CN118302194A

CN118302194A - Compositions, dosages and methods for treating thyroid eye disease

Info

Publication number: CN118302194A
Application number: CN202280056115.5A
Authority: CN
Inventors: D·奥肖内西; B·卡兹
Original assignee: Viridian Treatment Co
Current assignee: Viridian Treatment Co
Priority date: 2021-08-10
Filing date: 2022-08-10
Publication date: 2024-07-05

Abstract

Provided herein are antibodies and compositions directed against IGF-1R and uses thereof.

Description

Compositions, dosages and methods for treating thyroid eye disease

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application number 63/260,133 filed on 8-10-2021 and U.S. provisional application number 63/261,744 filed on 9-28-2021, each of which is hereby incorporated by reference in its entirety.

Reference to an electronically submitted sequence Listing

The present application comprises a sequence listing that has been electronically submitted in the form of an XML file and is hereby incorporated by reference in its entirety. The XML copy was created at month 8 and 10 of 2022, named "257635_000402_ST26.XML" and was 12,855 bytes in size.

Background

Thyroid-related eye diseases (TAOs) (also known as Thyroid Eye Diseases (TED), graves' eye diseases or orbital diseases (GO), thyrotoxic eye herniation (exophthalmos), thyroid-dysfunction eye diseases and several other terms) are orbital diseases associated with thyroid dysfunction. TAOs fall into two categories. Active TAO generally lasts 1-3 years and is characterized by a sustained autoimmune/inflammatory response in the orbital soft tissue. Active TAO causes soft tissue of the eye to dilate and remodel. Autoimmune/inflammatory responses of active TAO spontaneously subside, and the condition translates to inactive TAO. Inactivity TAO is a term used to describe the long-term/permanent sequelae of activity TAO. The etiology of TAO is not yet clear. TAOs are often associated with graves hyperthyroidism, but may also occur as part of other autoimmune conditions that affect the thyroid gland and produce pathology in the orbit and periorbital tissues, but rarely in the pre-tibial skin (pre-tibial myxoedema) or the digit (thyroid acromegaly). TAO is an autoimmune orbital disease in which orbit and periocular soft tissues are primarily affected, and eyes and vision are secondarily affected. In TAO, the eye is forced to protrude forward (bulge) from its orbital socket due to inflammation and distension of the orbital soft tissue (mainly the eye muscles and fat) -this phenomenon is called protruding eye (proptosis) or protruding eyeball (exophthalmos). Although most TAO cases do not lead to blindness, this condition can lead to vision-threatening exposed keratopathy, troublesome double vision (ghosting), and pressing thyroid dysfunction optic neuropathy. TAO may occur before, simultaneously with, or after systemic complications of thyroid dysfunction. Ocular manifestations of TAO include upper eyelid recession, eyelid hysteresis, swelling, redness (erythema), conjunctivitis and eye bulge (herniation or abrupt eye), conjunctival edema, periorbital edema, and altered eye movement with significant functional, social, and cosmetic consequences. Many signs and symptoms of TAO (including protruding eye and ocular congestion) result from orbital adipose tissue and periocular muscle dilation. Adipose tissue volume increases, in part, due to new adipocyte development (lipogenesis) within orbital fat. Accumulation of hydrophilic glycosaminoglycans (mainly hyaluronic acid) in the perimuscular connective tissue between the orbital adipose tissue and the extraocular muscle fibers further expands the adipose compartments and enlarges the extraocular body. Hyaluronic acid is produced by fibroblasts located in orbital fat and extraocular muscles, and its synthesis in vitro is stimulated by a variety of cytokines and growth factors including IL-1 beta, interferon-gamma, platelet derived growth factors, thyroid Stimulating Hormone (TSH) and insulin-like growth factor I (IGF-I).

Antibodies that activate insulin-like growth factor I receptor (IGF-IR) have also been detected and are involved in activity TAO. Without being bound by any theory, it is believed that TSHR and IGF-IR form an organism functional complex in orbital fibroblasts, and blocking IGF-IR appears to attenuate IGF-1 and TSH dependent signaling. Blocking IGF-IR with antibody antagonists has been shown to reduce TSHR and IGF-I dependent signaling, thus interrupting the pathological activity of autoantibodies as agonists of either receptor.

IGF-IR is a widely expressed heterotetrameric protein that is involved in the regulation of proliferation and metabolic function in a variety of cell types. It is a tyrosine kinase receptor comprising two subunits. IGF-IR alpha comprises a ligand binding domain, while IGF-IR beta is involved in signaling and comprises tyrosine phosphorylation sites.

Current therapies for hyperthyroidism due to graves' disease are inadequate because of the lack of therapies that target specific underlying pathogenic autoimmune mechanisms of the disease. Even more complex is the treatment of moderate to severe active TAO. Although their pathogenesis has been better understood in recent years, TAO remains a therapeutic challenge and problem. There is no approved drug for the treatment of active TAO. Intravenous injection of glucocorticoid (ivGC) and oral administration of glucocorticoid are used for treatment of moderately to severely active TAO patients, but the results are less satisfactory. Partial response is common and relapse (rebound) after withdrawal is not uncommon. Adverse events do occur and many patients eventually require rehabilitation surgery when their condition is converted to inactive TAO. Thus, there remains a need to provide alternative therapies for TAO and its related symptoms.

Disclosure of Invention

The present disclosure relates generally to IGF-1R antibodies and antigen-binding fragments thereof, and uses thereof. Certain IGF-1R antibodies and antigen binding fragments inhibit IGF-1R function or block IGF-I mediated IGF-1R signaling biological functions. In addition, the present invention relates generally to methods for treating thyroid-related eye diseases (TAOs) (also known as Thyroid Eye Diseases (TED), graves' eye diseases or orbital diseases (GO), thyroid toxic herniation, thyroid dysfunctional eye diseases) and other thyroid eye disorders associated with IGF-1R signaling.

In some embodiments, the present disclosure provides a method of treatment comprising treating thyroid-related eye disease in a subject in need thereof, the method of treatment comprising: administering a first dose of an antibody intravenously or subcutaneously to the subject, wherein the first dose is selected from the group consisting of: about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg, and administering one or more subsequent doses of antibody intravenously or subcutaneously to the subject, wherein each subsequent dose is selected from the group consisting of: about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg, wherein the antibody is as provided herein.

In some embodiments, the present disclosure provides a method of treating thyroid-related eye disease in a subject in need thereof, comprising: administering a first dose of an antibody intravenously or subcutaneously to the subject, wherein the first dose is selected from the group consisting of: about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg, and administering one or more subsequent doses of antibody intravenously or subcutaneously to the subject, wherein each subsequent dose is selected from the group consisting of: about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg, wherein the antibody is provided herein, wherein one or more subsequent doses are administered when the subject is determined to not have adequate relief (adequate response) from one or more previous doses by clinical activity scoring and/or measurement of the eye-burst.

In some embodiments, the present disclosure provides a first dose of about 2mg/kg, about 3mg/kg, about 2.5mg/kg, about 5mg/kg, about 7.5mg/kg, about 10mg/kg, about 15mg/kg, or about 20 mg/kg.

In some embodiments, at least one of the one or more subsequent doses is about 2mg/kg, about 3mg/kg, about 5mg/kg, about 7.5mg/kg, about 10mg/kg, about 15mg/kg, or about 20mg/kg.

In some embodiments, the first dose is about 10mg/kg. In some embodiments, the one or more subsequent doses are about 10mg/kg.

In some embodiments, the present disclosure provides methods comprising further administering one or more loading doses of the antibody to the subject prior to administering the first dose.

In some embodiments, the disclosure provides methods comprising further administering a first loading dose of antibody to the subject prior to administering the first dose, wherein the first loading dose is selected from the group consisting of: about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 12.5mg/kg, or about 12.5mg/kg to about 15mg/kg.

In some embodiments, the first loading dose is about 5mg/kg, 7.5mg/kg, 10mg/kg, 12.5mg/kg, 15mg/kg, or 20mg/kg.

In some embodiments, the second loading dose is about 5mg/kg, 7.5mg/kg, 10mg/kg, 12.5mg/kg, 15mg/kg, or 20mg/kg.

In some embodiments, the present disclosure provides a method of improving treatment of thyroid-related eye disease in a subject who had previously received administration of one or more treatments, comprising: administering intravenously or subcutaneously to the subject at least one dose of an antibody consisting of about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg, wherein the antibody is an antibody as provided herein, such as, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises HCDR1 of SEQ ID NO:7, HCDR2 of SEQ ID NO:8, and HCDR3 of SEQ ID NO:9, and the light chain comprises LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5, and LCDR3 of SEQ ID NO: 6; or wherein the antibody comprises a light chain comprising a variable region having the amino acid sequence of SEQ ID NO. 2 and a heavy chain comprising a variable region sequence having the amino acid sequence of SEQ ID NO. 3; or wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO. 11 and a heavy chain comprising the amino acid sequence of SEQ ID NO. 10, wherein the at least one dose results in an improvement in one or more measurements relative to one or more measurements prior to the at least one dose.

In some such embodiments, the one or more measurements are selected from the group consisting of a sudden eye, a CAS, a level of exacerbation of the other eye, a GO-QoL score, and combinations thereof.

In some embodiments, if the subject does not have satisfactory relief after at least one dose (satisfactory response), one or more subsequent doses of antibody, each selected from the group consisting of: about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg.

In some embodiments, the one or more subsequent doses improve one or more of the glaring, CAS, exacerbation level of the other eye, GO-QoL score, and combinations thereof, as compared to before the one or more subsequent doses.

The present disclosure provides a method of treating thyroid-related eye disease in a subject in need thereof, comprising: administering a first dose of an antibody intravenously or subcutaneously to a subject, wherein the first dose is selected from the group consisting of: about 250mg, about 300mg, about 350mg, or about 400mg, and administering intravenously or subcutaneously to the subject one or more subsequent doses of the antibody, wherein each subsequent dose is selected from the group consisting of: about 250mg, about 300mg, about 350mg, or about 400mg, wherein the antibody is as provided herein.

As used herein, antibodies provided herein can include heavy and light chains. In some embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO. 11 and a heavy chain comprising the amino acid sequence of SEQ ID NO. 10. In some embodiments, the heavy chain may include Fc mutations in the Fc domain, such as the M252Y, S254T and T256E mutations. In some embodiments, the heavy chain comprises a VH having the amino acid sequence of SEQ ID NO. 3. In some embodiments, the light chain comprises a VL comprising the amino acid sequence of SEQ ID NO. 2. Thus, in some embodiments, the antibody comprises the VL of SEQ ID NO. 2 and the VH of SEQ ID NO. 3. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising HCDR1 of SEQ ID NO. 7, HCDR2 of SEQ ID NO. 8 and HCDR3 of SEQ ID NO. 9 and a light chain variable region (VL) comprising LCDR1 of SEQ ID NO. 4, LCDR2 of SEQ ID NO. 5 and LCDR3 of SEQ ID NO. 6.

In some embodiments, the first dose is about 250mg, 300mg, 350mg, or 400mg.

In some embodiments, one or more subsequent doses are about 250mg, 300mg, 350mg, or 400mg.

In some embodiments, the present disclosure provides methods comprising further administering a first loading dose of antibody to the subject prior to administering the first dose, wherein the first loading dose is selected from the group consisting of about 250mg, 300mg, 350mg, or 400 mg.

In some embodiments, the first loading dose is about 250mg, 300mg, 350mg, or 400mg.

In some embodiments, the second loading dose is about 250mg, 300mg, 350mg, or 400mg.

In some embodiments, one or more subsequent doses are the same as the first dose.

In some embodiments, the amount of one or more subsequent doses is different from the amount of the first dose.

In some embodiments, at least one of the one or more subsequent doses is administered one week, two weeks, three weeks, four weeks, five weeks, six weeks, or eight weeks after the first dose.

In some embodiments, only one, two, three, four, five, six, or seven subsequent doses are administered to the subject.

In some embodiments, a total of two, three, four, five, six, seven, or eight doses are administered to the subject.

In some embodiments, the subject's clinical activity score decreases after two or three doses of antibody.

In some embodiments, one week, two weeks, three weeks, four weeks, five weeks, a prior dose is followed,

Each subsequent dose was administered six weeks, seven weeks or eight weeks.

In some embodiments, at least one dose is administered by intravenous infusion over 45 minutes to about 90 minutes, or over 60 minutes to about 90 minutes.

In some embodiments, at least one dose is administered subcutaneously. In some such embodiments, the subcutaneous administration is self-administration.

In some embodiments, the second loading dose of antibody is administered to the subject after the first loading dose, and wherein the first and second loading doses are administered prior to the first dose.

In some embodiments, the first loading dose and the second loading dose are the same dose amounts.

In some embodiments, the first loading dose and the second loading dose are different dose amounts.

In some embodiments, the first loading dose is administered to the subject one, two, three, or four weeks prior to the administration of the first dose.

In some embodiments, the antibody is administered as part of a pharmaceutically acceptable composition comprising the antibody and at least one pharmaceutically acceptable excipient, wherein the antibody has a solubility of at least about 150mg/ml in the pharmaceutically acceptable composition.

In some embodiments, the subject has unsatisfactory relief from prior treatment of a thyroid-related eye disease (unsatisfactory response). In some such embodiments, the unsatisfactory relief is one or more of the following: failing to alleviate the abrupt eye by 2mm or more; failing to reduce CAS on one or more components or failing to reduce 2 points or more; the other eye deteriorates by 2mm or more; the multiple vision is not alleviated; failing to continue improving the compound for a period of time; failure to improve the score of the graves' quality of life (GO-QoL) assessment by 8 points or more; and combinations thereof.

In some embodiments, the first loading dose and the second loading dose are administered between about one week, about two weeks, or about three weeks apart.

In some embodiments, the second loading dose is administered about one week, about two weeks, or about three weeks prior to the first dose.

In some embodiments, there is provided a method of treating thyroid-associated eye disease in a subject in need thereof, the method comprising intravenously administering to the subject a first dose of 10mg/kg of an antibody, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises HCDR1 of SEQ ID NO:7, HCDR2 of SEQ ID NO:8, and HCDR3 of SEQ ID NO:9, and the light chain comprises LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5, and LCDR3 of SEQ ID NO: 6.

In some embodiments, the method further comprises administering a subsequent dose of about 10 mg/kg. In some embodiments, the subsequent dose is administered about 3 weeks after the first dose. In some embodiments, the method further comprises administering a subsequent dose of about 10mg/kg every 3 weeks after the first dose. In some embodiments, subsequent doses are administered every 3 weeks for a total of 4 subsequent doses. In some embodiments, subsequent doses are administered every 3 weeks for a total of 7 subsequent doses. In some embodiments, the subject's eyes are relieved within 3 weeks or within 6 weeks after the first dose and the CAS score is improved.

Drawings

Figures 1A-D illustrate various embodiments as provided herein.

Fig. 2A-B illustrate various embodiments as provided herein.

Figures 3A-F illustrate various embodiments as provided herein.

Fig. 4A-C illustrate various embodiments as provided herein.

Fig. 5A-B illustrate various embodiments as provided herein.

Fig. 6A-B illustrate various embodiments as provided herein.

Fig. 7 illustrates various embodiments as provided herein.

Fig. 8 illustrates various embodiments as provided herein.

Fig. 9 illustrates various embodiments as provided herein.

Fig. 10A-C illustrate various embodiments as provided herein.

Fig. 11 illustrates various embodiments as provided herein.

Fig. 12A-C illustrate various embodiments as provided herein.

Fig. 13A-B illustrate various embodiments as provided herein.

Fig. 14A-B illustrate various embodiments as provided herein.

Detailed Description

Provided herein are antibodies that bind to and modulate IGF-1R activity. For example, the antibodies may be used to treat thyroid eye disease.

As used herein, "thyroid-related eye disease" (TAO), "thyroid eye disease" (TED), "graves eye disease" or "graves orbital disease" (GO) refer to the same condition or disorder, and may be used interchangeably. They all refer to inflammatory orbital pathologies associated with some autoimmune thyroid disorders (most commonly associated with "graves 'disease" (GD), but sometimes with other diseases such as Hashimoto' sthyroiditis).

The terms "protruding eye" and "eye protrusion" (also referred to as exophthalmus, exophthalmia or exorbitism) refer to an organ protruding forward, shifting, bulging or protruding. As used herein, these terms refer to the eye protruding, shifting, bulging or protruding forward from the orbit. Some of the skilled in the art consider that the salient eye and the eyeball protrusion have the same meaning and are often used interchangeably, while others attribute subtle differences to their meaning. Some people use the protrusion of eyeballs to refer to severe protruding eyes; or endocrine related abrupt eyes. Other people use the term eye relief when describing eye-related presbyopia of a subject, e.g., with TAO (TED or GO).

As used herein, the terms "protruding eye" and "protruding eyeball" are used interchangeably and refer to an eye protruding, displacing, bulging, or protruding forward from the orbit. Any increase in soft tissue content of the orbit, occurring laterally or posteriorly, will move the eyeball anteriorly due to the rigid bone structure of the orbit with only the anterior opening for dilation. The protruding eye or eyeball herniation may be the result of a variety of disease processes including infection, inflammation, tumors, trauma, cancer metastasis, endocrine lesions, vascular disease, and extraorbital lesions. TAO (TED or GO) is currently considered to be the most common cause of eye-spurs in adults. The herniation may be bilateral, as is common in TAO (TED or GO), or unilateral (as is common in orbital tumors).

The measurement of the degree of protrusion of the eyeball may be performed using, for example, an eyeball salimeter (an instrument for measuring the degree of forward displacement of the eye). The device allows measuring the anterior distance of the lateral orbital margin to the anterior cornea. Computed Tomography (CT) scanning and Magnetic Resonance Imaging (MRI) can also be used to evaluate the extent of herniation or protruding eyes. CT scanning is an excellent imaging modality for diagnosing TAO. In addition to allowing visualization of the enlarged extraocular muscles, CT scanning may also provide a description of the orbital bone anatomy to a surgeon or clinician when orbital decompression is desired. MRI, by virtue of its multiplanar and inherent contrast capabilities, can provide excellent imaging of the orbital contents without the radiation exposure associated with CT scan studies. MRI provides better imaging of the optic nerve, orbital fat, and extraocular muscles, but CT scanning provides a better view of the orbital bone architecture. Orbital ultrasound examination can also be used to diagnose and evaluate TAO because it can be performed quickly and with high confidence. The high reflectivity and enlargement of the extraocular muscles are readily assessed, and continuous ultrasound examination can also be used to assess the progression or stability of ocular disease. Based on the techniques currently available or that will become available in the future, one of skill in the art will be able to determine the best modality for diagnosing and evaluating the degree of protrusion of the eye or eyeball.

As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in its broadest sense and specifically covers but is not limited to monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies. A "parent antibody" is an antibody obtained by exposing the immune system to an antigen prior to modification of the antibody for its intended use, such as humanization of the antibody for use as a human therapeutic antibody.

As used herein, unless otherwise indicated, "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen to which a full-length antibody binds, e.g., a fragment that retains one or more CDR regions. Examples of antibody binding fragments include, but are not limited to: fab, fab ', F (ab') ₂, and Fv fragments; a diabody; a linear antibody; single chain antibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.

"Fab fragment" consists of one light chain, the C _H region and the variable region of one heavy chain. The heavy chain of a Fab molecule is unable to form disulfide bonds with another heavy chain molecule.

The "Fc" region comprises two heavy chain fragments, including the C _H and C _H 2 domains of an antibody. The two heavy chain fragments are held together by hydrophobic interactions of two or more disulfide bonds and the C _H domain.

In some embodiments, an antibody or antigen fragment herein comprises an Fc region. In some embodiments, the Fc region comprises a mutation that when linked to the Fc region increases the half-life of the antibody. In some embodiments, the Fc region comprises the S228P, L235E, M Y, S254T, T256E, M428L, N434S, L234F, P331S mutation or any combination thereof. In some embodiments, the Fc region comprises M252Y, S T and T256E mutations. In some embodiments, the Fc region comprises S228P and L235E mutations. { GOODW-24002-CNPT/01912999v1}10 in some embodiments, the antibody comprises L234F, L235E and P331S mutations. In some embodiments, the Fc region comprises M252Y, S254T, T256E, S P and L235E mutations. In some embodiments, the Fc region comprises the S228P, L235E, M428L and N434S mutations. In some embodiments, the Fc region comprises M428L and N434S mutations. In some embodiments, the Fc region comprises L234F, L235E, P331S, M252Y, S254T and T256E mutations. Mutations in the Fc region are also described in US2007041972A1, EP2235059B1, U.S. patent No. 8,394,925 and Mueller et al, mol Immunol 1997Apr;34 441-52, each of which is incorporated by reference in its entirety. The numbering referred to herein refers to the Kabat numbering system of the Fc region.

"Fab ' fragments" comprise a light chain and a portion or fragment of a heavy chain comprising the V _H domain and the C _H 1 domain, and further comprise a region between the C _H 1 and C _H 2 domains, such that an interchain disulfide bond can form between the two heavy chains of two Fab ' fragments to form the F (ab ') ₂ molecule.

The "F (ab') ₂ fragment" comprises two light chains and two heavy chains, wherein the two heavy chains comprise a portion of the constant region between the C _H and C _H ² domains such that an interchain disulfide bond is formed between the two heavy chains. Thus, the F (ab ') ₂ fragment consists of two Fab' fragments which are held together by disulfide bonds between the two heavy chains.

"Fv region" includes variable regions from the heavy and light chains, but lacks constant regions.

The term "single chain Fv" or "scFv" antibody refers to an antibody fragment comprising the V _H and V _L domains of the antibody, wherein these domains are present in a single polypeptide chain. Generally, fv polypeptides further comprise a polypeptide linker between the V _H and V _L domains that enables the scFv to form the structure required for antigen binding. For reviews of scFv see Pluckthun(1994)The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds.Springer-Verlag,New York,pp.269-315., see also International patent application publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203.

A "domain antibody" is an immunologically functional immunoglobulin fragment that comprises only either a heavy chain variable region or a light chain variable region. In some cases, two or more V _H regions are covalently linked to a peptide linker to produce a bivalent domain antibody. The two V _H regions of a bivalent domain antibody may target the same or different antigens.

"Bivalent antibody" includes two antigen binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies may be bispecific (see below).

In certain embodiments, monoclonal antibodies herein also include camelized single domain antibodies. See, e.g., ,Muyldermans et al.(2001)Trends Biochem.Sci.26:230;Reichmann et al.(1999)J.Immunol.Methods 231:25;WO 94/04678;WO 94/25591;U.S.Pat.No.6,005,079). in one embodiment, the invention provides a single domain antibody comprising two V _H domains, which are modified such that a single domain antibody is formed.

As used herein, the term "diabody" refers to a small antibody fragment having two antigen binding sites, the fragment comprising a heavy chain variable domain (V _H) linked to a light chain variable domain (V _L) in the same polypeptide chain (V _H-V_L or V _L-V_H). By using a linker that is too short to allow pairing between two domains on the same strand, these domains are forced to pair with the complementary domain of the other strand and create two antigen binding sites. Diabodies are more fully described, for example, in EP 404,097; WO 93/11161; and Holliger et al (1993) Proc.Natl. Acad.Sci.USA 90:6444-6448. For reviews of engineered antibody variants, see Holliger and Hudson (2005) Nat. Biotechnol.23:1126-1136.

Typically, a variant antibody or antigen-binding fragment of an antibody provided herein retains at least 10% of its IGF-1R binding activity (when compared to the modified parent antibody) when the activity is expressed on a molar basis. In some embodiments, a variant antibody (or antigen fragment thereof) or antigen binding fragment of an antibody provided herein retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of IGF-1R binding affinity as the parent antibody. As described herein, it is also contemplated that an antibody or antigen binding fragment of the invention may include conservative or non-conservative amino acid substitutions that do not substantially alter its biological activity, which may also be referred to as "conservative variants" or "functional conservative variants" of the antibody.

An "isolated antibody" refers to a purified state of the bound compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "isolated" does not mean that such materials are completely absent or that water, buffer or salt are absent unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compounds described herein.

As used herein, the term "monoclonal antibody" refers to a substantially homogeneous population of antibodies, i.e., the amino acid sequences of the antibody molecules comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a plurality of different antibodies whose variable domains, particularly their CDRs, have different amino acid sequences, which are typically specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be prepared by the hybridoma method described for the first time by Kohler et al (1975) Nature 256:495, or may be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al (1991) Nature 352:624-628and Marks et al (1991) J.mol. Biol. 222:581-597. See also Presta (2005) J.allergy Clin.Immunol.116:731.

As used herein, a "chimeric antibody" is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein the first and second antibodies are from different species. (U.S. Pat. No.4,816,567; and Morrison et al, (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855). Typically, the variable domain is obtained from an antibody ("parent antibody") of an experimental animal (such as a rodent) and the constant domain sequence is obtained from a human antibody, and thus the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human subject than the parent (e.g., rodent) antibody.

As used herein, the term "humanized antibody" refers to a form of antibody that comprises sequences from both human and non-human (e.g., murine, rat) antibodies. Typically, a humanized antibody will comprise substantially at least one, and typically all, of two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the Framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).

The term "fully human antibody" refers to an antibody that includes only the protein sequence of a human immunoglobulin. If the fully human antibody is produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell, it may comprise a murine carbohydrate chain. Similarly, "mouse antibody" refers to an antibody that includes only mouse immunoglobulin sequences. Alternatively, if the fully human antibody is produced in a rat, rat cell or hybridoma derived from a rat cell, it may comprise a rat carbohydrate chain. Similarly, "rat antibody" refers to an antibody that includes only rat immunoglobulin sequences.

Typically, the basic antibody structural units comprise tetramers. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Generally, human light chains are classified as kappa light chains and lambda light chains. Furthermore, human heavy chains are generally classified as μ, δ, γ, α or ε, and define the isotypes of antibodies as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See generally Fundamental Immunology ch.7 (Paul, W., ed.,2nd ed.Raven Press,N.Y (1989).

The variable regions of each light/heavy chain pair form an antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are typically identical.

Typically, the variable domains of the heavy and light chains comprise three hypervariable regions, also known as Complementarity Determining Regions (CDRs), which are located within relatively conserved Framework Regions (FR). CDRs are typically aligned by framework regions so as to be able to bind to specific epitopes. Typically, from N-terminus to C-terminus, both the light and heavy chain variable domains include FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Amino acid assignment to each domain is generally according to the definition ：Sequences of Proteins of Immunological Interest,Kabat,et al.;National Institutes of Health,Bethesda,Md.;5^th ed.;NIH Publ.No.91-3242(1991);Kabat(1978)Adv.Prot.Chem.32:1-75;Kabat,et al.,(1977)J.Biol.Chem.252:6609-6616;Chothia,et al.,(1987)J Mol.Biol.196:901-917 or Chothia, et al, (1989) Nature 342:878-883.

As used herein, the term "hypervariable region" refers to the amino acid residues in an antibody that are responsible for antigen binding. Hypervariable regions include amino acid residues from the "complementarity determining regions" or "CDRs" (i.e., residues 24-34 (CDRL 1), 50-56 (CDRL 2) and 89-97 (CDRL 3) in the light chain variable domain and residues 31-35 (CDRH 1), 50-65 (CDRH 2) and 95-102(CDRH3);Kabat et al.(1991)Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.) in the heavy chain variable domain and/or those residues from the "hypervariable loops" (i.e., residues 26-32 (CDRL 1), 50-52 (CDRL 2) and 91-96 (CDRL 3) in the light chain variable domain and 26-32 (CDRH 1), 53-55 (CDRH 2) and 96-101 (CDRH 3) in the heavy chain variable domain); chothia and Lesk (1987) J.mol. Biol.196:901-917) As used herein, the term "framework" or "FR" residues refer to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

Additionally, in some embodiments, the antibody may be in the form of: full length antibodies, single domain antibodies, recombinant heavy chain only antibodies (VHH), single chain antibodies (scFv), shark heavy chain only antibodies (VNAR), micro proteins (cysteine knot proteins, knottins), DARPin; tetranectin (TETRANECTIN); affibodies (affibodies); a transmembrane body (Transbody); anti-carrier protein (Anticalin); adNectin; affilin; a microbody; a peptide aptamer; alterase; a plastic antibody; phylomer; stradobody; giant body (maxibody); evibody; fynomer, armadillo-repeat protein, kunitz domain, avimer, atrimer, pre-antibody (probody), immunomer, trimab (triomab), troybody; pepbody; vaccine bodies (vaccibody), uniBody; affimer, duoBody, fv, fab, fab ', F (ab') 2, peptidomimetic or synthetic molecules, as described in U.S. patent No. US 7,417,130、US 2004/132094、US 5,831,012、US2004/023334、US 7,250,297、US 6,818,418、US2004/209243、US 7,838,629、US 7,186,524、US 6,004,746、US 5,475,096、US2004/146938、US2004/157209、US 6,994,982、US 6,794,144、US 2010/239633、US 7,803,907、US2010/119446 and/or US 7,166,697, the respective contents of which are incorporated herein by reference in their entirety. See also Storz MAbs.201mmay-Jun; 3 (3): 310-317, which are hereby incorporated by reference.

As used herein, the term "antigen" means any molecule that has the ability to directly or indirectly produce or bind to an antibody. Included within the definition of "antigen" are protein-encoding nucleic acids. An "antigen" may also refer to a binding partner of an antibody. In some embodiments, the antigen is an IGF-1R protein expressed on the surface of a cell. In some embodiments, the cell is an intact cell. Intact cells are cells that have not been lysed or ruptured using a detergent or other agent. Cells that have been treated with detergents or other agents that disrupt or punch on the cell membrane are not intact cells. For example, provided herein are methods for producing antibodies that bind to IGF-1R protein, comprising culturing cells comprising a nucleic acid molecule encoding an IGF-1R antibody.

As used herein, "specifically binds" or "immunospecifically binds" refers to the binding of an antibody to a predetermined antigen (e.g., IGF-1R) or an epitope present on an antigen. In some embodiments, the antibody binds to a predetermined antigen (e.g., BSA, casein, or another non-specific polypeptide) with a dissociation constant (K _D) of 10 ^-7 M or less, and at least twice as small K _D as K _D to which a non-specific antigen other than the predetermined antigen binds. The phrases "antibody that recognizes IGF-1R" and "IGF-1R specific antibody" are used interchangeably herein with the term "antibody that immunospecifically binds IGF-1R". IGF-1R may be referenced in this disclosure. The degree of specificity required for an anti-IGF-1R antibody may depend on the intended use of the antibody and is in any case defined by its suitability for the intended purpose. In some embodiments, an antibody of the contemplated method or a binding compound derived from an antigen binding site of an antibody binds to its antigen (IGF-1R) with an affinity that is at least two times greater, at least ten times greater, at least 20 times greater, or at least 100 times greater than the affinity to any other antigen.

Methods for determining mAb specificity and affinity by competitive inhibition can be found in Harlow,et al.,Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1988),Colligan et al.,eds.,Current Protocols in Immunology,Greene Publishing Assoc.and Wiley Interscience,N.Y.,(1992,1993), and Muller, meth. Enzymol.92:589 601 (1983), which references are incorporated herein by reference in their entirety.

The term "homolog" means a protein sequence that has 40% to 100% sequence homology or identity to a reference sequence. The percent identity between two peptide chains can be determined by comparison using the default set of AlignX modules of Vector NTI v.9.0.0 (Invitrogen corp., carslbad, calif.). In some embodiments, the antibody or antigen binding fragment thereof has at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology or identity to a sequence described herein. In some embodiments, the antibody has a conservative substitution compared to the sequences described herein. Exemplary conservative substitutions are shown in table 1 and are encompassed within the scope of the disclosed subject matter. Conservative substitutions may be in the framework region or antigen binding site as long as the properties of the antibody are not adversely affected. Substitutions may be made to improve antibody properties, such as stability or affinity. Conservative substitutions will result in a molecule having similar functional and chemical characteristics as the molecule to which such modifications are made. Exemplary amino acid substitutions are shown in the following table.

In some embodiments, variants of the proteins and peptides provided herein are provided. In some embodiments, the variant comprises a substitution, deletion, or insertion. In some embodiments, the variants include 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 (e.g., 1-10) substitutions. As described herein, a substitution may be a conservative substitution. In some embodiments, the substitutions are non-conservative. In some embodiments, the variants include 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 (e.g., 1-10) deletions. In some embodiments, the variants include 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 (e.g., 1-10) insertions. In some embodiments, substitutions, deletions, or insertions are present in the CDRs provided herein. In some embodiments, no substitution, deletion, or insertion is present in the CDRs provided herein.

As used herein, the term "in combination with … …" means that the described agents can be administered to an animal or subject together in the form of a mixture, simultaneously as a single agent, or sequentially in any order as a single agent.

Techniques for generating antibodies to small peptide sequences are well known in the art, and these antibodies recognize and bind those small peptide sequences in free or conjugated form or when presented as native sequences in the context of large proteins. Such antibodies include murine, murine-human, and human-human antibodies produced by hybridoma or recombinant techniques known in the art. Antibodies may also be produced in humans, mice, sheep, rats, rabbits, sharks, llamas or chickens. In some embodiments, the antibody is produced in chicken. Antibodies can also be produced in other small animals.

The term "epitope" means the portion of any molecule that is capable of being recognized and bound by an antibody at the antigen binding region of one or more abs. Epitopes are generally composed of chemically active surface groups of molecules (such as amino acids or sugar side chains) and have specific three-dimensional structural features as well as specific charge features. Examples of epitopes include, but are not limited to, residues described herein that form IGF-1R epitopes. In some embodiments, the epitope is present only in non-denatured proteins. In some embodiments, the epitope is present only in denatured proteins.

In some embodiments, the source of DNA encoding the non-human antibody includes an antibody-producing cell line, such as a hybrid cell line commonly referred to as a hybridoma.

Hybrid cells are formed by fusion of non-human antibody-producing cells (typically spleen cells of animals immunized against a natural or recombinant antigen, or peptide fragments of an antigen protein sequence). Alternatively, the non-human antibody-producing cells may be B lymphocytes obtained from the blood, spleen, lymph nodes or other tissues of an animal immunized with the antigen.

The second fusion partner providing immortalization may be lymphoblastoid cells or plasmacytoma or myeloma cells, which are not themselves antibody-producing cells, but rather malignant. Fusion partner cells include, but are not limited to, hybridomas SP2/0-Ag14 (abbreviated as SP2/0 (ATCC CRL 1581)) and myeloma P3X63Ag8 (ATCC TIB 9) or derivatives thereof. See, for example, ausubel, harlow, and Colligan, infra, the contents of which references are incorporated herein by reference in their entirety.

Antibodies can be generated according to the examples provided herein. Once the sequence is known, antibodies can also be produced according to known methods. Antibodies can also be converted to different types, such as to human IgG, and the like. By converting an antibody to a human antibody, the human subject should not recognize the antibody as an extraneous antibody. The conversion of non-human IgG antibodies to human IgG antibodies is well known and can be routinely performed once the native sequence is known. As discussed herein, antibodies can be modified according to known methods. Such methods are described, for example, in that ,Riechmann L,Clark M,Waldmann H,Winter G(1988).Reshaping human antibodies for therapy".Nature 332(6162):332–323;Tsurushita N,Park M,Pakabunto K,Ong K,Avdalovic A,Fu H,Jia A,Vásquez M,Kumar S.(2004). contributes to antibody-producing cells encoding the nucleotide sequence of the antigen-binding region of a chimeric antibody can also be produced by transforming non-human (such as primate) or human cells. For example, antibody-producing B lymphocytes can be infected and transformed with a virus such as Epstein-Barr virus to produce immortalized antibody-producing cells (Kozbor et al., immunol. Today 4:72 79 (1983)). Alternatively, B lymphocytes may be transformed by providing a transgene or a transgene product, as is well known in the art. See, for example, ausubel, harlow, and Colligan, infra, the contents of which references are incorporated herein by reference in their entirety. Cell fusion is accomplished by standard procedures well known to those skilled in the immunological arts. Fusion partner cell lines and methods for fusion and selection of hybridomas and selection of mabs are well known in the art. See, for example, ausubel, harlow, and Colligan, infra, the contents of which references are incorporated herein by reference in their entirety.

In some embodiments, the antibody is a MAb that binds IGF-1R. In some embodiments, the antibody binds to an amino acid of an IGF-1R epitope.

In some embodiments, the antibodies comprise the sequences provided herein.

The sequence of the antibody may be modified to produce a human IgG antibody. The transformation of the sequences provided herein may be modified to produce other types of antibodies. The CDRs may also be linked to other antibodies, proteins, or molecules to produce antibody fragments that bind IGF-1R. This may be in the form of an antibody drug conjugate ("ADC"), a multispecific molecule, or a chimeric antigen receptor. The CDR and antibody sequences provided herein are also humanized or prepared to be fully human according to known methods. The sequences may also be prepared as chimeric antibodies as described herein.

In some embodiments, an antibody comprises an amino acid sequence comprising a sequence provided herein or a fragment thereof. In some embodiments, the antibody comprises one or more of the amino acid sequences provided herein, antigen binding fragments thereof, or human IgG variants thereof. "human IgG variant thereof" refers to an antibody that has been modified to human IgG when the starting antibody is not a human IgG antibody.

As described herein, the production of antibodies having known sequences is conventional and can be performed by any method. Thus, in some embodiments, nucleic acids encoding antibodies or fragments thereof are provided. In some embodiments, the nucleic acid encodes a sequence provided herein. Antibodies may also be modified to chimeric or human antibodies. Antibodies may also be used in injectable pharmaceutical compositions. As also described herein, the antibody may be an isolated antibody or an engineered antibody.

In some embodiments, provided are "derivatives" of antibodies, fragments, regions or derivatives thereof, which term includes those proteins encoded by truncated or modified genes to produce molecular species functionally similar to immunoglobulin fragments. Modifications include, but are not limited to, the addition of gene sequences encoding cytotoxic proteins such as plant and bacterial toxins. Modifications may also include reporter proteins, such as fluorescent or chemiluminescent tags. Fragments and derivatives may be produced in any manner.

The identification of these antigen binding regions and/or epitopes recognized by the abs described herein provides information necessary to produce additional monoclonal antibodies having similar binding characteristics and therapeutic or diagnostic utility as embodiments of the application.

The nucleic acid sequence encoding an antibody described herein may be genomic DNA or cDNA, or RNA (e.g., mRNA) encoding at least one of the variable regions described herein. A convenient alternative to using chromosomal gene segments as a source of DNA encoding the V region antigen binding fragments is to use cDNA to construct chimeric immunoglobulin genes, for example, as reported by Liu et al (Proc.Natl.Acad.Sci., USA 84:3439 (1987) and J.Immunology 139:3521 (1987), which references are incorporated herein by reference in their entirety.

For example, a cDNA encoding a V region antigen binding region capable of detecting, binding to, or neutralizing IGF-1R antigen may be provided using known methods based on the use of the amino acid sequences provided herein. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson et al, see below). Using the genetic code, one or more different oligonucleotides can be identified, each of which will be capable of encoding an amino acid. The probability that a particular oligonucleotide will in fact constitute an actual XXX coding sequence can be estimated by taking into account the abnormal base pairing relationships in eukaryotic or prokaryotic cells expressing the antibody or fragment and the frequency with which particular codons are actually used (to encode particular amino acids). Such "rules of codon usage" are disclosed by Lathes et al, J.molecular.biol.183:1.12 (1985). Using the "codon usage rule" of Lathes, a single oligonucleotide or set of oligonucleotides comprising the theoretical "most probable" nucleotide sequence capable of encoding an antibody variable or constant region sequence was identified.

The variable regions described herein can be combined with any type of constant region including human constant regions or murine constant regions. Human genes encoding the constant (C) regions of antibodies, fragments and regions can be derived from human fetal liver libraries by known methods. The human C region gene may be derived from any human cell, including those that express and produce human immunoglobulins. The human C _H region may be derived from any of the known classes or isoforms of the human H chain, including gamma, mu, alpha, delta, or epsilon, and subtypes thereof, such as G1, G2, G3, and G4. Since the H chain isotype is responsible for the various effector functions of the antibody, the selection of the C _H region will be guided by the desired effector functions such as complement fixation or antibody-dependent cellular cytotoxicity (ADCC) activity. Preferably, the C _H region is derived from γ1 (IgG 1), γ3 (IgG 3), γ4 (IgG 4) or μ (IgM). The human C _L region may be derived from the human L chain isoform kappa or lambda. In some embodiments, the antibody comprises an Fc domain. In some embodiments, the Fc domain comprises a mutation to extend the half-life of the antibody. In some embodiments, the Fc domain includes mutations, such as those described in U.S. patent No. 7,670,600, which is hereby incorporated by reference in its entirety. In some embodiments, the constant region comprises a mutation at amino acid residue 428 relative to a wild-type human IgG constant domain, numbered according to the EU numbering index of Kabat. Without being bound by any particular theory, antibodies comprising mutations corresponding to residue 428 may have an extended half-life compared to the half-life of IgG having wild-type human IgG constant domains. In some embodiments, the mutation is a substitution of a natural residue with threonine, leucine, phenylalanine, or serine. In some embodiments, the antibody further comprises one or more amino { GOODW-24002-CNPT/01912999v1}21 acid substitutions at one or more of amino acid residues 251-256, 285-290, 308-314, 385-389, and 429-436, relative to the corresponding wild-type human IgG constant domain, numbered according to the EU numbering index of Kabat. Specific mutations or substitutions at these positions are described in U.S. patent No. 7,670,600, which is hereby incorporated by reference in its entirety.

Genes encoding the human immunoglobulin C region can be obtained from human cells by standard cloning techniques (Sambrook,et al.(Molecular Cloning:A Laboratory Manual,2nd Edition,Cold Spring Harbor Press,Cold Spring Harbor,N.Y.(1989) and Ausubel et al, eds. The human C region gene can be readily obtained from known clones containing genes representing two classes of L chains, five classes of H chains, and subclasses thereof. Chimeric antibody fragments such as F (ab') ₂ and Fab can be prepared by designing a suitably truncated chimeric H chain gene. For example, a chimeric gene encoding the H chain portion of the F (ab') ₂ fragment will include a DNA sequence encoding the CH ₁ domain and hinge region of the H chain, followed by a translation stop codon to produce a truncated molecule.

In some embodiments, antibodies, murine, human, humanized or chimeric antibodies, fragments and regions of antibodies described herein are produced by: DNA segments encoding the H and L chain antigen binding regions of IGF-1R antigen specific antibodies are cloned and ligated to DNA segments encoding the C _H and C _L regions, respectively, to produce murine, human or chimeric immunoglobulin encoding genes.

Thus, in some embodiments, a fusion chimeric gene is constructed comprising at least a first DNA segment encoding an antigen binding region of non-human origin, such as a functionally rearranged V region with a junction (J) segment, linked to a second DNA segment encoding at least a portion of a human C region.

Thus, the cDNA encoding the V and C regions of an antibody, the method of producing an antibody according to some embodiments described herein, involves several steps, as exemplified below: 1. isolating messenger RNA (mRNA) from a cell line producing antibodies to IGF-1R antigen and from an optional additional antibody providing heavy and light constant regions; cloning and generating cDNA therefrom; 2. a full-length cDNA library is prepared from the purified mRNA, wherein appropriate V and/or C region gene segments of the L and H chain genes can be: (i) identification with an appropriate probe, (ii) sequencing, and (iii) compatibility with a C or V gene segment of another antibody from the chimeric antibody; 3. as described above, the complete H or L chain coding sequence is constructed by ligating a cloned specific V region gene segment to a cloned C region gene; 4. the L and H chains are expressed and produced in selected hosts (including prokaryotic and eukaryotic cells) to provide murine-murine, human-human or human murine antibodies.

Two coding DNA sequences are said to be "operably linked" if the ligation results in sequences that can be translated serially without altering or interrupting the triplet reading frame. The DNA coding sequence is operably linked to a gene expression element if the linkage results in the gene expression element functioning properly to cause expression of the coding sequence.

As used herein and unless otherwise indicated, the term "about" is intended to mean ± 5% of the value to which it is modified. Thus, about 100 represents 95 to 105.

In some embodiments, the antibodies described herein are used to detect the presence of an antigen. The antibodies of the invention may be used in any device or method to detect the presence of an antigen.

The term "purified" with respect to an antibody refers to an antibody that is substantially free of other substances associated with the molecule in its natural environment. For example, the purified protein is substantially free of cellular material or other proteins from cells or tissues from which it is derived. The term refers to a protein wherein the isolated protein is sufficiently pure to be analyzed, or at least 70% to 80% (w/w) pure, at least 80% -90% (w/w) pure, 90-95% pure; and, at least 95%, 96%, 97%, 98%, 99% or 100% (w/w) pure formulation. In some embodiments, the antibody is purified.

As an alternative to preparing monoclonal antibody secreting hybridomas, monoclonal antibodies directed against a polypeptide can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptides described herein, thereby isolating immunoglobulin library members that bind to the polypeptide. Techniques and commercially available kits for generating and screening phage display libraries are well known to those skilled in the art. In addition, examples of methods and reagents particularly suitable for generating and screening antibody or antigen binding protein display libraries are found in the literature. Thus, the epitopes described herein can be used to screen for other antibodies that can be used therapeutically, diagnostically, or as research tools.

Antibody conjugates

The antibodies provided herein can also be conjugated to a chemical moiety. The chemical moiety may in particular be a polymer, a radionuclide or a cytotoxic factor. In some embodiments, this may be referred to as an antibody drug conjugate. In some embodiments, the chemical moiety is a polymer that extends the half-life of the antibody molecule in the subject. Suitable polymers include, but are not limited to, polyethylene glycol (PEG) (e.g., PEG having a molecular weight of 2kDa, 5kDa, 10kDa, 12kDa, 20kDa, 30kDa, or 40 kDa), dextran, and monomethoxy polyethylene glycol (mPEG). Lee, et al, (1999) (bioconj.chem.10:973-981) discloses PEG conjugated single chain antibodies. Wen, et al, (2001) (bioconj.chem.12:545-553) discloses conjugated antibodies with PEG attached to a radiometal chelator (diethylenetriamine pentaacetic acid (DTPA)). Examples of chemical moieties include, but are not limited to, antimitotic agents such as calicheamicin (e.g., octogamicin), monomethyl auristatin E (monomethyl auristatin E), maytansine (mertansine), and the like. Other examples include, but are not limited to, bioactive anti-microtubule agents, alkylating agents, and DNA small groove binding agents. Other examples are provided herein and below. The chemical moiety may be linked to the antibody through a linking group (maleimide), a cleavable linker, such as a cathepsin cleavable linker (valine-citrulline), and in some embodiments through one or more spacers (e.g., p-aminobenzyl carbamate). Without being bound by any particular theory, once the antibody conjugate binds IGF-1R, it can be internalized, and the chemical moiety can kill the cell or otherwise inhibit its growth. In some embodiments, the cell is a thyroid cell.

Antibodies and antibody fragments of the invention may also be conjugated to tags, such as ⁹⁹Tc、⁹⁰Y、¹¹¹In、³²P、¹⁴C、¹²⁵I、³H、¹³¹I、¹¹C、¹⁵O、¹³N、¹⁸F、³⁵S、⁵¹Cr、⁵⁷To、²²⁶Ra、⁶⁰Co、⁵⁹Fe、⁵⁷Se、¹⁵²Eu、⁶⁷CU、²¹⁷Ci、²¹¹At、²¹²Pb、⁴⁷Sc、¹⁰⁹Pd、²³⁴Th, and ⁴⁰K、¹⁵⁷Gd、⁵⁵Mn、⁵² Tr and ⁵⁶ Fe.

Antibodies and antibody fragments may also be conjugated to fluorescent or chemiluminescent tags, including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrins, phycocyanins, allophycocyanins, phthaldehyde, fluorescamine, ¹⁵² Eu, dansyl, umbelliferone, fluorescein, luminol (luminal) tags, isoluminol tags, aromatic acridinium ester (acridinium ester) tags, imidazole tags, acridinium salt tags, oxalate tags, aequorin (aequorin) tags, 2, 3-dihydro phthalazindione, biotin/avidin, spin tags, and stable free radicals.

The antibody molecules may also be conjugated to cytotoxic factors such as diphtheria toxin, pseudomonas aeruginosa (Pseudomonas aeruginosa) exotoxin a chain, ricin a chain, abrin a chain, mo Disu (modeccin) a chain, alpha-sarcin (alpha-sarcin), aleurites fordii proteins and compounds (e.g., fatty acids), caryophyllin protein, pokeweed (Phytoiacca americana) protein PAPI, PAPII and PAP-S, balsam pear (momordica charantia) inhibitors, curcin, crotonin, soapbark (saponaria officinalis) inhibitors, mitogens, restrictocins, phenomycin and enomycin (enomycin).

Any method known in the art for conjugating an antibody molecule of the invention to each moiety may be employed, including those described by Hunter,et al.,(1962)Nature 144:945;David,et al.,(1974)Biochemistry13:1014;Pain,et al.,(1981)J.Immunol.Meth.40:219; and Nygren, j. (1982) histochem.and cytochem.30:407. Methods for conjugating antibodies are conventional and well known in the art.

Chimeric antigen receptor

The antibodies provided herein can also be incorporated into chimeric antigen receptors ("CARs"), which can be used, for example, in CAR-T cells. In some embodiments, the extracellular domain of the CAR can be an antibody provided herein. In some embodiments, the antibody is in the form of an scFv. CAR-T cells are one type of treatment in which the T cells of the patient are modified so that they will attack cells expressing IGF-1R. T cells are taken from the patient's blood. Genes for specific receptors that bind to a protein on the patient's cells are then added in the laboratory. In some embodiments, the receptor binds to IGF-1R using the binding regions of antibodies provided herein. CAR-T cells comprising IGF-1R antibodies can then be used to treat disorders, such as those provided herein.

In some embodiments, provided herein are antibodies (e.g., anti-IGF-1R antibodies). In some embodiments, the antibody is a recombinant antibody that binds to IGF-1R protein. In some embodiments, the IGF-1R protein is a human IGF-1R protein. In some embodiments, the IGF-1R protein recognized by the antibody is in its native (non-denatured) conformation. In some embodiments, the antibody does not specifically bind to denatured IGF-1R protein. As used herein, the term "recombinant antibody" refers to an antibody that does not occur in nature. In some embodiments, the term "recombinant antibody" refers to an antibody that is not isolated from a human subject.

In some embodiments, the antibody comprises one or more peptides or variants thereof having the sequence:

The VH and VL sequences may be in any form, including but not limited to scFv forms wherein the VH and VL regions are linked to a peptide linker. Examples of peptide linkers that may be used to attach the various peptides provided herein include, but are not limited to: (GGGGS) _n(SEQ ID NO:12);(GGGGA)_n (SEQ ID NO: 13) or any combination thereof, wherein each n is independently 1-5. In some embodiments, the variable region is not linked to a peptide linker. In some embodiments, the antibodies comprise or consist of the polypeptides set forth in SEQ ID NOs 10 and 11. In some embodiments, the antibodies comprise polypeptides comprising SEQ ID NOs 3, 4,5, 6, 7, 8 and 9.

In some embodiments, antibodies or antigen binding fragments thereof are provided, wherein the antibodies or antibody fragments comprise a peptide selected from the following table.

In some embodiments, the antibody comprises one or more peptides or variants thereof having the following sequences, including one or more variable domains (italics), CDRs (italics bold) and human IgG 1/kappa constant domains (underlined):

In some embodiments, the antibody or antibody binding fragment thereof comprises a heavy or light chain having the sequences of SEQ ID NOs 10 and 11. In some embodiments, the antibody or antibody binding fragment thereof comprises a heavy chain having the sequence of SEQ ID NO. 10. In some embodiments, the antibody or antibody binding fragment thereof comprises a heavy chain having a sequence with 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or substantially 100% identity to the sequence of SEQ ID NO. 10. In some embodiments, sequences having 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or substantially 100% identity to the sequence of SEQ ID NO. 10 include the CDRs of SEQ ID NO. 7, 8, and/or 9 as described above.

In some embodiments, the antibody or antibody binding fragment thereof comprises a light chain having the sequence of SEQ ID NO. 11. In some embodiments, the antibody or antibody binding fragment thereof comprises a light chain having a sequence that is 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or substantially 100% identical to the sequence of SEQ ID NO. 11. In some embodiments, sequences having 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or substantially 100% identity to SEQ ID NO. 11 comprise the CDRs of SEQ ID NO. 4, 5 and/or 6 as described above.

In some embodiments, the antibody or antibody binding fragment thereof comprises a light chain CDR having the sequence of SEQ ID NO. 4, 5 or 6. In some embodiments, the antibody or antibody binding fragment thereof comprises heavy chain CDRs with a sequence of SEQ ID No. 7, 8 or 9.

In some embodiments, the antibody or antibody binding fragment thereof comprises a light chain having LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the sequence of SEQ ID No. 4, LCDR2 has the sequence of SEQ ID No. 5, and LCDR3 has the sequence of SEQ ID No. 6.

In some embodiments, the antibody or antibody binding fragment thereof comprises a heavy chain having HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the sequence of SEQ ID No. 7, HCDR2 has the sequence of SEQ ID No. 8, and HCDR3 has the sequence of SEQ ID No. 9.

The different CDR motifs may be combined in any combination, including those not described in the table above. For example, the following embodiments are provided as non-limiting examples of such combinations.

In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising light chain CDR1, CDR2 and CDR3 sequences, wherein the light chain CDR1 sequence has the amino acid sequence of SEQ ID No. 4; the light chain CDR2 has the amino acid sequence of SEQ ID NO. 5; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 6; and (ii) a heavy chain variable region comprising heavy chain CDR1, CDR2 and CDR3 sequences, wherein the heavy chain CDR1 sequence has the amino acid sequence of SEQ ID No. 7; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO. 8; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO. 9; or a variant of any of the foregoing.

In some embodiments, the antibody or antigen binding fragment thereof comprises: (i) A light chain variable region comprising light chain CDRl, CDR2 and CDR3 sequences, wherein the light chain CDRl sequence has an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or substantially 100% identical to the amino acid sequence of SEQ ID No. 4; light chain CDR2 has an amino acid sequence having 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or substantially 100% identity to the amino acid sequence of SEQ ID NO. 5; and the light chain CDR3 sequence has an amino acid sequence having 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or substantially 100% identity to the amino acid sequence of SEQ ID NO. 6; and (ii) a heavy chain variable region comprising heavy chain CDR1, CDR2 and CDR3 sequences, wherein the heavy chain CDR1 sequence has an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or substantially 100% identical to the amino acid sequence of SEQ ID No. 7; the heavy chain CDR2 sequence has an amino acid sequence having 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or substantially 100% identity to the amino acid sequence of SEQ ID NO. 8; and the heavy chain CDR3 sequence has an amino acid sequence that has 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or substantially 100% identity to the amino acid sequence of SEQ ID No. 9; or a variant of any of the foregoing.

In some embodiments, antibodies or antigen binding fragments or proteins thereof are provided that include peptides having the sequence set forth in any one of SEQ ID NOs 1,2, 3,4, 5, 6, 7, 8, 9, 10 or 11.

In some embodiments, the antibody or antigen binding fragment thereof comprises a sequence or variant of any of the foregoing.

Pharmaceutical composition

In some embodiments, to prepare a pharmaceutical or sterile composition of an anti-IGF-1R antibody or other protein provided herein, an antibody or antigen-binding fragment thereof or other protein provided herein is admixed with a pharmaceutically acceptable carrier or excipient. See, for example ,Remington's Pharmaceutical Sciences and U.S.Pharmacopeia:National Formulary,Mack Publishing Company,Easton,PA(1984).

Formulations of therapeutic and diagnostic agents may be prepared by mixing with an acceptable carrier, excipient or stabilizer, for example, in the form of a lyophilized powder, slurry, aqueous solution or suspension (see, e.g., ,Hardman,et al.(2001)Goodman and Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill,New York,NY;Gennaro(2000)Remington:The Science and Practice of Pharmacy,Lippincott,Williams,and Wilkins,New York,NY;Avis,et al.(eds.)(1993)Pharmaceutical Dosage Forms:Parenteral Medications,Marcel Dekker,NY;Lieberman,et al.(eds.)(1990)Pharmaceutical Dosage Forms:Tablets,Marcel Dekker,NY;Lieberman,et al.(eds.)(1990)Pharmaceutical Dosage Forms:Disperse Systems,Marcel Dekker,NY;Weiner and Kotkoskie(2000)Excipient Toxicity and Safety,Marcel Dekker,Inc.,New York,NY). in some embodiments, diluting the antibody to an appropriate concentration in a sodium acetate solution at pH 5-6, and adding NaCl or sucrose to maintain tonicity, additional agents, such as polysorbate 20 or polysorbate 80, may be added to enhance stability.

Toxicity and therapeutic efficacy of antibody compositions administered alone or in combination with another agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD ₅₀ (lethal dose of 50% of the population) and ED ₅₀ (therapeutically effective dose of 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD ₅₀/ED₅₀). In particular aspects, antibodies exhibiting high therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds is preferably within a circulating concentration range that includes ED ₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

In some embodiments, the compositions of the invention are administered to a subject according to Physics' DESK REFERENCE 2003 (Thomson Healthcare;57th edition (November 1,2002)).

The mode of administration may vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intraarterial.

In some embodiments, the antibody or antigen binding fragment thereof may be administered by an invasive route (such as by injection). In some embodiments, the antibody or antigen-binding fragment thereof or pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intraarterially, intra-articular (e.g., in an arthritic joint), or by inhalation, aerosol delivery. Administration by a non-invasive route (e.g., oral; e.g., in a pill, capsule, or tablet) is also within the scope of embodiments of the present invention.

In some embodiments, the anti-IGF-1R antibody or antigen-binding fragment thereof is administered in combination with at least one additional therapeutic agent, such as, but not limited to, any therapeutic agent for treating thyroid-eye disease. For example, in some embodiments, an anti-IGF-1R antibody or antigen-binding fragment thereof is administered in combination with at least one additional therapeutic agent such as, but not limited to, a therapeutic agent for treating thyroid-eye disease or a disorder associated therewith. Examples of such treatments and therapies include, but are not limited to, antithyroid drugs, diabetic drugs, beta-blockers, propylthiouracil, methimazole, propranolol, atenolol (atenolol), metoprolol (metoprolol), nadolol (nadolol), corticosteroids, metformin, sulfonylurea, mevalonate (MEGLITINIDES), thiazolidinedione, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, common insulin, insulin aspart, insulin glulisine, insulin lispro, low-precision zinc insulin (insulin isophane), deglupine, diltiazem, glargine, acarbose (acerbose), miglol (miglol), acebutolol (acebutolol), atenolol, betaxolol (betaxolol), bisoprolol (betaxolol), cartalol (cartelol), carvedilol (arvedilol), esmolol (esmolol), labalol (labetalol), metolol, dol (nadolol), pranolol (sotalol), flugliptin (sotalol), and the peptides (sotalol), flugliptin (sotalol), and the peptides (sotalol) Risinatide (lixisenatide), canagliflozin (canagliflozin), dapagliflozin (dapagliflozin), enggliflozin (empagliflozin), or any combination thereof.

The composition may be administered with medical devices known in the art. For example, the pharmaceutical compositions of the present invention may be administered by injection with a hypodermic needle (including, for example, a pre-filled syringe or an auto-injector).

The pharmaceutical composition may also be administered using a needleless subcutaneous injection device; such as 6,620,135 in U.S. patent No.; 6,096,002;5,399,163;5,383,851;5,312,335;5,064,413;4,941,880;4,790,824 or 4,596,556.

The pharmaceutical composition may also be administered by infusion. Examples of well known implants and modular forms for administration of pharmaceutical compositions include: U.S. patent No. 4,487,603, which discloses an implantable micro infusion pump for dispensing a drug at a controlled rate; U.S. Pat. No. 4,447,233, which discloses a drug infusion pump that delivers a drug at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196 discloses an osmotic drug delivery system having multiple compartment compartments. Many other such implants, delivery systems and modules are well known to those skilled in the art.

Alternatively, the antibody may be administered in a local rather than systemic manner, for example, by direct injection of the antibody into the arthritic joint or pathogen-induced lesions characterized by immunopathology, typically in the form of a depot or sustained release formulation. Furthermore, antibodies may be administered in targeted drug delivery systems (e.g., in liposomes coated with tissue specific antibodies), targeting pathogen-induced lesions such as arthritic joints or characterized by immunopathology. Liposomes will target and be selectively absorbed by diseased tissue.

The dosing regimen will depend on several factors including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the dosing regimen delivers sufficient therapeutic antibody to achieve an improvement in the targeted disease state while minimizing undesirable side effects. Thus, the amount of biologic delivered will depend in part on the particular therapeutic antibody and the severity of the condition being treated. Guidance for selection of appropriate doses of therapeutic antibodies can be obtained (see, e.g. ,Wawrzynczak(1996)Antibody Therapy,Bios Scientific Pub.Ltd,Oxfordshire,UK;Kresina(ed.)(1991)Monoclonal Antibodies,Cytokines and Arthritis,Marcel Dekker,New York,NY;Bach(ed.)(1993)Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases,Marcel Dekker,New York,NY;Baert,et al.(2003)New Engl.J.Med.348:601-608;Milgrom et al.(1999)New Engl.J.Med.341:1966-1973;Slamon et al.(2001)New Engl.J.Med.344:783-792;Beniaminovitz et al.(2000)New Engl.J.Med.342:613-619;Ghosh et al.(2003)New Engl.J.Med.348:24-32;Lipsky et al.(2000)New Engl.J.Med.343:1594-1602).

The appropriate dosage is determined by the clinician, for example, using parameters or factors known or suspected to affect treatment in the art. Typically, the amount at the beginning of the dose is slightly less than the optimal dose and is then increased in small increments until the desired or optimal effect is achieved with respect to any negative side effects. Important diagnostic measures include, for example, those measures of symptoms of inflammation or the level of inflammatory cytokines produced. In general, it is desirable that the biologic to be used be derived from the same species as the treatment target animal, thereby minimizing any immune response to the agent. In the case of human subjects, for example, chimeric antibodies, humanized antibodies, and fully human antibodies may be desirable.

The antibody or antigen binding fragment thereof may be provided by continuous infusion or by a dose administered, for example, once daily, 1-7 times weekly, once weekly, biweekly, monthly, bi-monthly, quarterly, semi-annual, yearly, etc. The dosage may be provided, for example, intravenously, subcutaneously, topically, orally, intranasally, intrarectally, intramuscularly, intracranially, intraspinal, or by inhalation. The total weekly dose is typically at least 0.05 μg/kg body weight, more typically at least 0.2μg/kg、0.5μg/kg、1μg/kg、10μg/kg、100μg/kg、0.25mg/kg、1.0mg/kg、2.0mg/kg、5.0mg/ml、10mg/kg、25mg/kg、50mg/kg or more (see, e.g., ,Yang,et al.(2003)New Engl.J.Med.349:427-434;Herold,et al.(2002)New Engl.J.Med.346:1692-1698;Liu,et al.(1999)J.Neurol.Neurosurg.Psych.67:451-456;Portielji,et al.(20003)Cancer Immunol.Immunother.52:133-144)., a dose may also be provided to achieve a predetermined target concentration of antibody in the serum of a subject, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/ml or more. In other embodiments, fully human antibody is administered subcutaneously or intravenously at 10, 20, 50, 80, 100, 200, 500, 1000, or 2500 mg/subject once weekly, once every two weeks, "once every 4 weeks", once monthly, once every two months, or once quarterly, or other doses as provided herein.

As used herein, "inhibiting" or "treating" or "treatment" includes delaying the development of symptoms associated with a disorder and/or reducing the severity of symptoms of such disorder. These terms further include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying cause of such symptoms. Thus, these terms represent beneficial results that have been brought about in vertebrate subjects suffering from, or having the potential to develop, a disorder, disease or condition.

As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of an antibody or antigen-binding fragment thereof that is effective to cause a measurable improvement in one or more symptoms of a disease or disorder or progression of such disease or disorder when administered to a cell, tissue, or subject, alone or in combination with an additional therapeutic agent. A therapeutically effective dose further refers to an amount of a binding compound that is sufficient to cause at least a partial improvement in symptoms, e.g., treat, cure, prevent, or ameliorate a related medical condition, or increase the rate at which such condition is treated, cured, prevented, or ameliorated. When applied to a single active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to the combined amount of the active ingredients that produces a therapeutic effect, whether administered in combination, serially or simultaneously. An effective amount of the therapeutic agent will result in at least a 10% improvement in the diagnostic measure or parameter; typically at least 20%; preferably at least about 30%; more preferably at least 40%, most preferably at least 50%. In cases where subjective measures are used to assess disease severity, an effective amount may also result in an improvement in subjective measures. In some embodiments, an amount is a therapeutically effective amount if the amount is an amount that can be used to treat or ameliorate a condition provided herein.

As used herein, the term "subject" includes any organism, such as animals, including mammals (e.g., rats, mice, dogs, cats, rabbits), and, for example, humans. The subject may also be referred to as a patient. In some embodiments, the subject is a subject in need thereof. A subject "in need" refers to a subject who has been identified as in need of treatment for a disorder to be treated and who is treated for a particular purpose of treating such disorder. The condition may be, for example, any of the conditions described herein.

However, the isolated antibodies bind to epitopes on IGF-1R proteins or other proteins described herein and exhibit IGF-1R inhibiting or therapeutic activity in vitro and/or in vivo, and antibodies or antigen binding fragments thereof capable of inhibiting IGF-1R function are suitable as therapeutic agents for the treatment of IGF-1R related disorders in humans and animals. These disorders include thyroid eye disease. Thus, also provided are methods of treating such disorders, wherein the methods comprise administering an antibody or antigen-binding fragment thereof to a subject suffering from such disorder.

In some embodiments, the method comprises administering to a susceptible subject or to a subject exhibiting a condition in which IGF-1R is known or suspected to have caused the observed pathology, a therapeutically or prophylactically effective amount of one or more monoclonal antibodies or antigen binding fragments of antibodies described herein. Antibodies can be administered in any active form, including, but not limited to, scFV, fab, and F (ab') 2 fragments, as well as other forms of antibodies provided herein.

As used herein, IGF-1R-related pathology refers to a condition caused by modulation of IGF-1R. These disorders include, but are not limited to, thyroid eye disease and other disorders provided herein.

In some embodiments, the antibodies used are compatible with the recipient species such that the immune response to the MAb does not result in an unacceptably short circulation half-life or induce an immune response to the MAb in the subject.

Treatment of an individual may include administering a therapeutically effective amount of an antibody described herein. Antibodies may be provided in a kit, such as those provided herein. The antibodies may be used or administered alone or in combination with another therapeutic, analgesic or diagnostic agent, such as provided herein. In providing an antibody or fragment thereof capable of binding to IGF-1R to a patient, or an antibody capable of providing protection against IGF-1R pathology in a recipient patient, the dosage of the agent administered will vary depending on: age, weight, height, sex, general medical condition, prior medical history, etc. of the patient.

It is intended to provide an antibody capable of treating a disorder associated with IGF-1R activity or for treating an IGF-1R related pathology to a subject in an amount sufficient to affect a reduction, elimination or amelioration of an IGF-related symptom or pathology. Such pathologies include thyroid eye diseases and the like.

Thus, in some embodiments, methods of treating a subject having an IGF-1R mediated disorder are provided. In some embodiments, the method comprises administering a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as provided herein. In some embodiments, the disorder is thyroid eye disease. As provided herein, the antibody or antigen binding fragment thereof may be administered with other therapeutic agents. These may be administered simultaneously or sequentially.

In some embodiments, the antibody or antigen binding fragment thereof may be used to treat thyroid eye disease. In some embodiments, the antibodies or antigen binding fragments thereof may be used to treat or reduce the severity of thyroid-related eye disease (TAO) or symptoms thereof.

In some embodiments, methods or uses are provided to alleviate ocular process in a subject suffering from thyroid-related eye disease (TAO).

In some embodiments, the subject is a subject who has previously received an antibody treatment different from the antibodies provided herein.

In some embodiments, methods or uses are provided for Clinical Activity Scoring (CAS) of a subject having or suspected of having a thyroid-related eye disease (TAO).

In some embodiments, methods or uses are provided to alleviate a protruding eye by at least 2mm. In some embodiments, methods or uses are provided to alleviate a protruding eye by at least 3mm. In some embodiments, methods or uses are provided to alleviate a protruding eye by at least 2-3mm or 2-4mm. In some embodiments, the eye relief is at least 2, 3, or 4mm. In some embodiments, the relief of the eye from the protrusion is observed within 3 weeks after the administration of the first dose. In some embodiments, the relief of the eye from the protrusion is observed within 6 weeks after the administration of the first dose.

In some embodiments, in a subject with thyroid-related eye disease (TAO), the subject's Clinical Activity Score (CAS) is decreased.

As used herein, the term Clinical Activity Score (CAS) refers to a regimen described and scored according to table 2. According to this scheme, a score is obtained for each of the parameters evaluated in the table below. The sum of all scores defines clinical activity and provides CAS, with 0 or 1 constituting inactive disease and 7 constituting severe active eye disease.

As provided in table 2, CAS is composed of seven components: spontaneous retrobulbar pain, pain in attempts at eye movement (up, left, right, and down gaze), conjunctival redness, eyelid redness, bulbar conjunctival edema, mons/fold swelling, and eyelid swelling. Each component was scored with (1 score) or without (0 score). The score for each efficacy assessment is the sum of all the existing items; a range of 0-7 is obtained, where 0 or 1 constitutes an inactive disease and 7 constitutes a severely active ocular disease. A score of >2 change is considered clinically significant. In some embodiments, the score of the subject is improved by at least 2, 3, or 4 points. In some embodiments, the score of the subject improves within 3 weeks after the first dose. In some embodiments, the score of the subject improves within 6 weeks after the first dose.

The method of claim 1, wherein the spontaneous orbital pain is pain sensation or pressure sensation on or behind the eyeball. This pain may be caused by elevated intra-orbital pressure as the orbital tissue volume increases through excessive synthesis of extracellular matrix, fluid accumulation, and cellular infiltration and expansion. The gaze-induced orbital pain of item 2, which may be eye pain at the time of upward, downward or lateral vision or trial, i.e., eye movement upward, downward or lateral, or trial. This pain may be caused by stretching of one or more inflamed muscles, especially when attempting to look upward. Pressing the eyeball with a finger does not cause "stretching pain", and if this is an manifestation of elevated intra-orbital pressure, it is expected to occur. Both types of pain can be alleviated after anti-inflammatory treatment. Thus, these types of pain are believed to be directly related to orbital autoimmune inflammation and thus can be used to assess TAO activity.

Swelling in TAO is considered conjunctival edema (edema of conjunctiva), item 6 in table 1, and swelling of the mons and/or the semilunar folds. Both are signs of TAO activity. Eyelid swelling can be caused by edema, fat prolapse of the entire orbital membrane, or fibrosis. In addition to swelling, other symptoms indicative of active TAO include redness and/or pain of the conjunctiva, eyelid, mons and/or folds of the semilunar fold.

In some embodiments, the subject receiving treatment has a reduction in the protruding eye of at least 2mm. In some embodiments, the subject receiving treatment has a reduction in the protruding eye of at least 3mm. In some embodiments, the subject receiving treatment has a reduction in the protruding eye of at least 4mm.

In some embodiments, in a subject receiving treatment, the subject's Clinical Activity Score (CAS) is reduced by at least 2 points. In some embodiments, the subject's Clinical Activity Score (CAS) is reduced to one (1). In some embodiments, the subject's Clinical Activity Score (CAS) decreases to zero (0).

In some embodiments, there is provided a method of treating or lessening the severity of thyroid-related eye disease (TAO) in a subject, wherein treatment with said antibody (i) reduces the protrusion of the eye by at least 2mm; (ii) Not accompanied by a deterioration of the other eye (or contralateral eye) of 2mm or more; and (iii) reducing CAS of the subject to one (1) or zero (0).

In some embodiments, methods of improving the quality of life of a subject suffering from thyroid-related eye disease (TAO, also known as graves 'eye disease/graves' orbit disease) are provided. In some embodiments, quality of life is measured by a graves' eye disease quality of life (GO-QoL) assessment or a visual function or appearance score scale thereof. In some embodiments, the treatment results in a GO-QoL improvement of greater than or equal to 8 points. In some embodiments, the treatment results in an improvement in the functional component scale of GO-QoL. In some embodiments, the treatment results in an improvement in the appearance fraction scale of GO-QoL.

In some embodiments, methods of treating or lessening the severity of a double vision in a subject suffering from thyroid-related eye disease (TAO) are provided. In some embodiments, the review is persistent review. In some embodiments, the review is non-persistent review. In some embodiments, the review is intermittent review. In some embodiments, improvement in or reduction in severity of the double vision persists for at least 20 weeks after cessation of antibody administration. In some embodiments, improvement in or reduction in severity of the double vision persists for at least 50 weeks after cessation of antibody administration. In some embodiments, the subject's double vision improves within 3 weeks or within 6 weeks after the first dose.

The severity of the disease can be measured in the following non-limiting embodiments. For example, for eyelid aperture, the distance (in mm) between the eyelid edges is measured with the patient in the first eye position, relaxed sitting position, and looking at the object at distance. For eyelid swelling, the measurement/assessment is "absent/suspicious", "moderate" or "severe". There was no or no redness of the eyelid. Conjunctival redness is absent or present. In some embodiments, conjunctival edema is absent or present. In some embodiments, the inflammation of the caruncle or fold is absent or present. For individual patients, the herniation was measured in millimeters using the same Hertel tenon's and the same inter-canthus distance. Subjective review scores 0 to 3 (0 = no review; 1 = intermittent, i.e. review occurs at first eye gaze at tiredness or at first wake; 2 = non-persistent, i.e. review occurs at the extreme of gaze; 3 = persistent, i.e. continuous review occurs in first eye or reading position). For ocular muscular involvement, steering is measured in degrees (duction). Cornea involvement is absent/punctiform or keratopathy/ulceration. For optic nerve involvement, i.e., optimal correction of vision, color vision, optic disc, relative pupil afferent defects, the condition is absent or present. In addition, if optic nerve compression is suspected, the field of view is examined. In some embodiments, patients may be classified according to the following severity classifications. For example, thyroid eye disease that endangers vision: patients with thyroid Dysfunction Optic Neuropathy (DON) and/or cornea rupture. This class requires immediate intervention. Moderate to severe thyroid eye disease: patients who do not suffer from vision-threatening diseases whose ocular disease has sufficient impact on daily life to justify the risk of immunosuppression (if active) or surgical intervention (if inactive). Patients with moderate to severe thyroid eye disease typically have any one or more of the following: eyelid retraction greater than or equal to 2mm, moderate or severe soft tissue involvement, eye herniation greater than 3mm or 3mm above normal levels of race and gender, non-persistent or persistent presbyopia. Mild thyroid eye disease: the characteristics of thyroid eye disease in patients have little effect on daily life and are not sufficient to justify immunosuppression or surgical treatment. They typically have only one or more of the following: minimal eyelid retraction (< 2 mm), mild soft tissue involvement, eye herniation <3mm above normal levels of race and sex, transient or no presbyopia, and lubricant-responsive corneal exposure.

In some embodiments, the patient may be characterized by a graves' eye disease quality of life (GO-QoL) score. In addition to the salient eyes (or herniation) and CAS, a GO quality of life (GO-QoL) questionnaire was used to evaluate quality of life. The questionnaire was designed to determine the improvement in quality of life following treatment with the methods disclosed herein. In some embodiments, the questionnaire may determine that there is reduced or no side effect following treatment with an antibody or antigen-binding fragment thereof according to the methods disclosed herein as compared to treatment with a glucocorticoid. GO-QoL is a 16-item self-filling questionnaire, divided into 2 subsets, and used to evaluate the perceived impact of subjects on TED based on (i) daily physical activity related to visual function and (ii) psychosocial function. Quality of life was assessed using the GO QoL questionnaire. The GO-Qol questionnaire was completed on days 1 and weeks 6, 12 and 24 (or PW) during treatment and on months 7 and 12 (or PW) during follow-up [ C.B.Terwee et al,1998]. GO-Qol is a 16-item self-filling questionnaire divided into two self-assessment score tables; one covering the effect of visual function on daily activities and the other evaluating the effect of self-perceived appearance. The visual function component list encompasses activities such as driving, walking outdoors, reading, watching television, and the like. The appearance component table asks the subject questions, such as whether the eye disease has changed the subject's appearance, resulting in other people responding negatively to the subject, resulting in social isolation, and resulting in the subject attempting to mask his or her appearance. Each component table has 8 questions answered as follows: yes—very much so; yes—very few; or no-not at all. Each question was divided into 0-2 points, and then the total raw score was mathematically converted to a 0-100 scale, where 0 represents the greatest negative impact on quality of life and 100 represents no impact. Changes of > or equal to 8 points based on the 0-100 scale have been shown to be clinically significant. The combined scores take the original scores from both component tables and again convert them to a single 0-100 scale. The questionnaire has two self-assessment score scales. Each component table has 8 questions answered as follows: (i) is-very so; (ii) yes-little; or (iii) no-no at all. Each question was divided into 0-2 points, and then the total raw score was mathematically converted to a 0-100 scale, where 0 represents the greatest negative impact on quality of life and 100 represents no impact. Changes of >8 points on a scale of 0-100 are considered clinically significant. The combined scores take the original scores from both component tables and again convert them to a single 0-100 scale.

Patients may also be assessed by the presence or absence of a review Gorman scale. The Gorman assessment of subjective review includes four categories: no fixation (absence), fixation when the patient is tired or awake (intermittent), fixation when the patient is gazing extremely (non-persistent), and fixation when the patient is in the first eye position or reading position (persistent). Patients were scored according to the level at which they appeared to be double vision. Improvements of grade 1 or greater are considered clinically significant.

In some embodiments, the method comprises administering an antibody, such as those provided herein. In some embodiments, the antibody is administered as a first dose at a dose of about 1mg/kg to about 5mg/kg of antibody. In some embodiments, the antibody is administered as a first dose at a dose of about 5mg/kg to about 10mg/kg of antibody. In some embodiments, the antibody is administered in a subsequent dose at a dose of about 5mg/kg to about 20mg/kg of antibody. In some embodiments, the antibody is administered in the following amounts: about 10mg/kg antibody as the first dose; and about 20mg/kg antibody in subsequent doses. In some embodiments, the subsequent dose is administered every three weeks for at least 21 weeks.

In some embodiments, the antibody is administered in a pharmaceutical composition, such as those provided herein. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically active compounds for treating TAO. In some embodiments, the pharmaceutical composition further comprises a corticosteroid; rituximab or other anti-CD 20 antibody; tobrazumab or other anti-IL-6 antibodies; or selenium, infliximab or other anti-tnfα antibodies or Thyroid Stimulating Hormone Receptor (TSHR) inhibitors.

In some embodiments, the methods provided herein comprise administering to a subject an antibody or antigen-binding fragment thereof that specifically binds and inhibits IGF-IR. In some embodiments, the antibodies are as provided herein.

Kits useful for practicing embodiments described herein are also provided. The kit of the invention comprises a first container comprising or packaged in combination with an antibody as described above. The kit may also comprise a further container containing a solution necessary or appropriate for the implementation of the embodiment or a combined solution packaged therein. The container may be made of glass, plastic or foil and may be a vial, bottle, pouch, tube, bag, or the like. The kit may also contain written information, such as a program for implementing an embodiment, or analytical information, such as the amount of reagent contained in the first container means. The container may be located in another container means, such as a box or bag, together with the written information.

Yet another aspect provided herein is a kit for detecting IGF-1R protein in a biological sample. The kit includes a container containing one or more antibodies that bind to an epitope of an IGF-1R protein, and instructions for using the antibodies to correlate the presence or absence of an immune complex with the presence or absence of an IGF-1R protein in the sample for the purpose of binding to the IGF-1R protein to form an immune complex and detecting the formation of the immune complex. Examples of containers include multi-well plates that allow for simultaneous detection of IGF-1R protein in multiple samples.

In some embodiments, antibodies that bind to IGF-1R proteins are provided. In some embodiments, the antibody is isolated. In some embodiments, the antibody specifically binds. In some embodiments, the antibody binds to a properly folded IGF-1R protein. In some embodiments, the antibody is specific for a particular IGF-1R conformational state (open or closed). In some embodiments, the antibody binds to IGF-1R protein in the cell membrane. In some embodiments, the antibody binds to IGF-1R protein in the cell membrane of intact cells. In some embodiments, the antibody inhibits or neutralizes the function of IGF-1R protein. As used herein, the term "neutralizing" means that the activity or function of a protein is inhibited. Inhibition may be complete or partial. In some embodiments, the activity or function of the protein is inhibited by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. The percent inhibition may be based on the function or activity of the protein in the absence of antibodies. In some embodiments, the antibody inhibits glucose transport facilitated by IGF-1R. In some embodiments, the antibody inhibits internalization of IGF-1R protein.

In some embodiments, the antibody comprises a sequence provided herein or an antigen binding fragment thereof. In some embodiments, the antibody comprises a heavy chain CDR or antigen binding fragment thereof described herein. The heavy chain may be one or more of the heavy chains described herein. In some embodiments, the antibody comprises a light chain or antigen binding fragment thereof as described herein.

In some embodiments, methods of treating, inhibiting, or ameliorating an IGF-1R related pathology are provided. In some embodiments, the method comprises administering an antibody described herein or a pharmaceutical composition described herein to a subject to treat, inhibit, or ameliorate IGF-1R-related pathology. In some embodiments, the pathology is as described herein.

In some embodiments, methods of detecting the presence or absence of IGF-1R in a sample are provided, the methods comprising contacting the sample with one or more antibodies described herein, detecting binding of the antibodies to IGF-1R antigen. In some embodiments, detection of binding indicates the presence of IGF-1R antigen; or detection of no detection of binding to IGF-1R antigen indicates the absence of IGF-1R antigen. The detection may be performed by any known method, such as using a biosensor, ELISA, sandwich assay, or the like. However, in some embodiments, the method comprises detecting the presence of the protein under non-denaturing conditions. Non-denaturing conditions may be used so that the target protein is detected in its native or properly folded form.

In some embodiments, methods of identifying a test antibody that binds to an epitope on an IGF-1R protein are provided, the methods comprising contacting the test antibody with an epitope on the IGF-1R protein and determining whether the test antibody binds to the epitope. In some embodiments, determining comprises determining whether the test antibody binds to a protein and is competitively inhibited by an antibody comprising a sequence provided herein. In some embodiments, determining comprises mutating one or more residues of an epitope or protein and determining binding of the test antibody to the mutated epitope, wherein the test antibody is considered to bind to the epitope if the mutation reduces binding of the test antibody compared to the non-mutated epitope.

In some embodiments, methods of monitoring internalization of IGF-1R from the cell surface are provided. In some embodiments, the method comprises contacting the cell with an anti-IGF-1R antibody provided herein and detecting the presence of IGF-1R in or on the cell surface. Differences in cell surface expression can be measured and internalization can be monitored and measured. For example, this can be used to measure the effect of another molecule (such as a test agent) on modulation of IGF-1R protein internalization. Thus, the antibodies provided herein can be used to identify assays that modulate (increase or decrease) IGF-1R protein internalization. Test molecules that increase internalization, which would be measured as a decrease in binding of an anti-IGF-1R antibody to IGF-1R protein on the cell surface, can be identified according to the methods provided herein. Test molecules that reduce internalization, which would be measured as increased binding of an anti-IGF-1R antibody to an IGF-1R protein on the cell surface, can be identified according to the methods provided herein. Surface expression can be measured by fluorescence, which can be accomplished by recognizing a secondary antibody to IGF-1R antibody or by labeling the anti-IGF-1R antibody provided herein.

In some embodiments, methods of treating thyroid-related eye disease in a subject in need thereof are provided. In some embodiments, the method comprises periodically administering intravenously to the subject a dose of 10mg/kg of an anti-IGF-1R antibody for a period of time sufficient to alleviate one or more symptoms associated with thyroid-associated eye disease, wherein the anti-IGF-1R antibody comprises a heavy chain comprising HCDR1 of SEQ ID NO:7, HCDR2 of SEQ ID NO:8, and HCDR3 of SEQ ID NO:9 and a light chain comprising LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5, and LCDR3 of SEQ ID NO: 6. In some embodiments, the anti-IGF-1R antibody comprises a light chain and a heavy chain, wherein the light chain comprises a variable region having the amino acid sequence of SEQ ID NO. 2 and the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO. 3. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO. 11. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO. 10. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO. 10 and the light chain comprises the amino acid sequence of SEQ ID NO. 11. In some embodiments, the anti-IGF-1R antibody is administered by intravenous infusion. In some embodiments, the anti-IGF-1R antibody is administered every 3 weeks. In some embodiments, the anti-IGF-1R antibody is administered for a period of time sufficient to perform 5 doses. In some embodiments, the anti-IGF-1R antibody is administered for a period of time sufficient to perform 8 doses. In some embodiments, the anti-IGF-1R antibody is administered for a period of time selected from 3 weeks, 6 weeks, 9 weeks, 12 weeks, 15 weeks, 18 weeks, 21 weeks, 24 weeks, or more.

In some embodiments, antibodies for use in treating thyroid-related eye disease in a subject in need thereof are provided. In some embodiments, anti-IGF-1R antibodies are provided for use in treating thyroid-related eye diseases in a subject in need thereof.

In some embodiments, embodiments provided herein further include, but are not limited to:

1. A method of treating thyroid-related eye disease in a subject in need thereof, comprising:

Administering a first dose of an antibody intravenously or subcutaneously to the subject, wherein the first dose is selected from the group consisting of: about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg, and

Administering intravenously or subcutaneously to the subject one or more subsequent doses of antibody, wherein each subsequent dose is selected from the group consisting of: about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg,

Wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises HCDR1 of SEQ ID No. 7, HCDR2 of SEQ ID No. 8, and HCDR3 of SEQ ID No. 9, and the light chain comprises LCDR1 of SEQ ID No. 4, LCDR2 of SEQ ID No. 5, and LCDR3 of SEQ ID No. 6; or (b)

Wherein the antibody comprises a light chain comprising a variable region having the amino acid sequence of SEQ ID NO. 2 and a heavy chain comprising a variable region sequence having the amino acid sequence of SEQ ID NO. 3; or (b)

Wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO. 11 and a heavy chain comprising the amino acid sequence of SEQ ID NO. 10.

2. The method of claim 1, wherein the first dose is about 2mg/kg.

3. The method of claim 1, wherein the first dose is about 3mg/kg.

4. The method of claim 1, wherein the first dose is about 2.5mg/kg.

5. The method of claim 1, wherein the first dose is about 5mg/kg.

6. The method of claim 1, wherein the first dose is about 7.5mg/kg.

7. The method of claim 1, wherein the first dose is about 10mg/kg.

8. The method of claim 1, wherein the first dose is about 15mg/kg.

9. The method of claim 1, wherein the first dose is about 20mg/kg.

10. The method of any one of claims 2-9, wherein the amount of the one or more subsequent doses is the same as the amount of the first dose.

11. The method of any one of claims 2-9, wherein the amount of the one or more subsequent doses is different from the amount of the first dose.

12. The method of any one of claims 2-11, wherein at least one of the one or more subsequent doses is about 2mg/kg.

13. The method of any one of claims 2-11, wherein at least one of the one or more subsequent doses is about 3mg/kg.

14. The method of any one of claims 2-11, wherein at least one of the one or more subsequent doses is about 5mg/kg.

15. The method of any one of claims 2-11, wherein at least one of the one or more subsequent doses is about 7.5mg/kg.

16. The method of any one of claims 2-11, wherein at least one of the one or more subsequent doses is about 10mg/kg.

17. The method of any one of claims 2-11, wherein at least one of the one or more subsequent doses is about 15mg/kg.

18. The method of any one of claims 2-11, wherein at least one of the one or more subsequent doses is about 20mg/kg.

19. The method of any one of claims 1-18, wherein at least one of the one or more subsequent doses is administered one week, two weeks, three weeks, four weeks, five weeks, six weeks, or eight weeks after the first dose.

20. The method of any one of claims 1-19, wherein only one, two, three, four, five, six, or seven subsequent doses are administered to the subject.

21. The method of any one of claims 1-20, comprising administering a total of two, three, four, five, six, seven, or eight doses to the subject.

22. The method of any one of claims 1-21, wherein the subject's clinical activity score decreases after two or three doses of antibody.

23. The method of any one of claims 1-22, wherein each subsequent dose is administered one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, or eight weeks after the previous dose.

24. The method of any one of claims 1-23, comprising administering at least one dose by intravenous infusion over 45 minutes to about 90 minutes, or over 60 minutes to about 90 minutes.

25. The method of any one of claims 1-24, comprising administering at least one dose by subcutaneous administration.

26. The method of claim 25, wherein the subcutaneous administration is self-administration.

27. The method of any one of claims 1-26, further comprising administering one or more loading doses of antibody to the subject prior to administering the first dose.

28. The method of any one of claims 1-26, further comprising administering a first loading dose of antibody to the subject prior to administering the first dose, wherein the first loading dose is selected from the group consisting of: about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 12.5mg/kg, or about 12.5mg/kg to about 15mg/kg.

29. The method of claim 27 or 28, wherein a second loading dose of antibody is administered to the subject after the first loading dose, and wherein the first and second loading doses are administered prior to the first dose.

30. The method of claim 29, wherein the first loading dose and the second loading dose are the same dose amount.

31. The method of claim 29, wherein the first loading dose and the second loading dose are different dose amounts.

32. The method of any one of claims 27-31, wherein the first loading dose is about 5mg/kg.

33. The method of any one of claims 27-31, wherein the first loading dose is about 7.5mg/kg.

34. The method of any one of claims 27-31, wherein the first loading dose is about 10mg/kg.

35. The method of any one of claims 27-31, wherein the first loading dose is about 12.5mg/kg.

36. The method of any one of claims 27-31, wherein the first loading dose is about 15mg/kg.

37. The method of any one of claims 27-36, wherein the second loading dose is about 5mg/kg.

38. The method of any one of claims 27-36, wherein the second loading dose is about 7.5mg/kg.

39. The method of any one of claims 27-36, wherein the second loading dose is about 10mg/kg.

40. The method of any one of claims 27-36, wherein the second loading dose is about 12.5mg/kg.

41. The method of any one of claims 27-36, wherein the second loading dose is about 15mg/kg.

42. The method of any one of claims 27-41, wherein the first loading dose is administered to the subject one, two, three, or four weeks prior to the administration of the first dose.

43. The method of any one of claims 1-42, wherein the antibody is administered as part of a pharmaceutically acceptable composition comprising the antibody and at least one pharmaceutically acceptable excipient, wherein the antibody has a solubility of at least about 150mg/ml in the pharmaceutically acceptable composition.

44. The method of any one of claims 1-43, wherein the subject has unsatisfactory relief from prior treatment of a thyroid-related eye disease.

45. The method of claim 44, wherein the unsatisfactory relief is one or more of: failing to alleviate the abrupt eye by 2mm or more; failing to reduce CAS on one or more components or failing to reduce 2 points or more; the other eye deteriorates by 2mm or more; the multiple vision is not alleviated; failing to continue improving the compound for a period of time; failure to improve the score of the graves' quality of life (GO-QoL) assessment by 8 points or more; and combinations thereof.

46. A method of treating thyroid-related eye disease in a subject in need thereof, comprising:

Administering intravenously or subcutaneously to the subject one or more subsequent doses of antibody, wherein each subsequent dose is selected from the group consisting of: about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg, wherein the antibody is provided herein, including but not limited to:

Wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO. 11 and a heavy chain comprising the amino acid sequence of SEQ ID NO. 10,

Wherein one or more subsequent doses are administered when it is determined by measuring the clinical activity score and/or the eye burst that the subject has insufficient relief from one or more previous doses.

47. A method of improving treatment of thyroid-related eye disease in a subject previously administered one or more treatments, comprising:

Administering intravenously or subcutaneously to the subject at least one dose of antibody consisting of: about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg, wherein the antibody is represented by an antibody as provided herein, such as,

Wherein the at least one dose results in one or more measurements being improved relative to one or more measurements prior to the at least one dose.

48. The method of claim 47, wherein the one or more measurements are selected from the group consisting of a sudden eye, a CAS, a level of exacerbation of the other eye, a GO-QoL score, and combinations thereof.

49. The method of claim 47 or 48, wherein if the subject does not have satisfactory relief after at least one dose, one or more subsequent doses of antibody, each selected from the group consisting of: about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg.

50. The method of claim 49, wherein the one or more subsequent doses improve one or more of a protruding eye, CAS, a exacerbation level of the other eye, a GO-QoL score, and combinations thereof, as compared to before the one or more subsequent doses.

51. A method of treating thyroid-related eye disease in a subject in need thereof, comprising:

administering a first dose of an antibody intravenously or subcutaneously to the subject, wherein the first dose is selected from the group consisting of: about 250mg, about 300mg, about 350mg or about 400mg, and

Administering intravenously or subcutaneously to the subject one or more subsequent doses of antibody, wherein each subsequent dose is selected from the group consisting of: about 250mg, about 300mg, about 350mg or about 400mg, wherein the antibody is represented by SEQ ID NO. 1.

52. The method of claim 51, wherein the first dose is about 250mg.

53. The method of claim 52, wherein the first dose is about 300mg.

54. The method of claim 52, wherein the first dose is about 350mg.

55. The method of claim 52, wherein the first dose is about 400mg.

56. The method of any of claims 51-55, wherein the amount of the first dose and the amount of the one or more subsequent doses are the same dose amount.

57. The method of any of claims 51-55, wherein the amount of the first dose and the amount of the one or more subsequent doses are amounts of different doses.

58. The method of any one of claims 51-57, wherein at least one of the one or more subsequent doses is about 250mg.

59. The method of any one of claims 51-57, wherein at least one of the one or more subsequent doses is about 300mg.

60. The method of any one of claims 51-57, wherein at least one of the one or more subsequent doses is about 350mg.

61. The method of any one of claims 51-57, wherein at least one of the one or more subsequent doses is about 400mg.

62. The method of any one of claims 51-61, wherein at least one of the one or more subsequent doses is administered one week, two weeks, three weeks, four weeks, five weeks, six weeks, or eight weeks after the first dose.

63. The method of any one of claims 51-62, wherein only one, two, three, four, five, six, or seven subsequent doses are administered to the subject.

64. The method of any one of claims 51-63, comprising administering a total of two, three, four, five, six, seven, or eight doses to the subject.

65. The method of any one of claims 51-64, wherein the subject's clinical activity score decreases after two or three doses of antibody.

66. The method of any one of claims 51-65, wherein each subsequent dose is administered one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, or eight weeks after the previous dose.

67. The method of any one of claims 51-66, comprising administering at least one dose by intravenous infusion over 45 minutes to about 90 minutes, or over 60 minutes to about 90 minutes.

68. The method of any one of claims 51-67, comprising administering at least one dose by subcutaneous administration.

69. The method of claim 68, wherein the subcutaneous administration is self-administration.

70. The method of any one of claims 51-69, further comprising administering one or more loading doses of antibody to the subject prior to administering the first dose.

71. The method of any one of claims 51-69, further comprising administering a first loading dose of antibody to the subject prior to administering the first dose, wherein the first loading dose is selected from the group consisting of about 250mg, about 300mg, about 350mg, or about 400 mg.

72. The method of claim 70 or 71, wherein a second loading dose of antibody is administered to the subject after the first loading dose, and wherein the first and second loading doses are administered prior to the first dose.

73. The method of claim 72, wherein the first loading dose and the second loading dose are the same dose amount.

74. The method of claim 73, wherein the first loading dose and the second loading dose are different dose amounts.

75. The method of any one of claims 70-73, wherein the first loading dose is about 250mg.

76. The method of any one of claims 70-73, wherein the first loading dose is about 300mg.

77. The method of any one of claims 70-73, wherein the first loading dose is about 350mg.

78. The method of any one of claims 70-73, wherein the first loading dose is about 400mg.

79. The method of any one of claims 70-78, wherein the second loading dose is about 250mg.

80. The method of any one of claims 70-78, wherein the second loading dose is about 300mg.

81. The method of any one of claims 70-78, wherein the second loading dose is about 350mg.

82. The method of any one of claims 70-78, wherein the second loading dose is about 400mg.

83. The method of any one of claims 70-78, wherein the first loading dose is administered to the subject one, two, three, or four weeks prior to administration of the first dose.

84. The method of any one of claims 51-83, wherein the antibody is administered as part of a pharmaceutically acceptable composition comprising the antibody and at least one pharmaceutically acceptable excipient, wherein the antibody has a solubility of at least about 150mg/ml in the pharmaceutically acceptable composition.

85. The method of any one of claims 51-84, wherein the subject has unsatisfactory relief from prior treatment of a thyroid-related eye disease.

86. The method of claim 85, wherein the unsatisfactory relief is selected from failing to relieve a sudden eye by 2mm or more; failing to reduce CAS on one or more components or failing to reduce 2 points or more; the other eye deteriorates by 2mm or more; the multiple vision is not alleviated; failing to continue improving the compound for a period of time; failure to improve the score of the graves' quality of life (GO-QoL) assessment by 8 points or more; and combinations thereof.

87. The method of any one of claims 27-45 or 70-86, wherein the first loading dose and the second loading dose are administered between about one week, about two weeks, or about three weeks apart.

88. The method of claim 87, wherein the second loading dose is administered about one week, about two weeks, or about three weeks prior to the first dose.

89. A method of treating thyroid-related eye disease in a subject in need thereof, comprising:

Administering a first dose of an antibody intravenously or subcutaneously to the subject, wherein the first dose is selected from the group consisting of: about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg; and

Wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises HCDR1 of SEQ ID NO. 7, HCDR2 of SEQ ID NO. 8, and HCDR3 of SEQ ID NO. 9, and the light chain comprises LCDR1 of SEQ ID NO. 4, LCDR2 of SEQ ID NO. 5, and LCDR3 of SEQ ID NO. 6.

90. The method of embodiment 89, wherein the light chain comprises a variable region having the amino acid sequence of SEQ ID No.2 and the heavy chain comprises a variable region sequence having the amino acid sequence of SEQ ID No. 3.

91. The method of embodiment 89 or 90, wherein the light chain comprises the amino acid sequence of SEQ ID NO. 11.

92. The method of any of embodiments 89-91, wherein the heavy chain comprises the amino acid sequence of SEQ ID No. 10.

93. The method of embodiment 89, wherein the first dose is about 10mg/kg.

94. The method of embodiment 93, wherein the one or more subsequent doses is about 10mg/kg.

95. The method of any of embodiments 89-93, wherein the amount of the one or more subsequent doses is the same as the amount of the first dose.

96. The method of any of embodiments 89-94, wherein the amount of the one or more subsequent doses is different from the amount of the first dose.

97. The method of any of embodiments 89-96, wherein at least one of the one or more subsequent doses is administered one week, two weeks, three weeks, four weeks, five weeks, six weeks, or eight weeks after the first dose.

98. The method of embodiment 94, wherein one or more of the one or more subsequent doses is administered three weeks after the first dose.

99. The method of embodiment 94, wherein the subsequent dose is administered every three weeks after the first dose for 4, 5, 6, 7, or 8 cycles.

100. The method of embodiment 94, wherein the subsequent dose is administered every three weeks after the first dose for 5 or 8 cycles.

101. The method of embodiment 94, wherein the method comprises administering a total of five, six, seven, or eight doses to the subject.

102. The method of any of embodiments 89-101, wherein the subject's clinical activity score decreases after a first dose of antibody.

103. The method of any of embodiments 89-102, wherein the subject's clinical activity score decreases after two doses of antibody.

104. The method of embodiments 89-103, wherein the subject's clinical activity score decreases within 6 weeks of the first dose after the one or more subsequent doses.

105. The method of embodiments 89-103, wherein, after the one or more subsequent doses, the subject's clinical activity score decreases within 3 weeks of the initial one or more subsequent doses.

106. The method of any of embodiments 89-105, wherein the antibody is administered by intravenous infusion over 45 minutes to about 90 minutes, or over 60 minutes to about 90 minutes.

107. The method of embodiment 89, wherein the first dose is about 10mg/kg and the one or more subsequent doses are about 10mg/kg.

108. The method of any of embodiments 89-106, wherein the antibody is administered as part of a pharmaceutically acceptable composition comprising the antibody and at least one pharmaceutically acceptable excipient, wherein the pharmaceutical composition comprises the antibody at a concentration of 20mg/mL to about 30 mg/mL.

109. The method of embodiment 108, wherein the pharmaceutical composition comprises the antibody at a concentration of about 25 mg/mL.

110. The method of any of embodiments 89-109, wherein the treated subject has a reduction in the presbyopia of at least or about 1-4mm.

111. The method of embodiment 110, wherein the eye relief is at least or about 2-3mm.

112. The method of embodiment 110 or 111, wherein the protruding eye is reduced within 3 weeks of the first dose.

113. The method of embodiment 110 or 111, wherein the protruding eye is reduced within 6 weeks of the first dose.

114. The method of any of embodiments 89-113, wherein the treated subject has reduced double vision.

115. The method of embodiment 114, wherein the double vision is reduced within 3 weeks or 6 weeks of the first dose.

116. The method of any of embodiments 89-115, wherein the subject's Clinical Activity Score (CAS) improves within 3 weeks or 6 weeks.

117. The method of embodiment 37, wherein the CAS score is improved by at least-2, -3, or-4.

118. The method of any of embodiments 89-117, wherein the subject's eyes are relieved and CAS score is improved within 3 weeks or within 6 weeks of the first dose.

119. A method of treating thyroid-related eye disease in a subject in need thereof, comprising:

A first dose of 10mg/kg of antibody is administered intravenously to a subject,

Wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises HCDR1 of SEQ ID NO. 7, HCDR2 of SEQ ID NO. 8 and HCDR3 of SEQ ID NO. 9, and the light chain comprises LCDR1 of SEQ ID NO. 4, LCDR2 of SEQ ID NO. 5 and LCDR3 of SEQ ID NO. 6.

120. The method of embodiment 119, wherein the light chain comprises a variable region having the amino acid sequence of SEQ ID No. 2 and the heavy chain comprises a variable region sequence having the amino acid sequence of SEQ ID No. 3.

121. The method of embodiment 119, wherein the light chain comprises the amino acid sequence of SEQ ID NO. 11.

122. The method of embodiment 119, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO. 10.

123. The method of embodiment 119, wherein the heavy chain comprises the amino acid sequence of SEQ ID No. 10 and the light chain comprises the amino acid sequence of SEQ ID No. 11.

124. The method of any of embodiments 119-123, wherein the method further comprises administering a subsequent dose of about 10 mg/kg.

125. The method of embodiment 124, wherein a subsequent dose is administered about 3 weeks after the first dose.

126. The method of any of embodiments 119-125, wherein the method further comprises administering a subsequent dose of about 10mg/kg every 3 weeks after the first dose.

127. The method of embodiment 126, wherein subsequent doses are administered every 3 weeks for a total of 4 subsequent doses.

128. The method of embodiment 126, wherein subsequent doses are administered every 3 weeks for a total of 7 subsequent doses.

129. The method of any of embodiments 119-128, wherein the subject's eye relief is at least or about 1-4mm.

130. The method of embodiment 129, wherein the eye relief is at least or about 2-3mm.

131. The method of embodiment 129 or 130, wherein the protruding eye is reduced within 3 weeks of the first dose.

132. The method of embodiment 129 or 130, wherein the protruding eye is reduced within 6 weeks of the first dose.

133. The method of any of embodiments 119-132, wherein the treated subject has reduced double vision.

134. The method of embodiment 133, wherein the double vision is reduced within 3 weeks or 6 weeks of the first dose.

135. The method of any of embodiments 119-134, wherein the subject's Clinical Activity Score (CAS) improves within 3 weeks or 6 weeks.

136. The method of embodiment 135, wherein the CAS score is improved by at least-2, -3, or-4.

137. The method of any of embodiments 119-136, wherein the subject's eyes are relieved and CAS score is improved within 3 weeks or within 6 weeks of the first dose.

138. A method of treating thyroid-related eye disease in a subject in need thereof, comprising:

periodically administering intravenously to the subject a dose of 10mg/kg of an anti-IGF-1R antibody for a period of time sufficient to alleviate one or more symptoms associated with thyroid-associated eye disease,

Wherein the anti-IGF-1R antibody comprises a heavy chain comprising HCDR1 of SEQ ID NO. 7, HCDR2 of SEQ ID NO. 8 and HCDR3 of SEQ ID NO. 9 and a light chain comprising LCDR1 of SEQ ID NO. 4, LCDR2 of SEQ ID NO. 5 and LCDR3 of SEQ ID NO. 6.

139. The method of embodiment 138, wherein the anti-IGF-1R antibody comprises a light chain and a heavy chain, wherein the light chain comprises a variable region having the amino acid sequence of SEQ ID No. 2 and the heavy chain comprises a variable region having the amino acid sequence of SEQ ID No. 3.

140. The method of embodiment 138, wherein the light chain comprises the amino acid sequence of SEQ ID NO. 11.

141. The method of embodiment 138, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO. 10.

142. The method of embodiment 138, wherein the heavy chain comprises the amino acid sequence of SEQ ID No. 10 and the light chain comprises the amino acid sequence of SEQ ID No. 11.

143. The method of any of embodiments 138-142, wherein the anti-IGF-1R antibody is administered by intravenous infusion.

144. The method of any one of embodiments 138-143, wherein the anti-IGF-1R antibody is administered every 3 weeks.

145. The method of any of embodiments 138-144, wherein the anti-IGF-1R antibody is administered for a period of time sufficient to perform 5 doses.

146. The method of any of embodiments 138-144, wherein the anti-IGF-1R antibody is administered for a period of time sufficient to perform 8 doses.

147. The method of any one of embodiments 138-144, wherein the anti-IGF-1R antibody is administered for a period of time selected from 3 weeks, 6 weeks, 9 weeks, 12 weeks, 15 weeks, 18 weeks, 21 weeks, 24 weeks, or more.

148. The method of any of embodiments 138-147, wherein the subject's eye relief is at least or about 1-4mm.

149. The method of embodiment 148, wherein the eye relief is at least or about 2-3mm.

150. The method of embodiment 148 or 149, wherein the protruding eye is relieved within 3 weeks of the first dose.

151. The method of embodiment 148 or 149, wherein the protruding eye is relieved within 6 weeks of the first dose.

152. The method of any of embodiments 148-151, wherein the treated subject has reduced double vision.

153. The method of embodiment 152, wherein the double vision is reduced within 3 weeks or 6 weeks of the first dose.

154. The method of any of embodiments 138-153, wherein the subject's Clinical Activity Score (CAS) improves within 3 weeks or 6 weeks.

155. The method of embodiment 154, wherein the CAS score is improved by at least-2, -3, or-4.

156. The method of any of embodiments 138-155, wherein the subject's eyes are relieved and CAS score is improved within 3 weeks or within 6 weeks of the first dose.

157. An antibody for treating thyroid-associated eye disease in a subject in need thereof, the antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises HCDR1 of SEQ ID No. 7, HCDR2 of SEQ ID No. 8 and HCDR3 of SEQ ID No. 9, and the light chain comprises LCDR1 of SEQ ID No. 4, LCDR2 of SEQ ID No. 5 and LCDR3 of SEQ ID No. 6, wherein

Administering said antibody intravenously or subcutaneously to the subject as a first dose of antibody, wherein the first dose is selected from the group consisting of: about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg; and

Administering said antibody intravenously or subcutaneously to the subject as one or more subsequent doses of antibody, wherein each subsequent dose is selected from the group consisting of: about 1mg/kg to about 2mg/kg, about 2mg/kg to about 5mg/kg, about 3mg/kg to about 5mg/kg, about 5mg/kg to about 7.5mg/kg, about 7.5mg/kg to about 10mg/kg, about 10mg/kg to about 15mg/kg, or about 15mg/kg to about 20mg/kg.

158. The antibody of embodiment 157, wherein the light chain comprises a variable region having the amino acid sequence of SEQ ID No.2 and the heavy chain comprises a variable region sequence having the amino acid sequence of SEQ ID No. 3.

159. The antibody of embodiment 157 or 158, wherein the light chain comprises the amino acid sequence of SEQ ID NO. 11.

160. The antibody of any one of embodiments 157-159, wherein the heavy chain comprises the amino acid sequence of SEQ ID No. 10.

161. The antibody of embodiment 157, wherein the first dose is about 10mg/kg.

162. The antibody of embodiment 161, wherein the one or more subsequent doses is about 10mg/kg.

163. The antibody of any one of embodiments 157-161, wherein the amount of the one or more subsequent doses is the same as the amount of the first dose.

164. The antibody of any one of embodiments 157-161, wherein the amount of the one or more subsequent doses is different from the amount of the first dose.

165. The antibody of any one of embodiments 157-164, wherein at least one of the one or more subsequent doses is administered one week, two weeks, three weeks, four weeks, five weeks, six weeks, or eight weeks after the first dose.

166. The antibody of embodiment 165, wherein one or more of the one or more subsequent doses is administered three weeks after the first dose.

167. The antibody of embodiment 165, wherein the subsequent dose is administered every three weeks after the first dose for 4, 5, 6, 7, or 8 cycles.

168. The antibody of embodiment 165, wherein the subsequent dose is administered every three weeks after the first dose for 5 or 8 cycles.

169. The antibody of embodiment 165, wherein the treatment comprises administering a total of five, six, seven, or eight doses to the subject.

170. The antibody of any one of embodiments 157-169, wherein the subject's clinical activity score decreases after a first dose of antibody.

171. The antibody of any one of embodiments 157-170, wherein the subject has a reduced clinical activity score after two doses of antibody.

172. The antibody of embodiments 157-171, wherein the subject's clinical activity score decreases within 6 weeks of the first dose after the one or more subsequent doses.

173. The antibody of embodiments 1-171, wherein after the one or more subsequent doses, the subject's clinical activity score decreases within 3 weeks of the initial one or more subsequent doses.

174. The antibody of any one of embodiments 1-173, wherein the antibody is administered by intravenous infusion over 45 minutes to about 90 minutes, or over 60 minutes to about 90 minutes.

175. The antibody of embodiment 157, wherein the first dose is about 10mg/kg and the one or more subsequent doses are about 10mg/kg.

176. The antibody of any one of embodiments 157-19, wherein the antibody is administered as part of a pharmaceutically acceptable composition comprising the antibody and at least one pharmaceutically acceptable excipient, wherein the pharmaceutical composition comprises the antibody at a concentration of 20mg/mL to about 30 mg/mL.

177. The antibody of embodiment 176, wherein the pharmaceutical composition comprises the antibody at a concentration of about 25 mg/mL.

178. The antibody of any one of embodiments 157-177, wherein the treated subject has a reduction in the protruding eye of at least or about 1-4mm.

179. The antibody of embodiment 178, wherein the eye relief is at least or about 2-3mm.

180. The antibody of embodiment 178 or 179, wherein the protruding eye is reduced within 3 weeks of the first dose.

181. The antibody of embodiment 178 or 179, wherein the protruding eye is reduced within 6 weeks of the first dose.

182. The antibody of any one of embodiments 157-181, wherein the treated subject has reduced double vision.

183. The antibody of embodiment 182, wherein the double vision is reduced within 3 weeks or 6 weeks of the first dose.

184. The antibody of any one of embodiments 157-183, wherein the subject's Clinical Activity Score (CAS) improves within 3 weeks or 6 weeks.

185. The antibody of embodiment 184, wherein the CAS score is improved by at least-2, -3 or-4.

186. The antibody of any one of embodiments 157-185, wherein the subject's eyes are reduced and CAS score improved within 3 weeks or within 6 weeks of the first dose.

187. An anti-IGF-1R antibody for use in treating thyroid-associated eye disease in a subject in need thereof, the anti-IGF-1R antibody comprising a heavy chain comprising HCDR1 of SEQ ID No. 7, HCDR2 of SEQ ID No. 8 and HCDR3 of SEQ ID No. 9 and a light chain comprising the light chain of LCDR1 of SEQ ID No.4, LCDR2 of SEQ ID No. 5 and LCDR3 of SEQ ID No. 6, and wherein

The subject is periodically administered an anti-IGF-1R antibody intravenously at a dose of 10mg/kg for a period of time sufficient to alleviate one or more symptoms associated with thyroid-related eye disease.

188. The antibody of embodiment 187, wherein the anti-IGF-1R antibody comprises a light chain and a heavy chain, wherein the light chain comprises a variable region having the amino acid sequence of SEQ ID No. 2 and the heavy chain comprises a variable region having the amino acid sequence of SEQ ID No. 3.

189. The antibody of embodiment 187, wherein the light chain comprises the amino acid sequence of SEQ ID NO. 11.

190. The antibody of embodiment 187, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO. 10.

191. The antibody of embodiment 187, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO. 10 and the light chain comprises the amino acid sequence of SEQ ID NO. 11.

192. The antibody of any one of embodiments 187-191, wherein the anti-IGF-1R antibody is administered by intravenous infusion.

193. The antibody of any one of embodiments 187-192, wherein the anti-IGF-1R antibody is administered every 3 weeks.

194. The antibody of any one of embodiments 187-193, wherein the anti-IGF-1R antibody is administered for a period of time sufficient for 5 doses.

195. The antibody of any one of embodiments 187-193, wherein the anti-IGF-1R antibody is administered for a period of time sufficient for 8 doses.

196. The antibody of any one of embodiments 187-195, wherein the anti-IGF-1R antibody is administered for a period of time selected from 3 weeks, 6 weeks, 9 weeks, 12 weeks, 15 weeks, 18 weeks, 21 weeks, 24 weeks, or more.

197. The antibody of any one of embodiments 187-196, wherein the subject's eye process is reduced by at least or about 1-4mm.

198. The antibody of embodiment 197, wherein the eye relief is at least or about 2-3mm.

199. The antibody of embodiment 197 or 198, wherein the protruding eye is reduced within 3 weeks of the first dose.

200. The antibody of embodiment 197 or 198, wherein the protruding eye is reduced within 6 weeks of the first dose.

201. The antibody of any one of embodiments 187-200 wherein the double vision of the treated subject is reduced.

202. The antibody of embodiment 201, wherein the double vision is reduced within 3 weeks or 6 weeks of the first dose.

203. The antibody of any one of embodiments 187-202, wherein the Clinical Activity Score (CAS) of the subject improves within 3 weeks or 6 weeks.

204. The antibody of embodiment 203, wherein the CAS score is improved by at least-2, -3, or-4.

205. The antibody of any one of embodiments 187-204 wherein the subject's eyes are reduced and CAS score improved within 3 weeks or within 6 weeks of the first dose.

The present subject matter will now be described with reference to the following examples. These examples are provided for illustrative purposes only and the claims should in no way be construed as limited to these examples, but rather should be construed to encompass any and all modifications that are apparent from the teachings provided herein. Those skilled in the art will readily recognize various non-critical parameters that may be changed or modified to produce substantially similar results.

Examples

Example 1: VRDN-5000 bound better and showed increased antagonism compared to tetuzumab (teprotumumab)

Cell-based in vitro assays were performed in two different IFG1R expressing cell lines: a549 cells (fig. 1A and 1B) and human choroidal fibroblasts (HOCF) cells (fig. 1C and 1D).

In cell-based antibody binding assays, VRDN-5000 (which may also be referred to as VRDN-001) consistently showed increased binding levels (as measured by average fluorescence intensity) with increasing antibody concentration in a549 (fig. 1A; filled circles: VRDN-001; triangular: tetuzumab; open circles: igG) and HOCF (fig. 1C; filled circles = VRDN; triangular: tetuzumab; open circles: igG) cells compared to tetuzumab or IgG controls.

Similarly, VRDN-5000 consistently showed increased antagonism (as measured by average fluorescence intensity) with increasing antibody concentration in a549 (fig. 1B; filled circle: VRDN-001; triangle: tetuzumab) and HOCF (fig. 1D; filled circle = VRDN; triangle: tetuzumab) cells compared to tetuzumab.

VRDN-5000 bind to IGFR1 and exert a stronger antagonism than tetuzumab at picomolar and nanomolar antibody concentrations.

Example 2: treatment of patients with thyroid eye disease and clinical assessment of thyroid eye disease by IGF-1R antibodies.

The subject is provided with VRDN-5000 infusions as disclosed herein. The number of infusions per subject was individualized and based on the clinical judgment of the investigator. Day 1 visit occurred within 14 days after the final visit of the previous trial. The visit windows for weeks 1 and 4 were ±1 day, and the visit windows for weeks 3, 6, 9, 12, 15, 18, 21 and 24 were ±3 days. The follow-up period was only applicable to subjects who were Subjects with sudden eye non-responders in the previous trial; subjects who relapsed in the previous trial did not participate in the follow-up period. The visit window during the follow-up period was + -7 days.

The treatment period was 24 weeks (6 months) during which 8 infusions of tetuzumab were administered.

Subjects as synoptic non-responders were scheduled to participate in the 6 month follow-up period in the extension study; subjects who relapsed in the lead-in study and who were retreated in the current extension study will not participate in the follow-up period.

Efficacy assessment was performed on both eyes at each assessment time point. The "study eye" (i.e., the more affected eye) will remain the same as the eye identified at the baseline (day 1) visit of the previous study. Efficacy assessment was performed for both eyes, but the study eyes were used to assess the primary outcome measure.

Efficacy was assessed by means of the salient eye (measured using Hertel instruments as an eye herniation assessment for clinical measurement of severity), CAS (7 term scale), compound vision (measured as part of clinical measurement of severity), and clinical measurement of severity (including movement restriction assessment) for consistency of measurement.

Quality of life was assessed using the GO-QoL questionnaire.

Safety was assessed by AE and concomitant drug use monitoring, immunogenicity testing, physical and ophthalmic examinations, vital signs, clinical safety laboratory evaluations (whole blood count, chemistry including thyroid test combination (panel) and HbA 1C) and urine tests), pregnancy testing (if applicable) and Electrocardiogram (ECG). The study was also monitored by the data security Monitoring committee (DATA SAFETY Monitoring Board, DSMB).

For consistency of measurement, a salient eye assessment was performed using Hertel eye prominence meter, and (unless strictly unavoidable) the same Hertel instrument and the same observer were used in each assessment throughout the study. In addition, the same inner canthus spacing (ICD) was used each time.

The eye burst of each eye was measured on day 1 and weeks 6, 12, 18 and 24 (or exit ahead (premature withdrawal, PW)) during treatment, and on months 7, 9 and 12 (or PW) during follow-up. The measurement results of clinical measurement on the severity of eyeball herniation eCRF are recorded.

Antibodies were found to be effective in treating thyroid eye disease and improving the quality of life as provided herein.

Example 3: multiple dose escalation safety and efficacy studies (phase 1/2 study) of anti-IGF-1R in normal healthy volunteers and thyroid eye disease subjects

Normal Healthy Volunteers (NHV) and subjects with thyroid eye disease (TED subjects) were treated with VRDN-5000 to study safety, tolerability, primary efficacy, pharmacokinetic (PK) and Pharmacodynamic (PD) profiles.

NHV and subjects with TED were screened for eligibility using the inclusion and exclusion criteria for NHV and TED subjects prior to initiation of treatment. Intravenous infusion VRDN-5000 (SEQ ID NO: 71) was provided to NHV and TED subjects. Each subject received two infusions at doses of 3mg/kg, 10mg/kg or 20mg/kg, each infusion being spaced three weeks apart. Each infusion was administered over a period of 90 minutes. For a given subject, the amounts of the first dose and the second dose are the same (e.g., the subject receives a first dose of 3mg/kg and a second dose of 3mg/kg, 3 weeks apart, etc.). NHV subjects were monitored six weeks after the first dose, while TED subjects were monitored for 6 months after the first dose.

VRDN-5000 are provided in the form of 25mg/mL antibody solution in a 5.1mL fill volume in a 6mL clear single dose glass vial with a rubber septum and an aluminum seal and a plastic cap. Stored at 2-8deg.C or frozen at-20deg.C.

VRDN-5000 are administered in a dose in the range of 3mg/kg to 20 mg/kg. All subjects (NHV and subjects with TED) were monitored for safety, efficacy and other endpoints.

Safety endpoints included Adverse Events (AEs), severe Adverse Events (SAE) and laboratory evaluations, which were monitored and recorded throughout the study.

The primary efficacy endpoints included a rate of limp relief (Responder Rate) at 6 and 12 weeks (i.e., limp relief ≡2mm [ Hertel ]) relative to the baseline of the "study" [ more limp ] eyes.

Other endpoints include VRDN-001, IGF-1, and ADA blood levels at different time points before and after infusion, and changes in orbital fat volume from baseline as determined by Magnetic Resonance Imaging (MRI); extraocular muscle volume as determined by MRI; clinical Activity Score (CAS); a change in subjective review score; changes in objective assessment of eye movement measured at five basic gaze locations by prism deflection; and graves' orbital disease-a change in quality of life (GO-QoL) score.

Dosing was performed 21 days apart. Up to 48 subjects participated in the multiple dose escalation trial (12-16 NHV subjects and 16-32 subjects with TED in the multiple dose escalation study).

Single dose PK measurements were determined after the first of the two infusions, and repeat dose PK was determined after the second dose. PK and PD assays were performed in NHV subjects to minimize the number of study visits required for TED subjects. Preliminary efficacy data were collected from TED subjects 6, 12 and 24 weeks after the first of the two infusions. Safety and tolerability data were collected from NHV and TED subjects following VRDN-001 treatment. All measurements of NHV and TED subjects were performed as described herein.

The study was randomized, double-blind (sponsor excluded) and placebo-controlled. Subjects and study center staff were blinded to treatment. The pharmacist preparing the infusion bag is not blinded to the treatment and dispenses a 250mL saline bag with or without VRDN-5000 added according to the data of the interactive network response system (IWRS). The 250mL bag was infused over a 90 minute period.

Three dose levels were evaluated: 3mg/kg ("low"), 10mg/kg ("medium") and 20mg/kg ("high"). Each subject received two doses, each administered by intravenous infusion, three weeks apart. The amount of dose administered to each subject does not vary between administrations (e.g., the subject receives a first dose of 3mg/kg and a second dose of 3mg/kg, 3 weeks apart, etc.)

The low dose cohort included 4 NHV subjects randomly assigned at 3:1 (VRDN-5000 vs placebo). The medium and high dose cohorts included 4 NHV subjects and 8 TED subjects, each randomly assigned at 3:1 (VRDN-5000 vs placebo).

Two NHV subjects in the low dose cohort received treatment and were followed 1 week after their first infusion, then the remaining 2 subjects in the cohort (second) received treatment. Dose Limiting Toxicity (DLT) is a drug-related safety event whose severity requires cessation of treatment and/or prevention of VRDN-001 dose escalation. If the subject had developed DLT, another 4 NHV subjects were enrolled and only increased to the next dose when no other subjects developed DLT.

After the fourth NHV subject of the low dose cohort received one week of its second infusion, an increase to mid dose level was initiated after the data safety monitoring committee (DSMB) reviewed the safety data. Two NHV subjects were then enrolled into a medium dose cohort and were followed 1 week after their first infusion, and then enrolled into more NHV and TED subjects at that dose level.

Once the fourth NHV subject of the medium dose cohort was followed 1 week after its second infusion, and assuming that no more than 1 subject had developed DLT at that dose level, the dose was increased to the high dose level. This is done after the DSMB reviews the secure data. If the medium and high dose cohorts showed similar evidence of clinical activity in terms of rate of synaptic relief, group 8 TED subjects were dosed at low dose (3.0 mg/kg) and further group 12 subjects (4 NHV and 8 TED subjects) at medium dose (5.0 mg/kg) to establish a dose response curve for clinical activity.

The study procedure for NHV was as follows: all NHV subjects were screened within 28 days prior to treatment and complete physical examination and ECG were performed to rule out any abnormalities that prevented participation in the study. Subjects were admitted to the clinical pharmacology department 24 hours prior to each infusion and remained 7 days after the two infusions to collect PK samples. The subject's vital signs and ECG (telemetry) were continuously monitored during infusion and periodically examined for local tolerance at the skin infusion site. The subjects were returned to the department at the indicated time points for further blood sampling and evaluation, as described below. A network-based supervised audiometric test was performed 3 weeks before and after each infusion.

Blood samples for PK analysis and measurement of IGF-1 levels were collected by an indwelling venous cannula inserted into the opposite forearm of the infusion arm. PK samples were collected before the start of each infusion, 5 minutes before the end of the infusion, and 2, 4, 8, and 12 hours post-infusion, and more samples were collected 1, 3, 7, 14, and 21 days post-infusion; the final samples were collected 28 days after the second infusion. Blood samples were collected to detect IGF-1 levels before each infusion and 1, 2, 3, 7, 14 and 21 days after each infusion. Additional blood samples were collected for measurement of anti-drug antibodies (ADA) prior to each infusion VRDN-001 and 21 days after each infusion. At screening, immediately prior to each infusion and 7 days after each infusion, fasting blood and urine samples were obtained for hematology, chemistry and coagulation parameters and standard urine analysis. Full physical examination and ECG of NHV were performed at visit 7.

Study procedure for TED subjects were as follows: TED subjects were screened for qualification, medical history, and TED duration during a 28 day period prior to study entry into the group. The day prior to each infusion, subjects underwent a burst eye measurement, CAS evaluation, compound vision score evaluation, eye rotation prism measurement at 5 gaze locations, completed GO-QoL questionnaires, ophthalmoscopy, biomicroscopy, intraocular pressure (IOP), and audiometry. These evaluations will be repeated at the follow-up visit on days 43 and 85 (weeks 6 and 12). Subjects underwent a comprehensive physical examination and ECG was recorded at screening and repeated at the 6 th week follow-up visit. Orbital MRI was performed 3 days prior to two infusions and repeated for (±) 3 days on either side of the 6 th and 12 th week visits. Facial imaging was performed at screening and week 12 and 24 visits. The TED subjects were infused at the infusion clinic and the vital signs and ECG of the subjects were continuously monitored during the infusion. The skin infusion site was checked regularly for local tolerance. Study center staff telephone the TED subjects the next day after each infusion to ensure the health status of the subjects and ask if any AEs occurred since their day before discharge from the infusion clinic. The subject is informed that if there are any health problems, the study center is called on, and additional study visits are scheduled according to PI or subject requirements. All eye evaluations were performed on both eyes. These evaluations were performed immediately prior to each infusion on days 1 and 21, and again 3 weeks after the second infusion. Follow-up was performed 12 weeks and 24 weeks after the first infusion to evaluate the protruding eyes. A network-based supervised audiometric test was performed 3 weeks before and after each infusion.

Blood samples for PK and IGF-1 levels were obtained by an indwelling venous cannula inserted in the opposite forearm of the infusion arm before starting the first infusion, 5 minutes before the end of the infusion and at 2 and 4 hours after the infusion, and repeated at the same time point of the second infusion. Further samples were taken at each of the day 43 and 50 visits. Additional blood samples were again taken to measure ADA before each infusion and 3 weeks after the second infusion. At screening, 3 weeks after the first infusion (day before the second infusion) and 3 weeks after the second infusion, fasting blood and urine samples were obtained for hematology, chemistry and coagulation parameters and standard urine analysis.

The antibodies were found to be safe in NHV and safe and effective in treating thyroid eye disease, and also improve the quality of life as provided herein for subjects with TED.

Example 4: extended study of anti-IGF-1R in patients with thyroiditis

After completion of previous studies on safety, tolerability, primary efficacy, pharmacokinetic (PK) and Pharmacodynamic (PD) profile, subjects with thyroid eye disease (TED subjects) were treated with VRDN-5000 in an extended study.

Intravenous infusion VRDN-5000 is provided to the TED subject. Each subject received two infusions at doses of 3mg/kg, 10mg/kg or 20mg/kg, each infusion being spaced three weeks apart. Each infusion was administered over a period of 90 minutes. For a given subject, the first and second doses are the same (e.g., the first and second doses are 3mg/kg; the first and second doses are 10mg/kg; or the first and second doses are 20 mg/kg). Following the first dose, TED subjects were monitored for 6 months.

VRDN-5000 are administered in a dose in the range of 3mg/kg to 20 mg/kg. All subjects were monitored for safety, efficacy, and other endpoints.

The primary efficacy endpoint included a rate of eye relief at 24 weeks (i.e., eye relief of ≡2mm [ hertel ]) relative to the baseline of the eye of "study" [ more eye ].

Dosing was performed 21 days apart. Up to 48 subjects participated in the extension study. The total number of subjects depends on the results of the multi-dose escalation study in example 3. If all 48 subjects were included, the extension study included 3 randomized subjects (1:1:1; 16 per cohort/group (arm)) and two active treatment groups were compared (4 versus 8 infusions) to placebo group using a double blind (including sponsor) placebo control design.

The extended study had 80% confidence in testing each dose regimen relative to placebo, assuming a 50% difference in the relief of each active group relative to placebo. For this purpose, a side 1 type I error level of 0.025 was assigned on average (bonferoni) correction) by two comparisons, resulting in a paired type I error level of 0.0125.

The extension study was initiated after completion of the multi-dose escalation study described in example 3. The study continued to investigate the clinical activity of VRDN-5000 at the lowest dose, demonstrated clinically significant efficacy signaling in the multi-dose escalation study of example 3, and conducted dose response studies.

Specific parameters of dose and regimen in the expansion cohort were driven by data from the multi-dose escalation study in example 3, including PK data determined therein. If the test dose in the multi-dose escalation study in example 3 shows an equivalent efficacy signal, additional lower dose cohorts were explored. Otherwise, one expansion queue compares 4 infusions with 8 infusions received by the other expansion queue at the selected dose; the duration of treatment defined in all the expansion queues is fully supported by the toxicology data available at the time of the first infusion.

All three subjects in the cohort received the same number of infusions to maintain blindness. Eyes were evaluated for eyes, and subjects had the following evaluation: suspensation, CAS evaluation, review score evaluation, eye rotation prism measurement of 5 gaze locations, completion of GO-QoL questionnaire, ophthalmoscopy, biomicroscopy, and IOP. These evaluations were performed one day prior to each infusion and repeated at week 24 and 52 visits. A network-based supervised audiometric test was performed 1 day prior to each infusion and again at week 24 visit. Orbital MRI was performed 3 days prior to the first infusion and repeated for (±) 3 days of week 12, 24 and 52 visits. Facial imaging was performed at screening and at week 24 and 52 visits. The TED subjects were infused at the infusion clinic and the vital signs and ECG of the subjects were continuously monitored during the infusion. The skin infusion site was checked regularly for local tolerance.

Blood samples for PK and IGF-1 levels were obtained by an indwelling venous cannula inserted in the opposite forearm of the infusion arm before the start of the first infusion, 5 minutes before the end of the infusion and at 2 and 4 hours post-infusion, and further samples were obtained 1,3, 7, 14 and 21 days post-infusion. These sampling times are repeated immediately before and after the fourth infusion. Individual samples were taken before each infusion at weeks 3, 6, 12, 15, 18 and 21. More samples were taken at weeks 24, 25 and 52. If access to the research center is inconvenient due to other transactions, the subject may choose to have a blood sample taken by the phlebotomist/nurse at the subject's home or work site. Additional blood samples were taken to measure ADA prior to each infusion and at week 24, 25 and 52 visits.

Fasting blood and urine samples were again obtained at screening and 3 weeks after the fourth infusion (visit day prior to week 12) and eighth infusion (week 24) for hematology, chemistry and coagulation parameters and standard urine analysis. Study center staff telephone the TED subjects the next day after each infusion to ensure the health status of the subjects and ask if any AEs occurred since their day before discharge from the infusion clinic. Subjects were evaluated for any AE at all study visits and informed that if there were any health problems, please call the study center at any time during the study. Additional study visits were scheduled at the request of PI or subjects. The DSMB reviews safety and laboratory data every 6 month interval during the study.

Antibodies were found to be effective in treating thyroid eye disease and improving the quality of life as provided herein for subjects with TED.

Example 5: VRDN-5000 are insulin-like growth factor-1 receptor (IGF-1R) antagonist antibodies and are being developed for use in the treatment of Thyroid Eye Disease (TED). TED is driven by Thyroid Stimulating Hormone Receptor (TSHR) agonistic autoantibodies and interactions (cross talk) between TSHR and IGF-1R. TED is characterized by recruitment of fibroblasts expressing IGF-1R and TSHR in orbital tissues, where they mediate deposition of hyaluronic acid and expansion of orbital muscles and fat 1. IGF-1R antagonists were found to reverse this orbital tissue expansion and effectively alleviate symptom 2 in TED patients.

VRDN-5000 are humanized monoclonal antibodies targeting IGF-1R. VRDN-5000 IGF-1R binding and antagonist properties were analyzed.

Method of

Surface Plasmon Resonance (SPR) antibodies are captured by immobilized anti-Fc and recombinant IGF-1R extracellular domain (ECD) is mobilized as an analyte. The association and dissociation rate constants (ka and KD, respectively) and the equilibrium dissociation constant KD were obtained by global fitting of the data to a single-site model.

Epitope binding: VRDN-5000 are immobilized on the chip surface by amine coupling and are used to capture IGF-1R-ECD, after which tetuzumab is passed over the chip.

Cell binding: a549 human lung adenocarcinoma cells or primary human choroidal fibroblasts (HOCF) were incubated with VRDN-5000 or tetuzumab at different concentrations. A single dose of 50nm IgG1 isotype control was used as negative control. Unbound antibody was removed by washing and cells were incubated with Alexa Fluor 488-goat anti-human antibody and cell impermeable dye to gate living cells. The Median Fluorescence Intensity (MFI) of living cells was measured by flow cytometry and the data was analyzed using FlowJo software. Fitting a dose curve using a nonlinear regression model; log (agonist) versus response-variable slope (four parameters).

Internalizing: cells were incubated with different concentrations of the antibody of interest at 4℃and 37℃for 60 minutes. Cells were then washed 3 times and incubated with FITC-labeled goat anti-human Fc secondary for 30 minutes at 4 ℃. The MFI of living cells was measured by flow cytometry and the data was analyzed using FlowJo software.

Cell surface marker expression: HOCF cells were incubated with directly labeled 10ug/mL antibody or IgG isotype control. The Median Fluorescence Intensity (MFI) was measured by flow cytometry and the data was analyzed using FlowJo software.

Antagonism: serum-starved a549 or HOCF cells were pre-incubated with different concentrations of test antibody for one hour at 37 ℃ and then stimulated for 7 minutes at 37 ℃ by the addition of 100ng/mL (a 549) or 200ng/mL (HOCF) IGF-1. The biologically repeated phosphorylated IGF-1R (pIGF 1R) was measured using an R & DSYSTEMS PIGF-1R ELISA according to the manufacturer's protocol and the pIGF-1R concentration was normalized to the lowest test antibody concentration. Fitting a dose curve using a nonlinear regression model; log (inhibitor) versus reaction-variable slope (four parameters).

Results

VRDN-5000 bind IGF-1R with subnanomolar affinity. The following figure 2A shows that increasing concentrations of IGF-1R-ECD bound to VRDN-5000 or tetuzumab captured by anti-FC reveals a stepwise increase in SPR signal, enabling global fitting to the binding model. VRDN-5000 showed more durable binding interactions after IGF-1R elution. FIG. 2B shows IGF-1R-ECD stably bound to immobilized VRDN-5000. Tetuzumab showed no binding to IGF-1r: vrdn-5000 complex, indicating that tetuzumab and VRDN-5000 have overlapping epitopes.

VRDN-5000 bind with high affinity to IGF-1R on a549 cells. As shown in fig. 3A-C, the binding of VRDN-5000 to a549 cells was assessed by flow cytometry and found to have a similar binding profile to tetuzumab at three different concentrations. As shown in fig. 3D, the binding dose response curve demonstrates VRDN-5000EC50 = 0.1nM. As shown in FIG. 3E, VRDN-5000, VRDN-2700 (VRDN-5000 with M252Y, S T and T256E mutations in the Fc domain) and tetuzumab showed comparable binding at temperatures that block IGF-1R receptor internalization. FIG. 3F shows VRDN-5000 that VRDN-2700 and tetuzumab with M252Y, S254T and T256E mutations in the Fc domain resulted in a comparable level of internalization (-50%) as measured by a decrease in membrane IGF-1R receptor levels at 37℃versus 4 ℃.

HOCF was used as an in vitro model of TED pathology.

Cd34+, thy-1+ orbital fibroblasts are involved in extracellular matrix deposition in TED5 and in the development of { GOODW-24002-CNPT/01912999v1} 72-pathologic fibrosis. Here, HOFC is shown to express (A) IGF-1R and (B) TSHR, and (C) CD34 and Thy-1, indicating that they can be used as in vitro model systems for IGF-1R function in TED. The data are shown in FIGS. 4A-C.

VRDN-5000 bind IGF-1R on HOCF cells with high affinity.

Figures 5A-B show VRDN-5000 binding to HOCF cells, which were evaluated by flow cytometry and found to have a largely similar binding to tetuzumab at three different concentrations. Panel D (Panel D) shows a binding agent amount response curve E, indicating an EC 50=0.4 nM for VRDN-5000.

VRDN-5000 are subnanomolar IGF-1R antagonists. VRDN-5000 were effective in inhibiting IGF-1 stimulated receptor phosphorylation on a549 cells (ic50=0.09 nM) and HOCF cells (ic50=0.09 nM), as shown in fig. 6A-B.

Example 6. VRDN-5000 are more potent inhibitors of IGF-1 binding to IGF1R than tetuzumab.

IGF-1 binding to IGF-1R present on the cell surface is assayed. Briefly, labeled IGF1 was incubated with cells in the presence VRDN-5000, tetuzumab or no antibody (negative control). Cells were washed and then assayed for binding to IGF-1 by detecting the presence of IGF1 marker, as shown in fig. 7, VRDN-5000 were found to be more potent inhibitors. The maximum inhibition of VRDN-5000 was found to be 94% and the maximum inhibition of tetuzumab was found to be 48%.

Inhibition was also assessed in IGF-1 induced IGF1-R phosphorylation. The cell cultures were pre-incubated with antibodies (VRDN-5000 or tetuzumab) and IGF1 stimulation. Cells were lysed and pIGF1R was measured. The maximum inhibition of autophosphorylation was found to be 96% for VRDN-5000, whereas the maximum inhibition of autophosphorylation was found to be 76% for tetuzumab. These results are shown in FIG. 8.

Also found in measuring Akt phosphorylation was a more potent inhibition of IGF1 activity by VRDN-5000. Briefly, cell cultures were pre-incubated with antibodies (VRDN-5000 or tetuzumab) and stimulated with IGF 1. Cells were lysed and pAKT was measured using a standard assay. The maximum inhibition of Akt phosphorylation was found to be 93% for VRDN-5000, whereas the maximum inhibition of Akt phosphorylation by tetuzumab was found to be 66%.

These results indicate that VRDN-5000 on IGF-1R and tetuzumab epitopes overlap, VRDN-5000 bind to IGF-1R on cells with subnanomolar EC50, VRDN-5000 promotes IGF-1R internalization, and VRDN-5000 inhibits IGF-1R phosphorylation with subnanomolar IC 50. Thus VRDN-5000 bind, antagonize, and internalize IGF-1R at sub-nanomolar concentrations, suggesting that VRDN-5000 should be able to be used to potentially, potently inhibit the pathophysiology of driving TED.

Example 7 vrdn-5000 treatment of thyroid-related eye disease in subjects, a sudden eye relief occurred within 3 weeks after the first dose. Two infusions VRDN-5000 at 10mg/kg three weeks apart, at week 6, resulted in rapid and significant improvement in the presbyopia, CAS and compound vision. The results indicate that at week 6, patients receiving VRDN-5000 treatments developed a burst eye relief: 5/6 patients (83%); median time to burst relief: 3 weeks; CAS reaction: 6/6 (100%) CAS score 0 or 1:4/6 (67%); overall relief: 5/6 patients (83%); and a compound vision resolution: 3/4 (75%). The mean eye relief and improvement in double vision observed immediately 6 weeks after the first infusion VRDN-5000 was significantly faster than the published results (Smith et al.,Teprotumumab for Thyroid-Associated Ophthalmopathy,N Engl JMed 2017;376:1748-61;Douglas et al.,Teprotumumab for the Treatment of Active Thyroid Eye Disease,N.Engl J Med 2020;382:341-52; and Douglas et al.,Teprotumumab Efficacy,Safety,and Durability in Longer-Duration Thyroid Eye Disease and Re-treatment,Ophthalmology 2022,vol 129,no.4). for tetuzumab comparison data shown in fig. 9-14B.

As the data show, subjects on average showed a reduction or improvement in at least two of the presbyopia, and CAS scores, which were not observed in the placebo group. These data demonstrate the unexpected result of how fast VRDN-5000 can achieve therapeutic effects, which is unpredictable.

All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., genbank sequence or GeneID entry), patent application or patent was specifically and individually indicated to be incorporated by reference. The statement that is incorporated by reference is formulated by the applicant in accordance with 37c.f.r. ≡1.57 (b) (1) and relates to each and every individual publication, database entry (e.g. Genbank sequence or GeneID entry), patent application or patent, each of which is specifically identified in accordance with 37c.f.r. ≡1.57 (b) (2) even though such reference is not immediately adjacent to the statement that is incorporated by reference as specified. The inclusion of a specified statement that is incorporated by reference (if any) in the specification does not in any way weaken the general statement that is incorporated by reference. Citation of a reference herein is not intended as an admission that such reference is prior art with respect to it, nor does it constitute any admission as to the contents or date of such publication or file.

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

The description is to be construed as sufficient to enable those skilled in the art to practice the embodiments. Various modifications other than those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the disclosure and any appended claims.

Claims

administering a first dose of the antibody to the subject intravenously or subcutaneously, wherein the first dose is selected from the group consisting of about 1 mg/kg to about 2 mg/kg, about 2 mg/kg to about 5 mg/kg, about 3 mg/kg to about 5 mg/kg, about 5 mg/kg to about 7.5 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 10 mg/kg to about 15 mg/kg, or about 15 mg/kg to about 20 mg/kg; and

administering one or more subsequent doses of the antibody to the subject intravenously or subcutaneously, wherein each subsequent dose is selected from the group consisting of about 1 mg/kg to about 2 mg/kg, about 2 mg/kg to about 5 mg/kg, about 3 mg/kg to about 5 mg/kg, about 5 mg/kg to about 7.5 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 10 mg/kg to about 15 mg/kg, or about 15 mg/kg to about 20 mg/kg,

Wherein, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises HCDR1 of SEQ ID NO:7, HCDR2 of SEQ ID NO:8 and HCDR3 of SEQ ID NO:9, and the light chain comprises LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5 and LCDR3 of SEQ ID NO:6.

2. The method of claim 1, wherein the light chain comprises a variable region having an amino acid sequence of SEQ ID NO: 2, and the heavy chain comprises a variable region sequence having an amino acid sequence of SEQ ID NO: 3.

3. The method of claim 1 or 2, wherein the light chain comprises the amino acid sequence of SEQ ID NO:11.

4. The method of any one of claims 1-3, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 10.

5. The method of claim 1, wherein the first dose is about 10 mg/kg.

6. The method of claim 5, wherein the one or more subsequent doses is about 10 mg/kg.

7. The method of any one of claims 1-5, wherein the amount of the one or more subsequent doses is the same as the amount of the first dose.

8. The method of any one of claims 1-5, wherein the amount of the one or more subsequent doses is different from the amount of the first dose.

9. The method of any one of claims 1-8, wherein at least one of the one or more subsequent doses is administered one, two, three, four, five, six, or eight weeks after the first dose.

10. The method of claim 6, wherein the one or more subsequent doses are administered three weeks after the first dose.

11. The method of claim 6, wherein the subsequent doses are administered every three weeks after the first dose for 4, 5, 6, 7, or 8 cycles.

12. The method of claim 6, wherein the subsequent doses are administered every three weeks after the first dose for 5 or 8 cycles.

13. The method of claim 6, wherein the method comprises administering a total of five, six, seven, or eight doses to the subject.

14. The method of any one of claims 1-13, wherein after a first dose of the antibody, the subject's clinical activity score decreases.

15. The method of any one of claims 1-14, wherein the subject's clinical activity score decreases after two doses of the antibody.

16. The method of claims 1-15, wherein after the one or more subsequent doses, the subject's clinical activity score decreases within 6 weeks of the first dose.

17. The method of claims 1-15, wherein after the one or more subsequent doses, the subject's clinical activity score decreases within 3 weeks of the initial one or more subsequent doses.

18. The method of any one of claims 1-17, wherein the antibody is administered by intravenous infusion over 45 minutes to about 90 minutes, or over 60 minutes to about 90 minutes.

19. The method of claim 1, wherein the first dose is about 10 mg/kg and the one or more subsequent doses are about 10 mg/kg.

20. The method of any one of claims 1-19, wherein the antibody is administered as part of a pharmaceutically acceptable composition comprising the antibody and at least one pharmaceutically acceptable excipient, wherein the pharmaceutical composition comprises the antibody at a concentration of 20 mg/mL to about 30 mg/mL.

21. The method of claim 19, wherein the pharmaceutical composition comprises the antibody at a concentration of about 25 mg/mL.

22. The method of any one of claims 1-21, wherein the treated subject has a reduction in proptosis by at least or about 1-4 mm.

23. The method of claim 22, wherein the proptosis is reduced by at least or about 2-3 mm.

24. The method of claim 22 or 23, wherein the proptosis is reduced within 3 weeks of the first dose.

25. The method of claim 22 or 23, wherein the proptosis is reduced within 6 weeks of the first dose.

26. The method of any one of claims 1-25, wherein the treated subject experiences reduced diplopia.

27. The method of claim 26, wherein the diplopia is reduced within 3 weeks or 6 weeks of the first dose.

28. The method of any one of claims 1-27, wherein the subject's Clinical Activity Score (CAS) improves within 3 weeks or 6 weeks.

29. The method of claim 28, wherein the CAS score improves by at least -2, -3, or -4.

30. The method of any one of claims 1-29, wherein the subject's proptosis is reduced and CAS score improves within 3 weeks or within 6 weeks of the first dose.

31. A method of treating thyroid-related eye disease in a subject in need thereof, comprising:

administering to the subject an anti-IGF-1R antibody intravenously at a dose of 10 mg/kg periodically for a period of time sufficient to reduce one or more symptoms associated with thyroid-related eye disease,

Wherein, the anti-IGF-1R antibody comprises a heavy chain and a light chain, the heavy chain comprises HCDR1 of SEQ ID NO:7, HCDR2 of SEQ ID NO:8 and HCDR3 of SEQ ID NO:9, and the light chain comprises LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5 and LCDR3 of SEQ ID NO:6.

32. The method of claim 31, wherein the anti-IGF-1R antibody comprises a light chain and a heavy chain, wherein the light chain comprises a variable region having an amino acid sequence of SEQ ID NO:2, and the heavy chain comprises a variable region having an amino acid sequence of SEQ ID NO:3.

33. The method of claim 31, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 11.

34. The method of claim 31, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 10.

35. The method of claim 31, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 10, and the light chain comprises the amino acid sequence of SEQ ID NO: 11.

36. The method of any one of claims 31-35, wherein the anti-IGF-1R antibody is administered by intravenous infusion.

37. The method of any one of claims 31-36, wherein the anti-IGF-1R antibody is administered every 3 weeks.

38. The method of any one of claims 31-37, wherein the anti-IGF-1R antibody is administered for a period of time sufficient for 5 doses.

39. The method of any one of claims 31-37, wherein the anti-IGF-1R antibody is administered for a period of time sufficient for 8 doses.

40. The method of any one of claims 31-37, wherein the anti-IGF-1R antibody is administered for a period of time selected from 3 weeks, 6 weeks, 9 weeks, 12 weeks, 15 weeks, 18 weeks, 21 weeks, 24 weeks, or longer.

41. The method of any one of claims 31-40, wherein the subject's proptosis is reduced by at least or about 1-4 mm.

42. The method of claim 41, wherein the proptosis is reduced by at least or about 2-3 mm.

43. The method of claim 41 or 42, wherein the proptosis is reduced within 3 weeks of the first dose.

44. The method of claim 41 or 42, wherein the proptosis is reduced within 6 weeks of the first dose.

45. The method of any one of claims 31-53, wherein the treated subject experiences reduced diplopia.

46. The method of claim 45, wherein the diplopia is reduced within 3 weeks or 6 weeks of the first dose.

47. The method of any one of claims 31-46, wherein the subject's Clinical Activity Score (CAS) improves within 3 weeks or 6 weeks.

48. The method of claim 47, wherein the CAS score improves by at least -2, -3, or -4.

49. The method of any one of claims 31-48, wherein the subject's proptosis is reduced and CAS score improves within 3 weeks or within 6 weeks of the first dose.