragment according to claim 1, wherein... The heavy chain variable domain comprises the polypeptide sequence of SEQ ID NO: 2; and / or The light chain variable domain contains the polypeptide sequence of SEQ ID NO:

6.

5. The antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment specifically binds to: a) An IGF1R protein having the polypeptide sequence of SEQ ID NO: 1, or a fragment thereof containing at least 20 consecutive amino acids; b) An α-chain of an IGF1R protein having a polypeptide sequence comprising positions 31-736 of SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids; and / or c) A β chain of an IGF1R protein having a polypeptide sequence comprising positions 741-1367 of SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids.

6. The antibody or antigen-binding fragment according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is a rabbit antibody or antigen-binding fragment, or a mouse antibody or antigen-binding fragment.

7. The antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment.

8. The antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment comprises a monoclonal antibody or antigen-binding fragment, a transplanted antibody or antigen-binding fragment, a chimeric antibody or antigen-binding fragment, a human antibody or antigen-binding fragment, or a humanized antibody or antigen-binding fragment.

9. The antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein the antigen-binding fragment comprises: Fab, Fab', F(ab')2, variable fragment (Fv), trifunctional antibody, tetrafunctional antibody, microantibody, bispecific F(ab')2, trispecific F(ab')2, bifunctional antibody, bispecific bifunctional antibody, single-chain variable fragment (scFv), scFv-Fc, Fab-Fc, VHH, or bispecific scFv.

10. The antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein it is an antigen-binding fragment comprising scFv.

11. The antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein the scFv comprises a light chain variable domain located at the N-terminus of the heavy chain variable domain, optionally wherein the light chain variable domain and the heavy chain variable domain are connected by a linker sequence.

12. The antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein the scFv comprises a heavy chain variable domain located at the N-terminus of the light chain variable domain, optionally wherein the heavy chain variable domain and the light chain variable domain are connected by a linker sequence.

13. The antibody or antigen-binding fragment according to any one of claims 9 to 11, wherein... The light chain variable domain comprises: a polypeptide sequence of SEQ ID NO: 6; a polypeptide sequence having 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 6; or a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to SEQ ID NO: 6; and / or The heavy chain variable domain comprises: a polypeptide sequence of SEQ ID NO: 2; a polypeptide sequence having 1, 2, 3, 4 or 5 amino acid substitutions compared to SEQ ID NO: 2; or a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID NO:

2.

14. The antibody or antigen-binding fragment according to any one of claims 9 to 11, wherein the antigen-binding fragment is an scFv comprising the following: The polypeptide sequence of SEQ ID NO: 43; a polypeptide sequence having 1, 2, 3, 4 or 5 amino acid substitutions compared to SEQ ID NO: 43; or a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID NO: 43; and / or The polypeptide sequence of SEQ ID NO: 44; a polypeptide sequence having 1, 2, 3, 4 or 5 amino acid substitutions compared to SEQ ID NO: 44; or a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID NO:

44.

15. The antibody or antigen-binding fragment according to any one of claims 9 to 13, wherein the light chain variable domain and the heavy chain variable domain are covalently linked by a linker sequence, wherein the linker sequence comprises the polypeptide sequence of SEQ ID NO: 42 or a sequence having 1, 2, 3, 4 or 5 amino acid substitutions compared to SEQ ID NO:

42.

16. The antibody or antigen-binding fragment according to any one of claims 1 to 14, operatively linked to a signal peptide.

17. The antibody or antigen-binding fragment according to any one of claims 1 to 15, operably linked to a signal peptide configured to cause the antibody or antigen-binding fragment to be secreted from human cells.

18. A nucleic acid molecule encoding an antibody or antigen-binding fragment according to any one of claims 1 to 16.

19. The nucleic acid molecule of claim 17, wherein the nucleic acid molecule further comprises a promoter or enhancer sequence operatively linked to the encoded antibody or antigen-binding fragment.

20. The nucleic acid molecule of claim 17 or 18, wherein the nucleic acid molecule further encodes a signal peptide sequence operatively linked to the encoded antibody or antigen-binding fragment.

21. A vector comprising a nucleic acid molecule according to any one of claims 17 to 19.

22. The vector of claim 20, wherein the vector comprises an adenovirus vector, an HIV vector, a lentiviral vector, an adeno-associated virus vector ("AAV"), a plasmid vector, or a C-type vector. 3 DNA vector.

23. The carrier according to claim 21, wherein the AAV comprises AAV2, AAV4, AAV8 or AAV9.

24. A liposome comprising a nucleic acid molecule according to any one of claims 17 to 19.

25. A recombinant adeno-associated virus ("rAAV") vector comprising an AAV capsid and an expression cartridge, the expression cartridge comprising a polynucleotide operatively linked to a promoter encoding an antibody or antigen-binding fragment according to any one of claims 1 to 16.

26. The rAAV vector according to claim 24, wherein the antibody or antigen-binding fragment is scFv.

27. The rAAV carrier of claim 24, wherein the scFv comprises: Heavy chain variable domains containing the polypeptide sequence of SEQ ID NO: 2; and / or The light chain variable domain contains the polypeptide sequence of SEQ ID NO:

6.

28. The rAAV carrier of claim 24, wherein the scFv comprises: a) Heavy-chain variable fields, which contain A polypeptide sequence having SEQ ID NO: 3 or a CDR1 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO:

3. A polypeptide sequence having SEQ ID NO: 4 or a CDR2 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 4, and / or A polypeptide sequence having SEQ ID NO: 5 or a CDR3 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 5; and / or b) Light chain variable fields, which contain A polypeptide sequence having SEQ ID NO: 7 or a CDR1 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO:

7. A polypeptide sequence having SEQ ID NO: 8 or a CDR2 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 8, and / or The polypeptide sequence having SEQ ID NO: 9 or the CDR3 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO:

9.

29. The rAAV vector according to any one of claims 24 to 27, wherein the expression cartridge is side-attached by two inverted terminal repeat sequences ("ITR").

30. The rAAV carrier of claim 28, wherein the ITR is an AAV2 ITR.

31. The rAAV vector or composition according to any one of claims 24 to 29, wherein the AAV shell comprises viral proteins having at least 85%, 90%, 95%, 98%, or 100% similarity to SEQ ID NO: 13, 15, 17, or 19.

32. The rAAV vector according to any one of claims 24 to 29, wherein the AAV shell comprises a viral protein having at least 85%, 90%, 95%, 98%, or 100% similarity to SEQ ID NO:

19.

33. A composition comprising an rAAV vector, wherein the rAAV vector comprises: (a) AAV2, AAV5, AAV8 or AAV9 housing, and (b) An expression cartridge, wherein the expression cartridge contains a polynucleotide having at least 90% or 95% identity with a nucleotide sequence containing SEQ ID NO:21 or 22, and wherein the polynucleotide is linked to a promoter.

34. A composition comprising an rAAV vector, wherein the rAAV vector comprises: (a) AAV2, AAV5, AAV8 or AAV9 housing, and (b) An expression cartridge, wherein the expression cartridge contains a polynucleotide sequence having at least 90% or 95% identity with SEQ ID NO: 21 or 22.

35. The composition according to claim 32 or 33, wherein the AAV housing is AAV2.

36. The composition according to claim 32 or 33, wherein the AAV housing is AAV5.

37. The composition according to claim 32 or 33, wherein the AAV housing is AAV8.

38. The composition according to claim 32 or 33, wherein the AAV housing is AAV9.

39. A composition comprising an rAAV carrier, wherein the rAAV carrier comprises: (a) AAV housing, and (b) An expression cartridge, wherein the expression cartridge contains a polynucleotide encoding a polypeptide sequence having at least 90% or 95% sequence identity with SEQ ID NO: 2 or 6, and wherein the polynucleotide is linked to a promoter.

40. A pharmaceutical composition comprising: an antibody or antigen-binding fragment according to any one of claims 1 to 16, a nucleic acid molecule according to any one of claims 17 to 19, an rAAV vector according to any one of claims 24 to 31, or a composition according to any one of claims 32 to 38, and a pharmaceutically acceptable carrier.

41. The pharmaceutical composition of claim 39, wherein the composition comprises about 1 × 10 7 To approximately 1×10 14 One genome copy per milliliter of the rAAV vector.

42. The pharmaceutical composition of claim 39, wherein the composition comprises about 1 × 10⁻⁶ 12 To approximately 6.5 × 10 12 One genome copy per milliliter of the rAAV vector.

43. A kit comprising: an antibody or antigen-binding fragment according to any one of claims 1 to 16, an rAAV according to any one of claims 24 to 31, a composition according to any one of claims 32 to 38 or a pharmaceutical composition according to any one of claims 39 to 41, and a pharmaceutically acceptable carrier, and instructions for use for treating an individual's eye disease, condition, or symptom, comprising administering the antibody, antigen-binding fragment, composition, or pharmaceutical composition to the individual's eye.

44. The kit according to claim 42, wherein the eye disease, condition or symptom is Graves' Orbitopathy or Thyroid Eye Disease ("TED").

45. A method for treating an eye disease, condition, or symptom in an individual in need, the method comprising administering to the tear film or ocular tissue of the individual an effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 16, rAAV according to any one of claims 24 to 31, a composition according to any one of claims 32 to 38, or a pharmaceutical composition according to any one of claims 39 to 41. Optionally, wherein (i) the ocular tissue comprises tissue of the individual’s primary lacrimal gland or at least one accessory lacrimal gland, meibomian gland, or trabecular meshwork; and (ii) the administration causes one or more cells of the individual to express an antibody or antigen-binding fragment according to any one of claims 1 to 16, or to deliver or diffuse an antibody or antigen-binding fragment according to any one of claims 1 to 16 into at least one extraorbital fat pad of the individual’s eye.

46. ​​The method of claim 44, wherein the application to the tear film or eye tissue of the individual comprises applying the antibody, antigen-binding fragment, rAAV, composition, or pharmaceutical composition to the at least one accessory lacrimal gland of the individual.

47. The method of claim 44 or 45, wherein the application to the tear film or eye tissue of the individual comprises applying the antibody, antigen-binding fragment, rAAV, composition, or pharmaceutical composition to the meibomian gland.

48. The method according to any one of claims 44 to 46, wherein the application to the tear film or the ocular tissue of the individual comprises applying the antibody, antigen-binding fragment, rAAV, composition, or pharmaceutical composition to the trabecular meshwork of the individual.

49. The method according to any one of claims 44 to 47, wherein the eye disease, condition or symptom is thyroid eye disease ("TED").

50. The method according to any one of claims 44 to 48, wherein the method causes: a) Improve at least one symptom of the said ocular condition; and / or b) To slow the progression of the aforementioned eye symptoms.

51. The method of claim 49, wherein the at least one symptom of the ocular symptom comprises: a) Swelling of one or more eyelids; and / or b) Redness and / or pain in the conjunctiva, eyelid, lacrimal caruncle, and / or semilunar fold of at least one eye of the individual.

52. The method according to any one of claims 44 to 49, wherein the individual is a human individual.

53. A cell transfected or transduced by a vector according to any one of claims 20 to 22.

54. A kit comprising a pharmaceutical composition according to any one of claims 39 to 41 and instructions for use for treating a condition in a human individual, wherein the instructions comprise applying the pharmaceutical composition to the tear film or ocular tissue of the human individual, optionally wherein the ocular tissue comprises a primary or accessory lacrimal gland, meibomian glands, or tissue of the trabecular meshwork of the individual.

55. The pharmaceutical composition according to any one of claims 39 to 41, used to manufacture a medicament for treating the condition of a human individual in need.

56. A fusion protein comprising a first variable domain and a second variable domain linked by a linker sequence, said fusion protein specifically binding to a human insulin-like growth factor 1 receptor ("IGF1R") protein, wherein Each of the first variable domain and the second variable domain includes: (i) Having a polypeptide sequence of SEQ ID NO: 3 or a CDR1 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO:

3. A polypeptide sequence having SEQ ID NO: 4 or a CDR2 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 4, and / or A polypeptide sequence having SEQ ID NO: 5 or a CDR3 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 5; or (ii) Having a polypeptide sequence of SEQ ID NO: 7 or a CDR1 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO:

7. A polypeptide sequence having SEQ ID NO: 8 or a CDR2 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 8, and / or The polypeptide sequence having SEQ ID NO: 9 or the CDR3 region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO:

9.

57. The fusion protein of claim 55, wherein the first variable domain is different from the second variable domain.

58. The fusion protein of claim 55, wherein the first variable domain is a heavy chain variable domain comprising (i) and the second variable domain is a light chain variable domain comprising (ii).

59. The fusion protein according to any one of claims 55 to 57, wherein the fusion protein is scFv.

60. The fusion protein according to any one of claims 55 to 58, wherein the fusion protein further comprises an N-terminal signal peptide sequence configured to be secreted from human cells.

61. The fusion protein according to any one of claims 57 to 59, wherein the first variable domain is located at the N-terminus of the second variable domain.

62. The fusion protein according to any one of claims 57 to 59, wherein the second variable domain is located at the N-terminus of the first variable domain.

63. A nucleic acid molecule encoding the fusion protein according to any one of claims 55 to 61.

64. A vector comprising the nucleic acid molecule according to claim 62.

65. The vector of claim 63, wherein the vector comprises an adenovirus vector, an HIV vector, a lentiviral vector, an adeno-associated virus vector ("AAV"), a plasmid vector, or a C-type vector. 3 DNA vector.

66. A pharmaceutical composition comprising: a fusion protein according to any one of claims 55 to 61, a nucleic acid molecule according to claim 62 or a carrier according to claim 63 or 64, and at least one pharmaceutically acceptable carrier.

67. A method of treating an individual's eye disease, condition, or symptom, comprising administering to the individual's eye a fusion protein according to any one of claims 55 to 61, a nucleic acid molecule according to claim 62, a carrier according to claim 63 or 64, or a pharmaceutical composition according to claim 65.

68. The method of claim 66, wherein the ocular disease, condition, or symptom is Graves' orbital lesion or thyroid ophthalmopathy ("TED"), and the treatment comprises administering an effective amount of the fusion protein of any one of claims 55 to 61, the nucleic acid molecule of claim 62, the carrier of claim 63 or 64, or the pharmaceutical composition of claim 65, thereby improving at least one symptom of the ocular condition and / or delaying the progression of the ocular condition.

69. The method of claim 66 or 67, wherein the application is applied to the tear film or ocular tissue of at least one eye of the individual.

70. The method according to any one of claims 66 to 68, wherein the fusion protein according to any one of claims 55 to 61 is applied, and the application causes the fusion protein to be delivered to or diffuse into at least one extraorbital fat pad of the individual's eye.

71. The method according to any one of claims 66 to 68, wherein the nucleic acid molecule according to claim 62 or the vector according to claim 63 or 64 is applied, and the application causes one or more cells of a human individual to express the fusion protein according to any one of claims 55 to 61, and the fusion protein is delivered or diffused into at least one extraorbital fat pad of the individual's eye.

72. The method according to any one of claims 66 to 68, wherein the pharmaceutical composition according to claim 65 is administered, and the administration causes (1) one or more cells of a human individual to express the fusion protein according to any one of claims 55 to 61, and / or (2) the fusion protein according to any one of claims 55 to 61 is delivered or diffused into at least one extraorbital fat pad of the eye of the individual.

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