HK40099096B - Compositions and methods for treatment of thyroid eye disease - Google Patents

Description

Compositions and methods for treating thyroid eye diseases

Cross-referencing of related applications:

This application claims priority to U.S. Provisional Application No. 63/091,839, filed October 14, 2020; U.S. Provisional Application No. 63/201,978, filed May 21, 2021; U.S. Provisional Application No. 63/260,130, filed August 10, 2021; and U.S. Provisional Application No. 63/261,742, filed September 28, 2021, each of which is incorporated herein by reference in its entirety.

Background Technology

Thyroid-associated eye disease (TAO) (also known as thyroid eye disease (TED), Graves' eye disease, or orbital disease (GO), thyrotoxic ophthalmopathy, thyroid dysfunction eye disease, and several other terms) is an orbital disease associated with thyroid dysfunction. TAO is classified into two types. Active TAO, which typically lasts 1 to 3 years, is characterized by a persistent autoimmune/inflammatory response in the orbital soft tissues. Active TAO causes expansion and remodeling of the ocular soft tissues. The autoimmune/inflammatory response of active TAO spontaneously resolves, and the condition transitions to inactive TAO. Inactive TAO is the term used to describe long-term/permanent sequelae of active TAO. The etiology of TAO is unknown. TAO is commonly associated with Graves' hyperthyroidism, but can also occur as part of other autoimmune conditions affecting the thyroid gland and producing pathologies in the orbital and periorbital tissues, and rarely in the pretibial skin (pretibial myxedema) or fingers (thyroid clubbing). Transocular optic apnea (TAO) is an autoimmune orbital disease that primarily affects the orbit and periorbital soft tissues, and secondarily the eyeball and vision. In TAO, inflammation and expansion of the orbital soft tissues (mainly the extraocular muscles and fat) force the eyeball forward (bulging) away from its socket—a phenomenon known as proptosis or exophthalmos. Although most cases of TAO do not cause vision loss, the condition can lead to vision-threatening exposure keratopathy, troublesome diplopia/double vision, and optic neuropathy caused by compressive thyroid dysfunction. TAO can precede, occur concurrently with, or follow systemic complications of thyroid dysfunction. The ocular clinical manifestations of TAO include upper eyelid retraction, eyelid lag, swelling, redness (erythema), conjunctivitis and bulging eye (proptosis or exophthalmos), bulbar conjunctival edema, periorbital edema, and altered eye movements, resulting in significant functional, social, and cosmetic consequences. Many signs and symptoms of TAO (including bulging eyes and conjunctival hyperemia) are caused by the expansion of orbital adipose tissue and periocular muscles. The increase in adipose tissue volume is partly due to the development of new adipocytes within the orbital fat (adipogenesis). The accumulation of hydrophilic glycosaminoglycans (primarily hyaluronic acid) in the perimuscular connective tissue between the orbital adipose tissue and extraocular muscle fibers further expands the adipose compartments and enlarges the extraocular muscles. Hyaluronic acid is produced by fibroblasts residing in the orbital fat and extraocular muscles, and its in vitro synthesis is stimulated by several cytokines and growth factors, including IL-1β, interferon-γ, platelet-derived growth factor, thyroid-stimulating hormone (TSH), and insulin-like growth factor I (IGF-I).

Antibodies activating the insulin-like growth factor I receptor (IGF-IR) and involving active TAO were also detected. Without being bound by any theory, it is believed that TSHR and IGF-IR form a physiological and functional complex in orbital fibroblasts, and blocking IGF-IR appears to attenuate IGF-1 and TSH-dependent signaling. It has been shown that blocking IGF-IR with antibody antagonists reduces TSHR and IGF-I dependent signaling, thereby interrupting the pathological activity of autoantibodies acting as agonists of either receptor.

IGF-IR is a widely expressed heterotetrameric protein involved in the regulation of proliferation and metabolic functions in many cell types. It is a tyrosine kinase receptor containing two subunits. IGF-IRα contains a ligand-binding domain, while IGF-IRβ is involved in signal transduction and contains a tyrosine phosphorylation site.

Current treatments for hyperthyroidism caused by Graves' disease are incomplete due to the lack of therapies targeting the specific underlying autoimmune mechanisms of the disease. Even more complex is the treatment of moderate to severe active TAO. Despite a deeper understanding of its pathogenesis in recent years, TAO remains a treatment challenge and problem. There are no approved drugs for the treatment of active TAO. Intravenous glucocorticoids (ivGC) and oral glucocorticoids have been used to treat patients with moderate to severe active TAO, but the results are rarely satisfactory. Some patients experience frequent responses, and relapse (rebound) is common after discontinuation of treatment. Adverse events occur, and many patients eventually require rehabilitative surgery when their symptoms have progressed to inactive TAO. Therefore, alternative therapies for TAO and its associated symptoms remain needed.

Summary of the Invention

The implementation scheme generally relates to IGF-1R antibodies and their antigen-binding fragments. Certain IGF-1R antibodies and antigen-binding fragments inhibit IGF-1R function or block the biological function of IGF-I-mediated IGF-1R signaling. Additionally, the present invention generally relates to a method for treating thyroid-associated eye disease (TAO), also known as thyroid eye disease (TED), Graves' eye disease or orbital disease (GO), thyrotoxic ophthalmopathy, thyroid dysfunctional eye disease, and other thyroid eye diseases associated with IGF-1R signaling.

In some embodiments, an antibody or antigen-binding fragment thereof comprising the sequence provided herein is provided. In some embodiments, the antibody comprises a VL sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 79 or 86; and a VH sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 80 or 83. In some embodiments, the antibody comprises an LCDR sequence as set forth in SEQ ID NO:17, 18, 19, 23, 24, 25, 29, 30, 31, 35, 36, 37, 41, 42, 43, 47, 48, 49, 53, 54, 55, 59, 60, 61 or 81, and an HCDR sequence as set forth in SEQ ID NO:20, 21, 22, 26, 27, 28, 32, 33, 34, 38, 39, 40, 44, 45, 46, 50, 51, 52, 56, 57, 58, 62, 63 or 64; or any combination or variant thereof.

In some embodiments, the antibody or its antigen-binding fragment comprises a V _L peptide, or any variant thereof, as set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 79, or 86. In some embodiments, the antibody or its antigen-binding fragment comprises a V _H peptide, or any variant thereof, as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 80, or 83.

In some embodiments, the antibody or antibody fragment comprises: (i) 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: 20, 26, 32, 38, 44, 50, or 56; the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 21, 27, 33, 39, 45, 51, or 57; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 22, 28, 34, 40, 46, 52, or 58; or any of the foregoing. (i) a variant of the former; and (ii) a light chain variable region comprising light chain CDR1, CDR2 and CDR3 sequences, wherein the light chain CDR1 sequence has an amino acid sequence of SEQ ID NO: 17, 23, 29, 35, 41, 47 or 53; the light chain CDR2 sequence has an amino acid sequence of SEQ ID NO: 18, 24, 30, 36, 42, 48 or 54; and the light chain CDR3 sequence has an amino acid sequence of SEQ ID NO: 19, 25, 31, 37, 43, 49, 55 or 81; or a variant of any of the foregoing.

In some embodiments, the antibody or antibody fragment comprises: (i) 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:20; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:21; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:22; or a variant thereof; and (ii) 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:17; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:18; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:19; or a variant thereof.

In some embodiments, the antibody or antibody fragment comprises: (i) 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:26; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:27; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:28; or a variant thereof; and (ii) 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:23; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:24; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:25; or a variant thereof.

In some embodiments, the antibody or antibody fragment comprises: (i) 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:32; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:33; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:34; or a variant of any of the foregoing; and (ii) 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:29; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:30; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:31; or a variant of any of the foregoing.

In some embodiments, the antibody or antibody fragment comprises: (i) 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:38; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:39; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:40; or a variant thereof; and (ii) 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:35; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:36; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:37; or a variant thereof.

In some embodiments, the antibody or antibody fragment comprises: (i) 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:44; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:45; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:46; or a variant thereof; and (ii) 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:41; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:42; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:43; or a variant thereof.

In some embodiments, the antibody or antibody fragment comprises: (i) 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:50; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:51; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:52; or a variant thereof; and (ii) 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:47; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:48; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:49; or a variant thereof.

In some embodiments, the antibody or antibody fragment comprises: (i) 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:56; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:57; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:58; or a variant of any of the foregoing; and (ii) 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:53; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:54; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:55; or a variant of any of the foregoing.

In some embodiments, the antibody or antibody fragment comprises: (i) 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:62; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:63; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:64; or a variant of any of the foregoing; and (ii) 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:59; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:60; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:61; or a variant of any of the foregoing.

In some embodiments, the antibody or antibody fragment comprises: (i) 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:38; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:39; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:40; or a variant of any of the foregoing; and (ii) 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:35; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:36; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:81; or a variant of any of the foregoing.

In some embodiments, the antibody comprises a _VL sequence, or a variant thereof, as set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 79, or 86. In some embodiments, the antibody comprises a _VH sequence, or a variant thereof, as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 80, or 83.

In some implementations, the antibody comprises the sequence of SEQ ID NO:65 to 72, 78, 82 or 85, or a variant thereof.

In some embodiments, the antibody comprises a light chain having the amino acid sequence of SEQ ID NO:3 and a heavy chain having the amino acid sequence of SEQ ID NO:83. In some embodiments, the antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO:13 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:14.

In some embodiments, the antibody comprises a light chain having the amino acid sequence of SEQ ID NO:93 and a heavy chain having the amino acid sequence of SEQ ID NO:92.

In some embodiments, the antibody comprises a light chain having the amino acid sequence of SEQ ID NO:93 and a heavy chain having the amino acid sequence of SEQ ID NO:94.

In some embodiments, the antibody comprises a light chain having the amino acid sequence of SEQ ID NO:93 and a heavy chain having the amino acid sequence of SEQ ID NO:95.

In some implementations, any variant of the antibody provided herein may be provided, as long as the CDR remains constant compared to the parental (non-variant) sequence provided herein.

In some embodiments, the antibody comprises an Fc region. In some embodiments, the Fc region is as described in SEQ ID NO:75 to 77, 84, 87, 88, 89, or 90. In some embodiments, the Fc region is as described in SEQ ID NO:75. In some embodiments, the Fc region is as described in SEQ ID NO:76. In some embodiments, the Fc region is as described in SEQ ID NO:77. In some embodiments, the Fc region is as described in SEQ ID NO:84. In some embodiments, the Fc region is as described in SEQ ID NO:87. In some embodiments, the Fc region is as described in SEQ ID NO:88. In some embodiments, the Fc region is as described in SEQ ID NO:89. In some embodiments, the Fc region is as described in SEQ ID NO:90.

In some embodiments, a pharmaceutical composition comprising an antibody as provided herein is provided.

In some embodiments, a method is provided for treating thyroid-associated eye disease (TAO) or its symptoms or for reducing its severity, said method comprising administering to a subject an antibody or a pharmaceutical composition containing such an antibody as provided herein.

In some embodiments, a method of treating a subject with thyroid ophthalmopathy is provided, the method comprising administering to the subject an antibody or a pharmaceutical composition containing such an antibody as provided herein.

In some implementations, a method is provided for reducing the clinical activity score (CAS) of thyroid-associated eye disease (TAO) in a subject, the method comprising administering to the subject an antibody or a pharmaceutical composition containing the antibody as provided herein.

In some embodiments, methods are provided to reduce a) bulging eye by at least 2 mm and b) clinical activity score (CAS) in subjects with thyroid-associated eye disease (TAO), said methods comprising administering to the subject an antibody or a pharmaceutical composition comprising thereto as provided herein.

In some embodiments, a method is provided for treating a subject with thyroid-associated eye disease (TAO) or reducing its severity, the method comprising administering to the subject an antibody as provided herein or a pharmaceutical composition comprising thereto, wherein treatment with the antibody (i) reduces bulging of one eye by at least 2 mm; (ii) without deterioration of the other eye (or contralateral eye) by 2 mm or more; and (iii) reduces the subject's CAS to one (1) or zero (0).

In some implementations, methods are provided to improve the quality of life of subjects suffering from thyroid-associated eye disease (TAO, also known as Graves' eye disease/Graves' orbital disease), the methods comprising administering to the subject an antibody or a pharmaceutical composition containing such an antibody as provided herein.

In some implementations, methods are provided for treating diplopia or reducing the severity of thyroid-associated eye disease (TAO) in subjects, the methods comprising administering to the subject an antibody or a pharmaceutical composition containing such an antibody as provided herein.

In some embodiments, a method for increasing IGF-1R internalization on cells is provided, the method comprising contacting the cells with an antibody or a pharmaceutical composition containing such an antibody as provided herein.

In some embodiments, a method is provided to inhibit IGF-1-stimulated receptor phosphorylation on cells, the method comprising contacting the cells with an antibody or a pharmaceutical composition comprising thereunder, as provided herein.

In some embodiments, a method of treating a subject with thyroid ophthalmopathy is provided, the method comprising administering to the subject an antibody as provided herein or a pharmaceutical composition comprising thereto, wherein, at least 1, 2, or 3 weeks after administration, the serum concentration of the antibody in the subject is at least or about 70 μg/ml, 75 μg/ml, 80 μg/ml, 85 μg/ml, 90 μg/ml, 95 μg/ml, 100 μg/ml, or 105 μg/ml.

In some embodiments, a method is provided to inhibit IGF-1-induced receptor autophosphorylation in cells by at least 95%, 96%, 97%, 98%, 99%, or 100%, said method comprising contacting said cells with an antibody or a pharmaceutical composition comprising thereunder as provided herein.

In some embodiments, embodiments for any of the methods provided herein are provided, wherein the antibody or its antigen-binding fragment is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable diluent or excipient or carrier. 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-CD20 antibody; tocilizumab or other anti-IL-6 antibody; or selenium, infliximab or other anti-TNFα antibody or thyroid-stimulating hormone receptor (TSHR) inhibitor.

Attached Figure Description

Figure 1 shows the NHP (non-human primate) serum concentrations for the various antibodies and implementation schemes provided herein.

Figure 2 illustrates the various properties of the antibodies as presented herein.

Figure 3 illustrates the various properties of the antibodies as presented in this article.

Figure 4 illustrates the various properties of the antibodies as presented in this article.

Figure 5 illustrates the various properties of the antibodies as presented in this article.

Figure 6 illustrates the various properties of the antibodies as presented in this article.

Figure 7 illustrates the various properties of the antibodies as presented in this article.

Figure 8 illustrates the various properties of the antibodies as presented in this article.

Detailed Implementation

This article presents antibodies that bind to and modulate the activity of IGF-1R. These antibodies can be used, for example, to treat thyroid eye diseases.

As used in this article, “thyroid-associated ophthalmopathy” (TAO), “thyroid eye disease” (TED), “Graves’ eye disease”, or “Graves’ orbital disease” (GO) refer to the same condition or symptom and are used interchangeably. They all refer to inflammatory orbital pathologies associated with some autoimmune thyroid disorders, most commonly Graves’ disease (GD), but sometimes with other conditions (e.g., Hashimoto’s thyroiditis).

The terms “proptosis” and “exophthalmos” (also known as exophthalmos/exophthalmia/exorbitism) refer to the forward protrusion, displacement, bulging, or projection of an organ. As used herein, the term refers to the forward protrusion, displacement, bulging, or projection of the eye away from the orbit. Some skilled in the art consider proptosis and exophthalmos to have the same meaning and are often used interchangeably, while others consider there to be subtle differences in their meaning. Some use exophthalmos to refer to severe proptosis; or to endocrine-related proptosis. Others use the term exophthalmos when describing proptosis associated with, for example, the eyes of subjects with TAO (TED or GO).

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

The degree of proptosis can be measured using, for example, an exophthalmometer (an instrument used to measure the degree of forward displacement of the eye). This device allows measurement of the forward distance from the lateral margin of the orbit to the anterior cornea. Computed tomography (CT) scans and magnetic resonance imaging (MRI) can also be used to assess the degree of proptosis or bulging. CT scans are the best imaging modality for diagnosing TAO. In addition to allowing observation of enlarged extraocular muscles, CT scans provide surgeons or clinicians with a drawing of the orbital bony anatomy when orbital decompression is required. MRI provides excellent imaging of the orbital contents through its multiplanar and inherent contrast capabilities without the radiation exposure associated with CT scan studies. MRI provides better imaging of the optic nerve, orbital fat, and extraocular muscles, but CT scans provide a better view of the orbital bony architecture. Orbital ultrasound can also be used to diagnose and evaluate TAO because it can be performed quickly and with high reliability. It facilitates the assessment of the hyperreflectivity and enlargement of extraocular muscles, and sequential ultrasound examinations can also be used to assess the progression or stability of the eye disease. Based on currently available or future available technologies, those skilled in the art will be able to determine the optimal model for diagnosing and assessing the degree of bulging or exophthalmos.

As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Therefore, it is used in the broadest sense and specifically encompasses (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 camelified single-domain antibodies. "Parental antibody" is an antibody obtained by exposing the immune system to an antigen prior to modification of the antibody for an intended use (e.g., humanization of an antibody for use as a human therapeutic antibody).

As used herein, unless otherwise stated, "antibody fragment" or "antigen-binding fragment" means an antigen-binding fragment of an antibody, that is, an antibody fragment that retains the ability to specifically bind to an antigen bound by a full-length antibody, such as a fragment retaining one or more CDR regions. Examples of antibody-binding fragments include (but are not limited to) Fab, Fab', F(ab') ₂ , and Fv fragments; bispecific antibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.

The "Fab fragment" consists of a light chain and a heavy chain, along with a variable region ( _CH1 ). The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The “Fc” region contains two heavy chain segments with _CH1 and _CH2 domains containing the antibody. The two heavy chain segments are held together by two or more disulfide bonds and hydrophobic interactions with the _CH3 domain.

In some embodiments, the antibody or antigen fragment herein comprises an Fc region. In some embodiments, the Fc region contains a mutation that prolongs the half-life of the antibody when linked to the Fc region. In some embodiments, the Fc region comprises the S228P, L235E, M252Y, S254T, T256E, M428L, N434S, L234F, P331S mutations or any combination thereof. In some embodiments, the Fc region comprises the M252Y, S254T, and T256E mutations. Non-limiting examples of Fc regions containing the M252Y, S254T, and T256E mutations (collectively, “YTE mutations”) are found in the sequence of SEQ ID NO:89. In some embodiments, the Fc region containing the YTE mutation comprises the sequence of SEQ ID NO:90, which differs from SEQ ID NO:89 due to the presence of a C-terminal lysine (K) residue. The Fc region may be numbered according to the Kabat numbering system used for Fc regions.

In some embodiments, the Fc region contains S228P and L235E mutations. In some embodiments, the antibody contains L234F, L235E, and P331S mutations. In some embodiments, the Fc region contains M252Y, S254T, T256E, S228P, and L235E mutations. In some embodiments, the Fc region contains S228P, L235E, M428L, and N434S mutations. In some embodiments, the Fc region contains M428L and N434S mutations. In some embodiments, the Fc region contains L234F, L235E, P331S, M252Y, S254T, and T256E mutations. Mutations in the Fc region are also described in US2007041972A1, EP2235059B1, US Patent No. 8,394,925, and Mueller et al., Mol Immunol, April 1997; 34(6):441-52, each of which is incorporated herein by reference in its entirety. The numbers cited herein refer to the Kabat numbering system used for the Fc region.

In some implementations, the Fc region contains sequences selected from the following:

A “Fab’ fragment” is a portion or segment of a region containing a _VH domain and a _CH1 domain, as well as the region between the _CH1 and _CH2 domains, containing one light chain and one heavy chain, such that interchain disulfide bonds can be formed between the two heavy chains of two Fab’ fragments to form an F(ab’) ₂ molecule.

The “F(ab') ₂ segment” contains two light chains and two heavy chains containing a portion of the constant region between the _CH1 and _CH2 structural domains, such that interchain disulfide bonds are formed between ^the two heavy chains. Therefore, the F(ab') ₂ segment consists of two Fab' segments held together by the disulfide bonds between the two heavy chains.

The “Fv region” contains variable regions from both heavy and light chains, but lacks constant regions.

The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment containing both _VH and _VL domains, where these domains are contained within a single polypeptide chain. Generally, Fv polypeptides further include a polypeptide linker between the _VH and _VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun (1994), PHARMACOLOGY OF MONOCLONAL ANTIBODIES, Vol. 113, eds. Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315. See also International Patent Application Publication No. WO 88/01649 and U.S. Patents Nos. 4,946,778 and 5,260,203.

"Domain antibodies" are immunoglobulin fragments containing only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more _VH regions covalently bind to peptide linkers, producing bivalent domain antibodies. The two _VH regions of a bivalent domain antibody can target the same or different antigens.

A bivalent antibody contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific (see below).

In some embodiments, the monoclonal antibodies described herein also include camel-modified single-domain antibodies. See, for example, 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. Patent No. 6,005,079. In one embodiment, the present invention provides a single-domain antibody comprising two _VH domains, said two _VH domains being modified to form a single-domain antibody.

As used herein, the term "bispecific antibody" refers to a small antibody fragment having two antigen-binding sites, wherein the fragment contains a heavy chain variable domain ( _VH ) attached to a light chain variable domain (VL) within the same polypeptide chain ( _VH - _VL or _{VL - VH} ). Two antigen-binding sites are created by using a linker short enough that the two domains on the same chain cannot pair, forcing the domain to pair with a complementary domain of the other chain. Bispecific antibodies are described more fully, for example, in EP404,097; 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, when activity is expressed in molar quantities, the variant antibodies or antigen-binding fragments of the antibodies provided herein retain at least 10% of their IGF-1R binding activity (compared to the modified parent antibody). In some embodiments, the variant antibodies (or antigen fragments thereof) or antigen-binding fragments of the antibodies provided herein retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the IGF-1R binding affinity compared to the parent antibody. As described herein, it is also desirable that the antibodies or antigen-binding fragments of the present invention may include conserved or non-conserved amino acid substitutions that substantially do not alter their biological activity, which may also be referred to as “conserved variants” or “functionally conserved variants” of the antibody.

"Isolated antibody" refers to the purified state of a binding compound, meaning in this context 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" is not intended to mean the complete absence of said material or the absence of water, buffers, or salts, unless their amounts substantially interfere with the experimental or therapeutic use of the binding compound as described herein.

As used herein, the term "monoclonal antibody" refers to a substantially homogeneous population of antibodies, meaning that the amino acid sequences of the antibody molecules constituting the population are identical, except for the possibility of naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody formulations typically comprise numerous different antibodies with different amino acid sequences in their variable domains, particularly their CDRs, which are typically specific to different epitopes. The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used according to the invention can be produced by the hybridoma method first described in Kohler et al. (1975) Nature 256:495 or by a recombinant DNA method (see, for example, U.S. Patent 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 352:624-628 and 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 derived from different species (US Patent No. 4,816,567 and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855). Typically, the variable domain is derived from an antibody from a laboratory animal such as a rodent (“parental antibody”), and the constant domain sequence is derived from a human antibody, such that the resulting chimeric antibody is unlikely to elicit an adverse immune response in human subjects compared to the parental (e.g., rodent) antibody.

As used herein, the term "humanized antibody" refers to an antibody form containing sequences derived from both human and non-human (e.g., mouse, rat) antibodies. Generally, a humanized antibody will contain substantially all at least one and typically two variable domains, wherein all or substantially all hypervariable loops correspond to those regions of a non-human immunoglobulin, and all or substantially all framework (FR) regions are those regions of a human immunoglobulin sequence. A humanized antibody may optionally contain at least a portion of the constant region (Fc) of a human immunoglobulin.

The term "fully human antibody" refers to an antibody that contains only the sequence of human immunoglobulins. If produced in mice, mouse cells, or hybridomas derived from mouse cells, fully human antibodies may contain mouse carbohydrate chains. Similarly, "mouse antibody" refers to an antibody that contains only the sequence of mouse immunoglobulins. Alternatively, if produced in rats, rat cells, or hybridomas derived from rat cells, fully human antibodies may contain rat carbohydrate chains. Similarly, "rat antibody" refers to an antibody that contains only the sequence of rat immunoglobulins.

Typically, the basic antibody structural unit comprises a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50–70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition. The carboxyl-terminal portion of the heavy chain defines a constant region, primarily responsible for effector function. Human light chains are typically classified as κ light chains and λ light chains. Furthermore, human heavy chains are typically classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, the variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, with the heavy chain also including "D" regions of approximately 10 or more amino acids. See Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

The variable regions of each light chain/heavy chain pair form antibody binding sites. Therefore, generally speaking, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are generally the same.

Typically, the variable structural domains of heavy and light chains contain three hypervariable regions, also known as complementarity-determining regions (CDRs), located within relatively conservative framework regions (FRs). These CDRs are usually aligned through the framework regions to enable binding to specific epitopes. Generally, from the N-terminus to the C-terminus, the variable structural domains of light and heavy chains include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The allocation of amino acids to their respective domains is generally based on the definitions in Sequences of Proteins of Immunological Interest , Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th edition; NIH Publication 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 "hypervariate region" refers to the amino acid residues in an antibody responsible for antigen binding. The hypervariate region comprises amino acid residues from the "complementarity-determining region" or "CDR" (i.e., residues 24-34 (CDRL1), 50-56 (CDRL2), and 89-97 (CDRL3) in the light chain variable domain and residues 31-35 (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3) in the heavy chain variable domain; Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md.) and/or residues from the “hypervariant ring” (i.e., residues 26-32 (CDRL1), 50-52 (CDRL2), and 91-96 (CDRL3) in the light chain variable domain and 26-32 (CDRH1), 53-55 (CDRH2), and 96-101 (CDRH3) 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 hypervariant residues defined herein as CDR residues. The CDR provides most of the contact residues for antibody binding to antigens or epitopes. The CDRs of interest may be derived from the variable heavy and light chain sequences of donor antibodies and include analogs of naturally occurring CDRs that also share or retain the same antigen-binding specificity and/or neutralizing capacity as the donor antibodies from which they are derived.

In some implementation schemes, antibodies may take the following forms: full-length antibodies, single-domain antibodies, recombinant heavy chain-only antibodies (VHH), single-chain antibodies (scFv), shark heavy chain-only antibodies (VNAR), microproteins (cysteine knottin, knottin), ankyrin repeat protein (DARPin); tetratranectin; affibody; transbody; anticarrier protein; adNectin; affilin; microbody (M icrobody; peptide aptamer; alterase; plastic antibody; phylomer; stradobody; macrobody; evibody; fynomer; armadillo repeat protein; Kunitz domain; avimer; atrimer; probody; immunosome; triomab; troybody; pepbody; vaccine Vacibody, Unibody; Affimer, DuoBody, Fv, Fab, Fab', F(ab')2, peptide mimic molecules or synthetic molecules, such as US Patent Nos. 7,417,130, 2004/132094, 5,831,012, 2004/023334, 7,250,297, 6,818,418, 2004/209243, and 7,838. The contents of each of the following documents are incorporated herein by reference in their entirety: US No. 629, US No. 7,186,524, US No. 6,004,746, US No. 5,475,096, US No. 2004/146938, US No. 2004/157209, US No. 6,994,982, US No. 6,794,144, US No. 2010/239633, US No. 7,803,907, US No. 2010/119446 and/or US No. 7,166,697. See also Storz MAbs. May-June 2011; 3(3):310-317, which is incorporated herein by reference.

As used herein, the term "antigen" means any molecule capable of generating or binding to antibodies, directly or indirectly. The definition of "antigen" includes nucleic acids encoding proteins. "Antigen" may also refer to antibody conjugates. In some embodiments, the antigen is an IGF-1R protein expressed on the cell surface. In some embodiments, the cell is an intact cell. An intact cell is a cell that has not been dissolved or ruptured by the use of detergents or other reagents. Cells treated with detergents or other reagents that disrupt the cell membrane or pore the cell membrane are not intact cells. For example, this document provides a method for generating antibodies that bind to IGF-1R proteins, the method comprising culturing cells containing nucleic acid molecules encoding IGF-1R antibodies.

As used herein, “specific binding” or “immunospecific binding” or “immunospecifically binding” means that an antibody binds to a predetermined antigen (e.g., IGF-1R) or an epitope presented on said antigen. In some embodiments, the antibody binds with a dissociation constant (K_{_D} ) of 10 ^{^-7} M or less, and the K_{_D} of binding to the predetermined antigen is at least twice as small as the K_D of binding to a nonspecific antigen other than the predetermined _antigen (e.g., BSA, casein, or another nonspecific polypeptide). The phrases “antibody recognizing IGF-1R” and “antibody specific to IGF-1R” are used interchangeably herein with the term “antibody that binds immunospecifically to IGF-1R.” Reference may be made to IGF-1R. The degree of specificity required for an anti-IGF-1R antibody may depend on the intended use of the antibody and, at any rate, is defined by its suitability for the intended purpose. In some embodiments, the antibody or the binding compound derived from the antigen-binding site of the antibody in the covered methods has an affinity for its antigen (IGF-1R) that is at least two times, at least ten times, at least 20 times, or at least 100 times that for 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; Coligan 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), all of which are incorporated herein by reference in their entirety.

The term "homology" refers to a protein sequence that has between 40% and 100% sequence homology or identity with a reference sequence. The percentage of identity between two peptide chains can be determined by comparison-by-comparison alignment using the default settings of the AlignX module in Vector NTI v.9.0.0 (Invitrogen Corp., Carslbad, Calif.). In some embodiments, the antibody or its antigen-binding fragment has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with the sequence described herein. In some embodiments, the antibody has conserved substitutions relative to the sequence described herein. Exemplary conserved substitutions are illustrated in Table 1 and are covered within the scope of the disclosed subject matter. Conserved substitutions may be present in the framework region or the antigen-binding site, provided they do not adversely affect the properties of the antibody. Substitutions may be made to improve antibody properties, such as stability or affinity. Conservative substitutions will produce molecules with similar functions and chemical characteristics to those molecules that have undergone such modifications. Exemplary amino acid substitutions are shown in the table below.

In some embodiments, variants of the proteins and peptides provided herein are provided. In some embodiments, the variants comprise substitutions, deletions, or insertions. In some embodiments, the variants comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1 to 10) substitutions. As described herein, substitutions may be conserved. In some embodiments, substitutions are non-conserved. In some embodiments, the variants comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1 to 10) deletions. In some embodiments, the variants comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1 to 10) insertions. In some embodiments, substitutions, deletions, or insertions are present in the CDR provided herein. In some embodiments, substitutions, deletions, or insertions are not present in the CDR provided herein.

As used herein, the term “in combination with” means that the described agents may be administered to animals or subjects in any order, either as a mixture, as a single agent, simultaneously, or as single agents.

Techniques for generating antibodies against small peptide sequences are well known in the art, which recognize and bind to those sequences that are in free or conjugated form or, in the case of larger proteins, are native sequences. Such antibodies include mouse, mouse-human, and human-human antibodies generated using hybridoma or recombinant techniques known in the art. Antibodies can also be generated in humans, mice, sheep, rats, rabbits, sharks, llamas, or chickens. In some embodiments, antibodies are generated in chickens. Antibodies can also be generated in other small animals.

The term "epitope" refers to any part of a molecule that can be recognized and bound by an antibody at one or more of the antigen-binding regions of an antibody (Ab). Epitopes typically consist of chemically active surface groups, such as amino acid or sugar side chains, and possess specific three-dimensional structural features and specific charge characteristics. Examples of epitopes include (but are not limited to) the residues that form IGF-1R epitopes as described herein. In some embodiments, the epitope is present only in undenatured proteins. In some embodiments, the epitope is present only in denatured proteins.

In some implementations, the source of the DNA encoding non-human antibodies includes antibody-producing cell lines, such as hybridoma cell lines commonly known as hybridomas.

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

The second fusion conjugate providing immortalization can be a lymphoblastoid, plasmacytoma, or myeloma cell, which is not itself an antibody-producing cell but is malignant. Fusion conjugate cells include (but are not limited to) hybridoma SP2/0-Ag14, abbreviated as SP2/0 (ATCC CRL1581), and myeloma P3X63Ag8 (ATCC TIB9) or derivatives thereof. See, for example, Ausubel below, Harlow below, and Colligan below, the contents of which are incorporated herein by reference in their entirety.

Antibodies can be generated according to the embodiments provided herein. Antibodies can also be generated according to known methods once the sequence is known. Antibodies can also be converted into different types, such as human IgG. By converting antibodies into human antibodies, human subjects should not recognize the antibodies as foreign antibodies. The conversion of non-human IgG antibodies into human IgG antibodies is well known and can be performed conventionally once the natural sequence is known. As discussed herein, antibodies can be modified according to known methods. Such methods are described, for example, in 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). Antibody-producing cells that facilitate the nucleotide sequence encoding the antigen-binding region of chimeric antibodies can also be produced by transforming non-human (e.g., primate) or human cells. For example, antibody-producing B lymphocytes can be infected and transformed with a virus such as Epstein-Barr virus to obtain immortalized antibody-producing cells (Kozbor et al., Immunol. Today 4:72 79 (1983)). Alternatively, B lymphocytes can be transformed by providing a transforming gene or the product of a transforming gene, as is well known in the art. See, for example, Ausubel, Harlow, and Colligan hereinafter, the contents of which are incorporated herein by reference in their entirety. Cell fusion is achieved through standard procedures well known to those skilled in the art of immunology. Fusion-matching cell lines and methods for fusion and selection of hybridomas and screening of mAbs are well known in the art. See, for example, Ausubel, Harlow, and Colligan hereinafter, the contents of which are incorporated herein by reference in their entirety.

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

In some implementations, the antibody comprises a sequence as provided herein.

The antibody sequences can be modified to produce human IgG antibodies. The sequences provided herein can be transformed to produce other types of antibodies. The CDR can also be linked to other antibodies, proteins, or molecules to produce antibody fragments that bind to IGF-1R. These can be in the form of antibody-drug conjugates (“ADCs”), multispecific molecules, or chimeric antigen receptors. The CDR and antibody sequences provided herein have also been humanized or made fully human using known methods. The sequences can also be made into chimeric antibodies as described herein.

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

As described herein, generating antibodies with known sequences is conventional and can be performed by any method. Therefore, in some embodiments, a nucleic acid encoding an antibody or a fragment thereof is provided. In some embodiments, the nucleic acid encodes the sequence provided herein. Antibodies may also be modified to be chimeric antibodies or human antibodies. Antibodies may also be used in the form of injectable pharmaceutical compositions. Again, as described herein, antibodies may be isolated antibodies or engineered antibodies.

In some embodiments, antibodies, fragments, regions, or derivatives thereof are provided as “derivatives,” a term that includes those proteins encoded by genes of molecular species that have been truncated or modified to produce fragments functionally similar to immunoglobulin fragments. Modifications include (but are not limited to) the addition of genetic 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 can be produced in any manner.

Identifying these antigen-binding regions and/or epitopes recognized by the Abs described herein provides the information needed to generate additional monoclonal antibodies with similar binding characteristics and therapeutic or diagnostic utility compared to embodiments of this application.

The nucleic acid sequence encoding the 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 suitable alternative for using chromosomal gene fragments as the DNA source encoding the V region antigen-binding fragment is the use of cDNA for constructing chimeric immunoglobulin genes, as reported by Liu et al. (Proc. Natl. Acad. Sci., USA 84:3439 (1987) and J. Immunology 139:3521 (1987)), which are incorporated herein by reference in their entirety. Using cDNA requires gene expression elements and gene combinations suitable for the host cell to synthesize the desired protein. Using cDNA sequences is preferable to genomic sequences (which contain introns) because cDNA sequences can be expressed in bacteria or other hosts lacking a suitable RNA splicing system.

For example, cDNA encoding a V-region antigen-binding fragment capable of detecting, binding, or neutralizing IGF-1R antigens can be provided using known methods based on the amino acid sequence provided herein. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid (Watson et al., see below). The genetic code can be used to identify one or more different oligonucleotides, each of which will be able to encode an amino acid. The probability that a particular oligonucleotide will actually constitute the actual XXX-coding sequence can be estimated by taking into account the abnormal base pairings in eukaryotic or prokaryotic cells expressing the antibody or fragment and the frequency with which a particular codon (encoding a particular amino acid) is actually used. Such “codon usage rules” are disclosed by Lathe et al., J.Molec.Biol.183:112 (1985). Using Lathe’s “codon usage rules,” a single oligonucleotide or set of oligonucleotides containing the theoretically “most likely” nucleotide sequence capable of encoding the variable or constant region sequence of an antibody can be identified.

The variable regions described herein can be combined with any type of constant region, including human or mouse constant regions. Human genes encoding the constant (C) region, fragments, and regions of the antibody can be derived from a human fetal liver library using known methods. Human C region genes can be derived from any human cell, including human cells that express and produce human immunoglobulins. Human _CH regions can be derived from any known class or isotype of the human H chain, including γ, μ, α, δ, or ε, and its isotypes, such as G1, G2, G3, and G4. Because H chain isotypes are responsible for various effector functions of the antibody, the selection of _CH regions will be guided by the desired effector function (such as complement fixation) or activity in antibody-dependent cytotoxicity (ADCC). Preferably, _CH regions are derived from γ1 (IgG1), γ3 (IgG3), γ4 (IgG4), or μ (IgM). Human C _L regions can be derived from human L chain isotypes, κ, or λ. In some embodiments, the antibody comprises an Fc domain. In some embodiments, the Fc domain comprises a mutation that prolongs the antibody's half-life. In some embodiments, the Fc domain contains mutations, such as those described in U.S. Patent No. 7,670,600, which is incorporated herein by reference in its entirety. In some embodiments, the constant region contains a mutation at amino acid residue 428 relative to the constant domain of wild-type human IgG, the position being numbered according to the Kabat EU numbering index. Without being bound by any particular theory, antibodies containing the mutation corresponding to residue 428 may have an extended half-life compared to IgG having the constant domain of wild-type human IgG. In some embodiments, the mutation is a substitution of a native residue with threonine, leucine, phenylalanine, or serine. In some embodiments, the antibody further contains one or more amino acid substitutions relative to the corresponding constant domain of wild-type human IgG at one or more of amino acid residues 251-256, 285-290, 308-314, 385-389, and 429-436, the amino acid residues being numbered according to the Kabat EU numbering index. The specific mutations or substitutions at these locations are described in U.S. Patent No. 7,670,600, which is incorporated herein by reference in its entirety.

Genes encoding the human immunoglobulin C region can be obtained from human cells using standard cloning techniques (Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and Ausubel et al., eds., *Current Protocols in Molecular Biology* (1987-1993)). Human C region genes are readily obtained from known clones containing genes representing two classes of L chains, five classes of H chains, and their subclasses. Chimeric antibody fragments such as F(ab') ₂ and Fab can be prepared by designing appropriately truncated chimeric H chain genes. For example, a chimeric gene encoding the H chain portion of the F(ab') ₂ fragment would include the DNA sequence encoding the _CH1 domain and hinge region of the H chain, followed by a translation stop codon, thus yielding a truncated molecule.

In some embodiments, the antibodies described herein, mouse, human, humanized, or chimeric antibodies, antibody fragments and regions are generated by cloning DNA fragments encoding the H and L chain antigen-binding regions of IGF-1R antigen-specific antibodies, and by conjugating these DNA fragments to DNA fragments encoding the _CH and _CL regions, respectively, to produce mouse, human, or chimeric immunoglobulin-encoding genes.

Therefore, in some embodiments, a fusion chimeric gene is formed, comprising a first DNA segment encoding at least a non-human antigen-binding region (such as a functionally rearranged V region), which is linked to a second DNA segment encoding at least a portion of the human C region via a conjugation (J) segment.

Therefore, the method for generating cDNA encoding antibody V and C regions, and for generating antibodies according to some embodiments described herein, involves several steps, as illustrated below: 1. Isolating messenger RNA (mRNA) from cell lines that produce antibodies against IGF-1R antigens and from optional additional antibodies supplying constant regions of the heavy and light chains; cloning and generating cDNA from it; 2. Preparing a full-length cDNA library from the purified mRNA, wherein suitable V and/or C region gene fragments of the L and H chain genes can be: (i) identified with suitable probes, (ii) sequenced, and (iii) made compatible with C or V gene fragments from another antibody derived from a chimeric antibody; 3. Constructing a complete H or L chain coding sequence by ligating the cloned specific V region gene fragment to the cloned C region gene, as described above; 4. Expressing and generating the L and H chains in a selected host (including prokaryotic and eukaryotic cells) to provide mouse-mouse, human-mouse, human-human, or human-mouse antibodies.

Two coding DNA sequences are said to be "operably linked" if the ligation produces a continuous translatable sequence without changes or breaks in the triple reading frame. The coding sequence is operably linked to the gene expression element if the ligation results in the proper functioning of the gene expression element to induce expression of the DNA coding sequence.

As used herein and unless otherwise specified, the term “about” is intended to mean ±5% of the value it modifies. Thus, about 100 means 95 to 105.

In some embodiments, the antibodies described herein are used to detect the presence of antigens. The antibodies of this invention can be used in any apparatus or method to detect the presence of antigens.

The term "purified" as used to refer to antibodies means antibodies that are substantially free of other materials associated with molecules in their natural environment. For example, purified proteins are substantially free of cellular material or other proteins from cells or tissues from which they are derived. The term also refers to formulations in which the isolated proteins are of sufficient purity for analysis or are 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. In some embodiments, the antibody is purified.

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

Antibody conjugates

The antibodies described herein can also be conjugated with a chemical moiety. This chemical moiety may be, in particular, 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 increases the half-life of the antibody molecule in the subject. Suitable polymers include (but are not limited to) polyethylene glycol (PEG) (e.g., PEG with molecular weights of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa, or 40 kDa), dextran, and monomethoxy polyethylene glycol (mPEG). Lee et al., (1999) (Bioconj. Chem. 10:973-981) disclosed single-chain antibodies conjugated with PEG. Wen et al., (2001) (Bioconj. Chem. 12:545-553) disclosed conjugated antibodies having PEG attached to a radiometal chelating agent (diethylenetriaminepentaacetic acid (DTPA)). Examples of the chemical moiety include (but are not limited to) antimitotic agents such as chachiomycin (e.g., ozogamicin), monomethyl ozogamicin E, mertansine, etc. Other examples include (but are not limited to) bioactive antimicrotubule agents, alkylating agents, and DNA minor groove binding agents. Other examples are provided herein and below. The chemical moiety may be linked to the antibody via a linker group (maleimide), a cleavable adapter (such as a cathepsin-cleavable adapter (valine-citrulline)), and in some embodiments one or more spacers (e.g., p-aminobenzylcarbamate). Without being bound by any particular theory, once the antibody conjugate binds to 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.

The antibodies and antibody fragments of the present invention can also be conjugated with the following markers: ^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 , ^{52Tr and 56Fe .}

Antibodies and antibody fragments can also be conjugated with fluorescent or chemiluminescent labels, including fluorophores such as rare earth chelators, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, phthalaldehyde, fluorescamine, ^152Eu , dansyl, umbelliferone, luciferin, luminal label, isoluminal label, aromatic acridinium ester label, imidazole label, acridinium salt label, oxalate label, aequorin label, 2,3-dihydrophthalazinedione, biotin/antibiotin protein, spin labels, and stable free radicals.

Antibody molecules can also conjugate to cytotoxic factors such as diphtheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin toxin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids), carnation protein, Phytoiacca americana protein PAPI, PAPII and PAP-S, momordica charantia inhibitors, jatropha toxin, croton toxin, saponaria officinalis inhibitors, mitogens, localized aspergillin, phenolmycin and enomycin.

Any method known in the art for conjugating the antibody molecules of the present invention to various parts may be employed, including those described by Hunter et al., (1962) Nature 144:945; David et al., (1974) Biochemistry 13:1014; 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 described herein can also be incorporated into, for example, chimeric antigen receptors (“CARs”) that can be used in CAR-T cells. In some embodiments, the extracellular domain of the CAR may be an antibody as described herein. In some embodiments, the antibody is in the form of scFv. CAR-T cells are a type of therapy in which a patient’s T cells are modified to attack cells expressing IGF-1R. The T cells are obtained from the patient’s blood. Then, in the laboratory, a gene for a specific receptor that binds to a protein on the patient’s cells is added. In some embodiments, the receptor binds to IGF-1R using the binding region of the antibody provided herein. CAR-T cells containing the IGF-1R antibody can then be used to treat conditions such as those described herein.

In some embodiments, this document provides an antibody (e.g., an anti-IGF-1R antibody). In some embodiments, the antibody is a recombinant antibody that binds to the 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 said 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 is not naturally occurring. In some embodiments, the term "recombinant antibody" refers to an antibody that was not isolated from a human subject.

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

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

The columns indicating antibody sequences contain the VH and VL chains of the antibody. Where the VH chain is described using an Fc sequence, the Fc sequence may be modified or substituted for different Fc regions as provided herein. However, in some embodiments, the antibody may comprise the VH and VL sequences as provided in the various tables provided herein. For example, in some embodiments, the antibody comprises one or more VH, HC, LC, or VL sequences having the following sequences (those with constant domains are the complete light or heavy chain) or variants thereof:

In some embodiments, such as the variable light chain described in SEQ ID NO:13, the chain does not have a C-terminal arginine residue. This is illustrated, for example, in the following sequence:

Therefore, in some embodiments, where the variable light chain contains the sequence of SEQ ID NO:13, it may be substituted with the sequence of SEQ ID NO:97.

In some embodiments, such as those described in SEQ ID NO:14, the heavy chain variable region may contain C22S substitution. This is illustrated in the following sequence:

Therefore, in some embodiments, the antibody comprises the VH sequence of SEQ ID NO:96 and the VL sequence of SEQ ID NO:13 or SEQ ID NO:97.

In some implementations, the antibody comprises the VH sequence of SEQ ID NO:14 and the VL sequence of SEQ ID NO:97.

In some embodiments, the antibody comprises VL of SEQ ID NO:98 and VH of SEQ ID NO:99. In some embodiments, the antibody comprises VL of SEQ ID NO:98 and VH of SEQ ID NO:99, having an Fc region containing M252Y, S254T, and T256E mutations. In some embodiments, the antibody comprises VL of SEQ ID NO:98 and VH of SEQ ID NO:99, having an Fc region containing M428L and N434S mutations.

As described herein, heavy chains can be linked to the Fc region, including those with mutations that can affect the antibody's half-life. This article provides examples of non-restrictive mutations in the Fc region.

In the tables provided herein, LC and HC can be described using VH and VL domains with or without constant regions. Constant regions can be substituted as provided herein. VH and VL regions can be used to form antibodies as provided herein. VH and VL sequences can be in any form, including (but not limited to) scFv forms in which VH and VL regions are linked by peptide linkers. Examples of peptide linkers that can be used to link the various peptides provided herein include (but are not limited to): (GGGGS) _n (SEQ ID NO:73); (GGGGA) _n (SEQ ID NO:74) or any combination thereof, wherein each n is independently 1 to 5. In some embodiments, variable regions are not linked by peptide linkers. In some embodiments, the antibody comprises SEQ ID NO:1 and SEQ ID NO:2, or their CDR regions. In some embodiments, the antibody comprises SEQ ID NO:3 and SEQ ID NO:4, or their CDR regions. In some embodiments, the antibody comprises SEQ ID NO:5 and SEQ ID NO:6, or their CDR regions. In some embodiments, the antibody comprises SEQ ID NO:7 and SEQ ID NO:8, or their CDR regions. In some embodiments, the antibody comprises SEQ ID NO:9 and SEQ ID NO:10, or their CDR regions. In some embodiments, the antibody comprises SEQ ID NO:11 and SEQ ID NO:12, or their CDR regions. In some embodiments, the antibody comprises SEQ ID NO:13 and SEQ ID NO:14, or their CDR regions. In some embodiments, the antibody comprises SEQ ID NO:15 and SEQ ID NO:16, or their CDR regions.

In some embodiments, an antibody or an antigen-binding fragment thereof is provided, wherein the antibody or antibody fragment comprises peptides selected from the table below.

In some embodiments, the antibody or antibody-binding fragment thereof comprises a heavy chain or light chain CDR having the sequence of SEQ ID NO: 17 to 64 and 81. In some embodiments, the antibody or antibody-binding fragment thereof comprises a light chain CDR having the sequence of SEQ ID NO: 17, 18, 19, 23, 24, 25, 29, 30, 31, 35, 36, 37, 41, 42, 43, 47, 48, 49, 53, 54, 55, 59, 60, 61, or 81. In some embodiments, the antibody or antibody-binding fragment thereof comprises a heavy chain CDR having the sequence of SEQ ID NO: 20, 21, 22, 26, 27, 28, 32, 33, 34, 38, 39, 40, 44, 45, 46, 50, 51, 52, 56, 57, 58, 62, 63, or 64.

In some embodiments, the antibody or its antibody-binding fragment comprises a light chain having LCDR1, LCDR2 and LCDR3, wherein LCDR1 has a sequence of SEQ ID NO: 17, 23, 29, 35, 41, 47, 53 or 59, LCDR2 has a sequence of SEQ ID NO: 18, 24, 30, 36, 42, 48, 54 or 60, and LCDR3 has a sequence of SEQ ID NO: 19, 25, 31, 37, 43, 49, 55, 61 or 81.

In some embodiments, the antibody or its antibody-binding fragment comprises a heavy chain having HCDR1, HCDR2 and HCDR3, wherein HCDR1 has a sequence of SEQ ID NO: 20, 26, 32, 38, 44, 50, 56 or 62, HCDR2 has a sequence of SEQ ID NO: 21, 27, 33, 39, 45, 51, 57 or 63, and HCDR3 has a sequence of SEQ ID NO: 22, 28, 34, 40, 46, 52, 58 or 64.

Different CDR motifs can be combined in any combination, including those not depicted in the table above. For example, the following embodiments provide non-limiting examples of such combinations.

In some embodiments, the antibody or its antigen-binding fragment 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:17; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:18; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:19; 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:20; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:21; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:22; or a variant of any of the foregoing.

In some embodiments, the antibody or its antigen-binding fragment 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:23; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:24; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:25; 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:26; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:27; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:28; or a variant of any of the foregoing.

In some embodiments, the antibody or its antigen-binding fragment 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:29; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:30; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:31; 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:32; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:33; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:34; or a variant of any of the foregoing.

In some embodiments, the antibody or its antigen-binding fragment 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:35; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:36; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:37; 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:38; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:39; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:40; or a variant of any of the foregoing.

In some embodiments, the antibody or its antigen-binding fragment 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:41; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:42; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:43; 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:44; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:45; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:46; or a variant of any of the foregoing.

In some embodiments, the antibody or its antigen-binding fragment 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:47; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:48; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:49; 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:50; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:51; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:52; or a variant of any of the foregoing.

In some embodiments, the antibody or its antigen-binding fragment 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: 53; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 54; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 55; 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: 56; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 57; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 58; or a variant of any of the foregoing.

In some embodiments, the antibody or its antigen-binding fragment 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: 59; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 60; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 61; 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: 62; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 63; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO: 64; or a variant of any of the foregoing.

In some embodiments, the antibody or its antigen-binding fragment 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:35; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:36; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:81; 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:38; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:39; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:40; or a variant of any of the foregoing.

In some embodiments, the light chain variable region CDR1 is replaced by any of the other light chain CDR1 sequences. In some embodiments, the light chain variable region CDR2 is replaced by any of the other light chain CDR2 sequences. In some embodiments, the light chain variable region CDR3 is replaced by any of the other light chain CDR3 sequences. In some embodiments, the heavy chain variable region CDR1 is replaced by any of the other light chain CDR1 sequences. In some embodiments, the heavy chain variable region CDR2 is replaced by any of the other light chain CDR2 sequences. In some embodiments, the heavy chain variable region CDR3 is replaced by any of the other light chain CDR3 sequences.

In some embodiments, an antibody or its antigen-binding fragment or protein is provided, comprising a peptide having a sequence as set forth in any of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 79 or 86, and 2, 4, 6, 8, 10, 12, 14, 16, 80 or 83.

In some implementations, the antibody or its antigen-binding fragment contains a sequence or variant of any of the foregoing.

In some embodiments, the antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:65 or a variant of any of the foregoing. In some embodiments, the antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:66 or a variant of any of the foregoing. In some embodiments, the antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:67 or a variant of any of the foregoing. In some embodiments, the antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:68 or a variant of any of the foregoing. In some embodiments, the antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:69 or a variant of any of the foregoing. In some embodiments, the antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:70 or a variant of any of the foregoing. In some embodiments, the antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:71 or a variant of any of the foregoing. In some embodiments, the antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:72 or a variant of any of the foregoing. In some embodiments, the antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:78 or a variant of any of the foregoing. In some embodiments, the antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:82 or a variant thereof. In some embodiments, the antibody or its antigen-binding fragment comprises the sequence of SEQ ID NO:85 or a variant thereof.

In some embodiments, the _VL and/or _VH sequences are as provided herein. In some embodiments, the _VL sequence is provided as a light chain (LC) element. In some embodiments, the VL sequence provided as a light chain (LC) element is underlined within the LC sequence. In some embodiments, _{the VH sequence} provided as a heavy chain (LC) element is underlined within the HC sequence.

In some embodiments, the antibody or its antigen-binding fragment comprises a V _L peptide, or any combination thereof, as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 79 or 86. The V _L peptide may comprise a variant of any of these sequences provided herein.

In some embodiments, the antibody or its antigen-binding fragment comprises a V _H peptide, or any combination thereof, as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 80, or 83. The V _H peptide may comprise a variant of any of these sequences provided herein.

In some embodiments, the antibody or its antigen-binding fragment comprises a _VH peptide and a _VL peptide. The _VH peptide comprises the sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 80, or 83, and the _VL peptide comprises the sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 79, or 86.

In some embodiments, the antibody or its antigen-binding fragment comprises a _VH peptide and a _VL peptide, wherein the _VH peptide comprises the sequence set forth in SEQ ID NO:2 and the _VL peptide comprises the sequence set forth in SEQ ID NO:1. In some embodiments, the antibody or its antigen-binding fragment comprises a _VH peptide and a _VL peptide, wherein the _VH peptide comprises the sequence set forth in SEQ ID NO:4 and the _VL peptide comprises the sequence set forth in SEQ ID NO:3. In some embodiments, the antibody or its antigen-binding fragment comprises a _VH peptide and a _VL peptide, wherein the _VH peptide comprises the sequence set forth in SEQ ID NO:6 and the _VL peptide comprises the sequence set forth in SEQ ID NO:5. In some embodiments, the antibody or its antigen-binding fragment comprises a _VH peptide and a _VL peptide, wherein the _VH peptide comprises the sequence set forth in SEQ ID NO:8 and the _VL peptide comprises the sequence set forth in SEQ ID NO:7. In some embodiments, the antibody or its antigen-binding fragment comprises a _VH peptide and a _VL peptide, wherein the _VH peptide comprises the sequence set forth in SEQ ID NO:10 and the _VL peptide comprises the sequence set forth in SEQ ID NO:9. In some embodiments, the antibody or its antigen-binding fragment comprises a _VH peptide and a _VL peptide, wherein the _VH peptide comprises the sequence set forth in SEQ ID NO:12 and the _VL peptide comprises the sequence set forth in SEQ ID NO:11. In some embodiments, the antibody or its antigen-binding fragment comprises a _VH peptide and a _VL peptide, wherein the _VH peptide comprises the sequence set forth in SEQ ID NO:14 and the _VL peptide comprises the sequence set forth in SEQ ID NO:13. In some embodiments, the antibody or its antigen-binding fragment comprises a _VH peptide and a _VL peptide, wherein the _VH peptide comprises the sequence set forth in SEQ ID NO:16 and the _VL peptide comprises the sequence set forth in SEQ ID NO:15. In some embodiments, the antibody or its antigen-binding fragment comprises a _VH peptide and a _VL peptide, wherein the _VH peptide comprises the sequence set forth in SEQ ID NO:80 and the _VL peptide comprises the sequence set forth in SEQ ID NO:79. In some embodiments, the antibody or its antigen-binding fragment comprises a _VH peptide and a _VL peptide, wherein the _VH peptide comprises the sequence set forth in SEQ ID NO:83 and the _VL peptide comprises the sequence set forth in SEQ ID NO:3. In some embodiments, the antibody or its antigen-binding fragment comprises a _VH peptide and a _VL peptide, wherein the _VH peptide comprises the sequence set forth in SEQ ID NO:14 and the _VL peptide comprises the sequence set forth in SEQ ID NO:86.

In some embodiments, the antibody or its antigen-binding fragment comprises an LC peptide as set forth in SEQ ID NO: 1, 3, 5, 7, 9 or 11, or any combination thereof. The LC peptide may comprise a variant of any of these sequences provided herein.

In some embodiments, the antibody or its antigen-binding fragment comprises an HC peptide, or any combination thereof, as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12 or 83. The HC peptide may comprise a variant of any of these sequences provided herein.

In some embodiments, the antibody or its antigen-binding fragment comprises an HC peptide and an LC peptide, wherein the HC peptide comprises the sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, or 83 and the LC peptide comprises the sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, or 11. In some embodiments, the antibody or its antigen-binding fragment comprises an HC peptide and an LC peptide, wherein the HC peptide comprises the sequence set forth in SEQ ID NO:2 and the LC peptide comprises the sequence set forth in SEQ ID NO:1. In some embodiments, the antibody or its antigen-binding fragment comprises an HC peptide and an LC peptide, wherein the HC peptide comprises the sequence set forth in SEQ ID NO:4 and the LC peptide comprises the sequence set forth in SEQ ID NO:3. In some embodiments, the HC peptide comprising the sequence set forth in SEQ ID NO:4 has an additional C-terminal lysine (K) residue. In some embodiments, the antibody or its antigen-binding fragment comprises an HC peptide and an LC peptide, wherein the HC peptide comprises the sequence set forth in SEQ ID NO:6 and the LC peptide comprises the sequence set forth in SEQ ID NO:5. In some embodiments, the antibody or its antigen-binding fragment comprises an HC peptide and an LC peptide, wherein the HC peptide comprises the sequence set forth in SEQ ID NO:8 and the LC peptide comprises the sequence set forth in SEQ ID NO:7. In some embodiments, the antibody or its antigen-binding fragment comprises an HC peptide and an LC peptide, wherein the HC peptide comprises the sequence set forth in SEQ ID NO:10 and the LC peptide comprises the sequence set forth in SEQ ID NO:9. In some embodiments, the antibody or its antigen-binding fragment comprises an HC peptide and an LC peptide, wherein the HC peptide comprises the sequence set forth in SEQ ID NO:12 and the LC peptide comprises the sequence set forth in SEQ ID NO:11. In some embodiments, the antibody or its antigen-binding fragment comprises an HC peptide and an LC peptide, wherein the HC peptide comprises the sequence set forth in SEQ ID NO:83 and the LC peptide comprises the sequence set forth in SEQ ID NO:3.

In addition to these specific combinations, either _VH peptide or _VL peptide can be combined with each other.

In addition to these specific combinations, either HC peptide or LC peptide can be combined with each other.

In some embodiments, the antibody comprises a sequence of ATCC clone PTA-7444 or an antigen-binding fragment. The antibody sequence generated by ATCC clone PTA-7444 is incorporated herein by reference in its entirety, and the antibody sequence includes its antigen-binding fragment.

Additionally, as provided herein, the antibody may be a multispecific antibody because it has multiple binding regions of the same protein that target different proteins or different epitopes. In some embodiments, the antibody is a bispecific antibody.

As provided herein, the various peptides ( _VH or _VL ) described herein may be linked by or without peptide linkers and alternatively are continuous sequences. In some embodiments, the peptide linker comprises the following sequences: (GGGGS) _n (SEQ ID NO:73); (GGGGA) _n (SEQ ID NO:74), or any combination thereof, wherein each n is independently 1 to 5. The linked peptide form may be represented by the formula _VH - _ZVL or _VL - _ZVH , where Z is the peptide linker. In some embodiments, Z is (GGGGS) _n (SEQ ID NO:73); (GGGGA) _n (SEQ ID NO:74), or any combination thereof, wherein each n is independently 1 to 5.

As provided in this article, antibodies or their antigen-binding fragments may be sequence variants.

Other examples of antibodies include (but are not limited to) those provided below: US20160096894A1, EP1399483B1, EP2194067B1, US20040202651A1, US20110229933A1, US8137933B2, US8951790B2, US20190270820A1, US7572897B2, US20090275126A1, EP1959014B1, US20080014203A1, US20080226635A1, US20120076778A1, US20190153071A1, WO2011161119A1, US10 US20120237507A1, EP2681240B1, US9982036B2, US20180312573A1, EP2681239B1, US20160151487A1, US20190225696A1, WO2017011773A2, US20200023076A1, US20190153471A1, US20190194713A1, WO2020006486A1, US20080112888A1, US20150168424A1, EP2032989B2, and US9045536B2 are all incorporated herein by reference in their entirety. Other examples of antibodies include (but are not limited to) those provided below: US8153121B2, EP1469879B1, WO2016064716A1, US20190270820A1, US20180280527A1, US20190225696A1, US7998681B2, US20040202651A1, US2005013606 3A1, US20090285824A1, US20150274829A1, EP2322550B1, US20060286103A1, US20070071675 A1, US20100047239A1, US20130004416A1, US20080112888A1, US20150168424A1, US20100143 340A1, US20110014117A1, US20100260668A1, US20100074900A1, US20150017168A1, US20110 044980A1, US20130330323A1, US20120263722A1, US20120201746A1, US10519245B2, US20180 US20170218091A1, US20200115460A1, US20100104645A1, US20120065380A1, EP2970433B1, US20160289341A1, US20160289343A1, and US20190293656A1 are incorporated herein by reference in their entirety.

In some implementations, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the following sequence:

And the light chain contains the following sequence:

In some implementations, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the following sequence:

And the light chain contains the following sequence:

In some embodiments, the heavy chain of SEQ ID NO:94 includes a C-terminal lysine residue added to the C-terminus of SEQ ID NO:94.

In some implementations, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the following sequence:

Furthermore, the light chain contains the sequence of SEQ ID NO:93.

In some embodiments, the heavy chain of SEQ ID NO:95 includes a C-terminal lysine residue added to the C-terminus of SEQ ID NO:95.

In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the sequence of SEQ ID NO:83 and the light chain comprises the sequence of SEQ ID NO:3.

In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:96 and the VL sequence of SEQ ID NO:13 or SEQ ID NO:97. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:14 and the VL sequence of SEQ ID NO:97.

Pharmaceutical Composition

In some embodiments, to prepare pharmaceutical or sterile compositions of the anti-IGF-1R antibody or other protein provided herein, the antibody or its antigen-binding fragment or other protein provided herein is blended 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 can be prepared by mixing them with acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, or suspensions (see, for example, 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 al DosageForms: 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, the antibody is diluted to a suitable concentration in sodium acetate solution at pH 5 to 6, and NaCl or sucrose is added for tension. Additional agents such as polysorbate 20 or polysorbate 80 may be added to enhance stability.

The toxicity and therapeutic efficacy of antibody compositions, administered alone or in combination with another agent, can be determined in cell cultures or experimental animals using standard pharmaceutical procedures, such as determining the _LD50 (the dose that is lethal to 50% of the population) and _ED50 (the dose that is therapeutically effective to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index ( _LD50 / _ED50 ). In certain respects, antibodies exhibiting a high therapeutic index are desirable. Data obtained from these cell culture assays and animal studies can be used to formulate a range of doses for human use. The doses of such compounds are preferably within the range of circulating concentrations, including an _ED50 with low or no toxicity. The dose may vary within this range depending on the dosage form and route of administration.

In some embodiments, the compositions of the present invention are administered to subjects in accordance with Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (November 1, 2002)).

The route of administration can vary. Suitable routes of administration include oral, rectal, mucosal, enteral, and non-enteric; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, inhalation, local, skin, transdermal, or intra-arterial.

In some embodiments, the antibody or its antigen-binding fragment may be administered via an invasive route such as injection. In some embodiments, the antibody or its antigen-binding fragment or a pharmaceutical composition thereof may be administered intravenously, subcutaneously, intramuscularly, intra-arterially, intra-articularly (e.g., in arthritic joints), or by inhalation or aerosol delivery. Administration via non-invasive routes (e.g., oral; e.g., in pills, capsules, or tablets) is also within the scope of embodiments of the invention.

In some embodiments, the antibody or its antigen-binding fragment may be administered directly to the eye, the anterior chamber of the eye, the vitreous cavity of the eye, the suprachoroidal space, or the posterior orbital sinus. In some embodiments, it is administered by injection to the eye, the anterior chamber of the eye, the vitreous cavity of the eye, the suprachoroidal space, or the posterior orbital sinus. In some embodiments, the injection is intravitreal, intraorbital, posterior orbital, suprachoroidal, or intra-anterior chamber injection. In some embodiments, the injection is intravitreal. In some embodiments, the injection is intraorbital. In some embodiments, the injection is posterior orbital. In some embodiments, the injection is suprachoroidal. In some embodiments, the injection is intra-anterior chamber.

In some embodiments, an anti-IGF-1R antibody or its antigen-binding fragment 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 its antigen-binding fragment is administered in combination with at least one other therapeutic agent (such as, but not limited to, a therapeutic agent for treating thyroid eye disease or related symptoms). Examples of such treatments and therapeutic agents include, but are not limited to, antithyroid drugs, diabetes medications, beta-blockers, propylthiouracil, methimazole, propranolol, atenolol, metoprolol, nadolol, corticosteroids, metformin, sulfonylureas, meglitinide, thiazolidinediones, DPP-4 inhibitors, and GLP-1 receptor agonists. SGLT2 inhibitors, conventional insulin, insulin aspart, insulin lisgluten, insulin lispro, insulin isopranin, insulin degludec, insulin detemir, insulin glargine, acarbose, miglitol, acebutolol, atenolol, betaxolol, bisoprolol, cartelol, carvedilol l), esmolol, labetalol, metoprolol, nadolol, nebivolol, penbutolol, pindolol, propranolol, sotalol, timolol, tomolol ophthalmic solution, sitagliptin, saxagliptin, linagliptin, alogliptin, dulaglutide, exenatide, semaglutide, liraglutide, lixisenatide, canagliflozin, dapagliflozin, empagliflozin, or any combination thereof.

The composition can be administered using medical devices known in the art. For example, the pharmaceutical composition of the present invention can be administered by injection using a subcutaneous injection needle, including, for example, a pre-filled syringe or an autoinjector.

The pharmaceutical composition can also be administered using a needle-free subcutaneous injection device, such as those disclosed in U.S. Patent Nos. 6,620,135, 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 can also be administered by infusion. Examples of well-known implantable and modular forms for administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable-flow implantable infusion device for continuous drug delivery; and U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having a multi-chambered compartment. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

Alternatively, antibodies can be administered locally rather than systemically, for example, by direct injection into arthritic joints or pathogen-induced lesions characterized by immunopathology, typically in the form of reservoirs or sustained-release formulations. Furthermore, antibodies can be administered in the form of targeted drug delivery systems that target, for example, arthritic joints or pathogen-induced lesions characterized by immunopathology (e.g., liposomes coated with tissue-specific antibodies). The liposomes will target the diseased tissue and be selectively absorbed by it.

Administration regimens depend on several factors, including the seroconversion rate of the therapeutic antibody, the severity of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody to achieve improvement in the condition of the target disease while minimizing undesirable side effects. Therefore, the delivered dose of the biological agent depends in part on the specific therapeutic antibody and the severity of the condition being treated. Guidelines for selecting appropriate doses of therapeutic antibodies are available (see, for example, 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). kker, 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.

Clinicians determine the appropriate dosage, for example, using parameters or factors known or suspected in the art to affect treatment. Generally, the initial dose is slightly less than the optimal dose, subsequently increased in small increments until the desired or optimal effect (relative to any negative side effects) is achieved. Important diagnostic measures include, for example, those measuring the symptoms of inflammation or the levels of inflammatory cytokines produced. Generally, it is desirable that the biologics to be used be derived from the same species as the animal targeted for treatment, thereby minimizing any immune response to the agent. In the case of human subjects, chimeric antibodies, humanized antibodies, and fully human antibodies may be desirable.

Antibodies or their antigen-binding fragments can be delivered by continuous infusion or by dosage, for example, once daily, once to seven times weekly, weekly, every two weeks, monthly, bi-monthly, quarterly, semi-annually, or annually. Dosage can be delivered, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracranially, intraspinally, or by inhalation. In some embodiments, antibodies are administered every three, four, five, six, seven, or eight weeks. In some embodiments, antibodies are administered every four weeks. In some embodiments, antibodies are administered every five weeks. In some embodiments, antibodies are administered every seven weeks. In some embodiments, antibodies are administered every six weeks. In some embodiments, antibodies are administered every eight weeks. In some embodiments, antibody administration lasts for at least 21 to 52 weeks or longer. In some embodiments, antibodies are administered on this schedule for at least 21 weeks. In some embodiments, antibodies are administered on this schedule for at least 24 weeks. In some embodiments, antibodies are administered on this schedule for at least 32 weeks. In some embodiments, antibodies are administered on this schedule for at least 36 weeks. In some embodiments, the antibody is administered on this schedule for at least 40 weeks. In some embodiments, the antibody is administered on this schedule for at least 42 weeks. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) once. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) twice. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) three times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) four times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) five times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) six times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) seven times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) eight times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) nine times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) ten times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) eleven times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) 12 times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) 13 times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) 14 times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) 15 times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) 16 times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) 17 times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) 18 times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) 19 times. In some embodiments, the antibody is administered (e.g., by infusion or subcutaneous injection) 20 times. When the antibody is administered more than once, it may be administered according to a schedule such as the schedule provided herein.

The total weekly dose can be provided as in this article. In some implementations, the total weekly dose is at least 0.05 μg/kg body weight, and more generally at least 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/ml, 10 mg/kg, 25 mg/kg, 50 mg/kg or more (see, for example, 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). Doses may also be provided to achieve predetermined target concentrations of the antibody in the subject's serum, such as 0.1 μg/ml, 0.3 μg/ml, 1 μg/ml, 3 μg/ml, 10 μg/ml, 30 μg/ml, 100 μg/ml, 300 μg/ml, or greater than 300 μg/ml.

In some implementations, at least 1, 2, or 3 weeks after administration, the serum concentration of the antibody in the subject is at least or about 10 μg/ml or 20 μg/ml or 50 μg/ml, 70 μg/ml, 75 μg/ml, 80 μg/ml, 85 μg/ml, 90 μg/ml, 95 μg/ml, 100 μg/ml, or 105 μg/ml.

In some embodiments, an IV dose of 20 mg/kg is administered. In some embodiments, an antibody dose is used to provide 133 μg/mL Cmin after approximately 5 weeks. In some embodiments, the administered antibody dose provides 102 μg/mL Cmin after 6 weeks. In some embodiments, the antibody dose is as provided herein, such as 10 mg/mg as a starting dose, with subsequent doses being the same or lower. In some embodiments, the antibody is administered as provided herein to achieve a Cmin dose of at least or approximately 100 μg/mL.

As used herein, “suppression” or “treatment” includes delaying the development of symptoms associated with a condition and/or reducing the severity of such symptoms. The term further includes improving existing uncontrolled or unwanted symptoms, preventing additional symptoms, and improving or preventing the underlying cause of such symptoms. Therefore, the term indicates that a beneficial outcome has been achieved in vertebrate subjects who have or are likely to develop a condition, disease, or symptom.

As used herein, the terms “therapeutic effective amount,” “therapeutic effective dose,” and “effective amount” refer to the amount of an antibody or antigen-binding fragment thereof that, when administered alone or in combination with an additional therapeutic agent to cells, tissues, or a subject, effectively causes a measurable improvement in one or more symptoms of a disease or condition or the progression of such a disease or condition. A therapeutic effective dose further refers to the amount of a conjugate compound sufficient to cause at least partial improvement in symptoms, such as treatment, cure, prevention, or improvement of the associated medical condition, or to increase the rate of treatment, cure, prevention, or improvement of the condition. When applied to an individual active ingredient administered alone, the therapeutic effective dose refers only to that ingredient. When applied in combination, the therapeutic effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of therapeutic agent will improve a diagnostic measure or parameter by at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%. In cases where subjective measures are used to assess disease severity, the effective amount may also cause an improvement in the subjective measure. In some implementations, the amount is a therapeutically effective amount if it is an amount that can be used to treat or improve the symptoms as provided herein.

As used throughout, the term "subject" includes any living organism, such as animals, including mammals (e.g., rats, mice, dogs, cats, rabbits) and, for example, humans. A subject may also be referred to as a patient. In some embodiments, a subject is a subject in need. A "subject in need" is a subject who has been identified as requiring treatment for the condition to be treated and who is being treated with the specific intent to treat that condition. For example, a condition may be any of the conditions described herein.

Given that isolated antibodies bind to epitopes on IGF-1R proteins or other proteins described herein and exhibit in vitro and/or in vivo IGF-1R inhibitory or therapeutic activity, antibodies or antigen-binding fragments thereof capable of inhibiting IGF-1R function are suitable as therapeutic agents for treating IGF-1R-related conditions in humans and animals. These conditions include thyroid ophthalmopathy. Therefore, methods for treating such conditions are also provided, wherein the method comprises administering an antibody or antigen-binding fragment thereof to a subject suffering from such conditions.

In some implementations, the method includes administering a therapeutically or prophylactically effective amount of one or more monoclonal antibodies or antigen-binding fragments of antibodies described herein to a susceptible subject or a subject exhibiting symptoms in which IGF-1R is known or suspected to cause the observed pathology. Any active form of the antibody that may be administered includes (but is not limited to) scFv, Fab, and F(ab')2 fragments and other forms of antibodies provided herein.

As used in this article, IGF-1R-related pathology refers to symptoms caused by the regulation of IGF-1R. These symptoms include (but are not limited to) thyroid ophthalmopathy and other symptoms described in this article.

In some implementations, the antibodies used are species compatible with the recipient so that the immune response against the MAb does not result in an unacceptably short circulating half-life or induce an immune response against the subject's MAb.

Individual treatment may include administration of a therapeutically effective amount of the antibodies described herein. Antibodies may be available in kit form, as those provided herein. Antibodies may be used alone or in combination with another therapeutic agent, analgesic, or diagnostic agent as provided herein. In administering an antibody or fragment thereof capable of binding to IGF-1R or an antibody capable of preventing IGF-1R pathology in a recipient patient, the dosage of the administered agent will vary depending on factors such as the patient's age, weight, height, sex, general medical condition, and medical history.

Antibodies capable of treating symptoms associated with IGF-1R activity or intended to treat IGF-1R-related pathologies are intended to be delivered to subjects in an amount sufficient to affect the reduction, resolution, or improvement of IGF-1R-related symptoms or pathologies. Such pathologies include thyroid ophthalmopathy, etc.

Therefore, in some embodiments, a method is provided for treating a subject with an IGF-1R-mediated condition. In some embodiments, the method includes administering a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof, as provided herein. In some embodiments, the condition is thyroid ophthalmopathy. As provided herein, the antibody or antigen-binding fragment thereof may be administered together with other therapeutic agents. These therapeutic agents may be administered simultaneously or sequentially.

In some embodiments, the antibody or its antigen-binding fragment can be used to treat thyroid eye disease. In some embodiments, the antibody or its antigen-binding fragment can be used to treat thyroid-associated eye disease (TAO) or its symptoms or to reduce its severity.

In some implementations, methods or uses are provided to reduce bulging of the eyes in subjects with thyroid-associated eye disease (TAO).

In some implementations, the subjects are those who have previously been treated with antibodies different from those provided herein.

In some implementations, a method or use is provided for a Clinical Activity Score (CAS) for subjects who have or are suspected of having thyroid-associated eye disease (TAO).

In some implementations, methods or uses are provided to reduce bulging of the eye of a subject with thyroid-associated eye disease (TAO) by at least 2 mm and to reduce the clinical activity score (CAS).

As used herein, the term Clinical Activity Score (CAS) refers to the scoring scheme described and administered according to Table 2. According to this scheme, one score is given for each parameter assessed in the table below. The sum of all scores defines clinical activity and provides the CAS, where 0 or 1 constitutes inactive disease and 7 represents severe active eye disease.

As shown in Table 2, CAS consists of seven components: spontaneous retrobulbar pain, pain when attempting to move the eye (upward, leftward, rightward, and downward gaze), conjunctival redness, eyelid redness, bulbar conjunctival edema, lacrimal caruncle/fold swelling, and eyelid swelling. Each component is scored as present (1 point) or absent (0 points). The score for each efficacy assessment is the sum of all present items; resulting in a range of 0 to 7, where 0 or 1 constitutes inactive disease and 7 represents severe active eye disease. A change of >2 points is considered clinically significant.

First, spontaneous orbital pain can be a feeling of pain or pressure on or behind the eyeball. This pain can be caused by increased intraorbital pressure when the volume of orbital tissue increases through excessive synthesis of extracellular matrix, fluid accumulation, and cell infiltration and proliferation. Second, gaze-induced orbital pain can be eye pain when looking or attempting to look upward, downward, or sideways—that is, pain when moving the eye up, down, or sideways or attempting to move the eye. This type of pain can be caused by stretching of inflamed muscles, especially when attempting to gaze upward. 'Stretching pain' is not caused by pressing a finger on the eyeball and would be predictable if it were a manifestation of increased intraorbital pressure. Both types of pain can be reduced after anti-inflammatory treatment. Therefore, these types of pain are considered directly related to autoimmune inflammation of the orbit and are thus suitable for assessing TAO activity.

Swelling in TAO is considered to be item 6 in Table 1, bulbar conjunctival edema (conjunctival edema), and swelling of the caruncle and/or semilunar fold. Both are signs of active TAO. Eyelid swelling can be caused by edema, fat prolapse throughout the orbital septum, or fibrotic degeneration. In addition to swelling, other symptoms indicating active TAO include redness and/or pain of the conjunctiva, eyelids, caruncle, and/or semilunar fold.

In some embodiments, the treated subject's bulging eye decreased by at least 2 mm. In some embodiments, the treated subject's bulging eye decreased by at least 3 mm. In some embodiments, the treated subject's bulging eye decreased by at least 4 mm.

In some implementations, the clinical activity score (CAS) of the treated subjects decreased by at least 2 points. In some implementations, the clinical activity score (CAS) of the subjects decreased to one (1). In some implementations, the clinical activity score (CAS) of the subjects decreased to zero (0).

In some implementations, a method is provided for treating a subject with thyroid-associated ophthalmopathy (TAO) or reducing its severity, wherein the treatment with the antibody (i) reduces the bulging of one eye by at least 2 mm; (ii) without deterioration of the other eye (or contralateral eye) by 2 mm or more; and (iii) reduces the subject's CAS to one (1) or zero (0).

In some implementations, methods are provided to improve the quality of life of subjects with thyroid-associated eye disease (TAO, also known as Graves' eye disease/Graves' orbital disease). In some implementations, quality of life is measured using the Graves' Eye Disease Quality of Life (GO-QoL) assessment or its visual function or appearance subscales. In some implementations, treatment results in a GO-QoL improvement of 8 points or more. In some implementations, treatment results in improvement on the GO-QoL function subscale. In some implementations, treatment results in improvement on the GO-QoL appearance subscale.

In some implementations, a method is provided to treat diplopia or reduce its severity in subjects with thyroid-associated eye disease (TAO). In some implementations, the diplopia is constant. In some implementations, the diplopia is non-constant. In some implementations, the diplopia is intermittent. In some implementations, the improvement in diplopia or the reduction in its severity persists for at least 20 weeks after the discontinuation of antibody administration. In some implementations, the improvement in diplopia or the reduction in its severity persists for at least 50 weeks after the discontinuation of antibody administration.

The severity of the disease can be measured in the following non-limiting embodiments. For example, for the eyelid opening, the distance between the eyelid margins (in mm) is measured when the patient is in a primary gaze position, seated, relaxed, and looking at a distant object. For eyelid swelling, it is measured/assessed as “absent/unclear,” “moderate,” or “severe.” Eyelid redness is absent or present. Conjunctival redness is absent or present. In some embodiments, conjunctival edema is absent or present. In some embodiments, inflammation of the caruncle or fold is absent or present. Proptosis is measured for individual patients using the same Hertel exophthalmometer and the same intercanthal distance, in millimeters. Subjective diplopia is scored from 0 to 3 (0 = no diplopia; 1 = intermittent, i.e., diplopia in the primary gaze position when tired or upon first waking; 2 = non-constant, i.e., diplopia during extreme gaze; 3 = constant, i.e., continuous diplopia in the primary gaze position or reading position). For ocular muscle involvement, monocular reduction is measured, expressed in degrees. Corneal involvement is either absent/punctate or present with corneal lesions/ulcers. For optic nerve involvement, i.e., defects in best-corrected visual acuity, color vision, optic disc, and relative pupillary afferentity, the aforementioned symptoms are either absent or present. Additionally, visual field testing is performed when optic nerve compression is suspected. In some implementations, patients may be categorized according to the following severity levels. For example, vision-threatening thyroid eye disease: patients with thyroid dysfunctional optic neuropathy (DON) and/or corneal rupture. This category requires immediate intervention. Moderate to severe thyroid eye disease: patients without vision-threatening disease, but whose eye disease is sufficient to affect daily life, justifying a reasonable risk of immunosuppression (if active) or surgical intervention (if inactive). Patients with moderate to severe thyroid eye disease typically have one or more of the following: eyelid retraction greater than or equal to 2 mm, moderate or severe soft tissue involvement, exophthalmos exceeding the normal range by 3 mm or more for race and sex, and non-constant or constant diplopia. Mild thyroid ophthalmopathy: Patients whose thyroid ophthalmopathy features have only a minor impact on daily life, insufficient to justify immunosuppression or surgical treatment. They typically have only one or more of the following: mild eyelid retraction (<2 mm), mild soft tissue involvement, proptosis <3 mm above normal for race and sex, transient diplopia or no diplopia, and corneal exposure that responds to lubricants.

In some implementations, patient quality of life can be characterized by the Graves' Eye Disease Quality of Life (GO-QoL) score. In addition to exophthalmos (or proptosis) and CAS, the GO-QoL questionnaire is used to assess quality of life. This questionnaire is designed to measure improvements in quality of life following treatment with the methods disclosed herein. In some implementations, the questionnaire can measure the reduction or absence of side effects following treatment with antibodies or their antigen-binding fragments according to the methods disclosed herein compared to treatment with glucocorticoids. GO-QoL is a 16-item self-administered questionnaire divided into two subsets and used to assess the subject's perceived impact of TED on (i) their visual function-related daily physical activities and (ii) their psychosocial functioning. Quality of life is assessed using the GO-QoL questionnaire. The GO-QoL questionnaire is completed on day 1 and week 6, week 12 and week 24 (or post-treatment) during treatment and at month 7 and month 12 (or post-treatment) during follow-up [C.B. Terwee et al., 1998]. The GO-QoL is a 16-item self-administered questionnaire divided into two self-assessment subscales: one subscale covers the impact of visual function on daily activities, and the other assesses the impact on self-perceived appearance. The visual function subscale covers activities such as driving, walking outdoors, reading, and watching television. The appearance subscale asks participants questions such as whether eye disease alters their appearance, causes negative reactions from others, leads to social isolation, or causes them to try to cover their appearance. Each subscale has eight questions, answered in the form of "yes" (very much), "yes" (somewhat), or "no" (not at all). Each question is rated from 0 to 2, and the total raw score is then mathematically converted to a 0-100 scale, where 0 represents the greatest negative impact on quality of life and 100 represents no impact. A change of ≥8 points on the 0-100 scale is considered clinically significant. The pooled score uses the raw scores from both subscales and is then converted back to a single 0-100 scale. The questionnaire has two self-assessment subscales. Each subscale has eight questions, answered with (i) yes – very much; (ii) yes – a little; or (iii) no – not at all. Each question is rated from 0 to 2, and the total raw score is then mathematically converted to a 0-100 scale, where 0 represents the greatest negative impact on quality of life and 100 represents no impact. A change of >8 points on the 0-100 scale is considered clinically significant. The pooled score is the raw score from both subscales, which is then converted back into a single 0-100 scale.

Patients can also be assessed by the presence or absence of a Gorman grade for diplopia. The Gorman assessment of subjective diplopia includes four categories: no diplopia (absence), diplopia when the patient is tired or awake (intermittent), diplopia during extreme gaze (non-constant), and continuous diplopia in the primary gaze or reading position (constant). Patients are scored according to the level of diplopia they have experienced. An improvement of grade 1 or higher is considered clinically significant.

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

In some embodiments, the antibody is administered in the form of 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-CD20 antibody; tocilizumab or other anti-IL-6 antibody; or selenium, infliximab or other anti-TNFα antibody or thyroid-stimulating hormone receptor (TSHR) inhibitor.

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

Kits for carrying out the embodiments described herein are also provided. The kits of the present invention include a first container containing or co-packaged with the antibodies described above. The kits may also include another container containing or co-packaged with solutions necessary or suitable for carrying out the embodiments. The containers may be made of glass, plastic, or foil, and may be vials, bottles, pouches, tubes, bags, etc. The kits may also contain written information, such as procedures for carrying out the embodiments; or analytical information, such as the amount of reagents contained in the first container component. The containers may be located together with the written information in another container device (e.g., a box or bag).

Another aspect provided herein is a kit for detecting IGF-1R protein in biological samples. The kit includes a container holding one or more antibodies that bind to epitopes of the IGF-1R protein and instructions for using the antibodies for the purposes of: binding the antibodies to the IGF-1R protein to form an immune complex and detecting the formation of the immune complex such that the presence or absence of the immune complex is correlated with the presence or absence of IGF-1R protein in the sample. Examples of the container include multi-well culture plates that allow for the simultaneous detection of IGF-1R protein in multiple samples.

In some embodiments, an antibody is provided that binds to an IGF-1R protein. In some embodiments, the antibody is isolated. In some embodiments, the antibody binds specifically. In some embodiments, the antibody binds to a suitably folded IGF-1R protein. In some embodiments, the antibody is specific to a particular IGF-1R conformational state (open or closed). In some embodiments, the antibody binds to an IGF-1R protein in the cell membrane. In some embodiments, the antibody binds to an IGF-1R protein in the cell membrane of an intact cell. In some embodiments, the antibody inhibits or neutralizes the function of the IGF-1R protein. As used herein, the term “neutralizes” means that the activity or function of the 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 percentage of inhibition may be based on the function or activity of the protein in the absence of the antibody. In some embodiments, the antibody inhibits glucose transport facilitated by IGF-1R. In some implementations, the antibody inhibits the internalization of the IGF-1R protein.

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

In some embodiments, methods are provided for treating, inhibiting, or improving IGF-1R-related pathology. In some embodiments, the methods include administering to a subject an antibody or pharmaceutical composition described herein to treat, inhibit, or improve IGF-1R-related pathology. In some embodiments, the pathology is as described herein.

In some embodiments, a method is provided for detecting the presence or absence of IGF-1R in a sample, the method comprising contacting the sample with one or more antibodies described herein to detect binding of the antibody to an IGF-1R antigen. In some embodiments, detection of binding indicates the presence of IGF-1R antigen; or the absence of detected binding to the IGF-1R antigen indicates its absence. Detection can be performed by any known method, such as using a biosensor, ELISA, sandwich assay, etc. However, in some embodiments, the method includes detecting the presence of a protein under non-denaturing conditions. Non-denaturing conditions can be used to detect the protein of interest in its native or suitably folded form.

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

In some embodiments, methods are provided for monitoring the internalization of IGF-1R from the cell surface. In some embodiments, the method includes contacting cells with an anti-IGF-1R antibody as provided herein and detecting the presence of IGF-1R in the cells 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 role of another molecule (such as a test agent) in regulating the internalization of the IGF-1R protein. Therefore, the antibodies provided herein can be used to identify test agents that regulate (increase or decrease) the internalization of the IGF-1R protein. Test molecules that increase internalization can be identified according to the methods provided herein, the increase in internalization being measured as a decrease in the binding of the anti-IGF-1R antibody to the IGF-1R protein on the cell surface. Test molecules that decrease internalization can be identified according to the methods provided herein, the decrease in internalization being measured as an increase in the binding of the anti-IGF-1R antibody to the IGF-1R protein on the cell surface. Surface expression can be measured by fluorescence, which can be performed by a secondary antibody that recognizes the IGF-1R antibody or by labeling the anti-IGF-1R antibody provided herein.

In some embodiments, a method is provided to inhibit IGF-1-stimulated receptor phosphorylation on cells. In some embodiments, the method includes contacting cells with an antibody or a pharmaceutical composition comprising the antibody, as provided herein. In some embodiments, contact includes administering the antibody or a pharmaceutical composition comprising the antibody to a subject. In some embodiments, the cells are cells of the eye. In some embodiments, the subject has thyroid eye disease (TED) or is at risk of it. In some embodiments, the antibody has an IC50 of less than or equal to about 0.2 nm, 0.15 nm, 0.10 nm, or 0.09 nm. In some embodiments, the IC50 is measured in vitro, as in the assays provided herein, as illustrated in the examples. In some embodiments, the IC50 is measured in cells, said cells being A549 cells or HOCF cells.

In some embodiments, a method of treating a subject with thyroid ophthalmopathy is provided, the method comprising administering to the subject an antibody or a pharmaceutical composition comprising thereas provided herein, wherein, at least 1, 2, or 3 weeks after administration, the serum concentration of the antibody in the subject is at least or about 70 μg/ml, 75 μg/ml, 80 μg/ml, 85 μg/ml, 90 μg/ml, 95 μg/ml, 100 μg/ml, or 105 μg/ml. In some embodiments, the serum concentration is measured after administering one, two, or three doses of the antibody or the pharmaceutical composition comprising thereas to the subject.

In some embodiments, a method is provided to inhibit IGF-1-induced receptor autophosphorylation in a subject of need by at least 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the method includes administering to the subject an antibody or a pharmaceutical composition comprising therethe as provided herein. In some embodiments, IGF-1-induced receptor autophosphorylation in the eye or orbital region of a subject is inhibited. In some embodiments, IGF-1-induced receptor autophosphorylation is inhibited, thereby treating a subject with thyroid ophthalmopathy or improving symptoms as described herein.

Listed implementation schemes

In some implementations, the implementations provided herein also include (but are not limited to):

1. An antibody or an antigen-binding fragment thereof, comprising:

VL sequences as described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 79 or 86;

The VH sequences described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 80 or 83;

LCDR sequences as described in SEQ ID NO: 17, 18, 19, 23, 24, 25, 29, 30, 31, 35, 36, 37, 41, 42, 43, 47, 48, 49, 53, 54, 55, 59, 60, 61 or 81, or

HCDR sequences as described in SEQ ID NO: 20, 21, 22, 26, 27, 28, 32, 33, 34, 38, 39, 40, 44, 45, 46, 50, 51, 52, 56, 57, 58, 62, 63 or 64; and

Any combination or variation thereof.

2. The antibody or its antigen-binding fragment as described in Embodiment 1, wherein the antibody binds to IGF-1R.

3. The antibody as described in Implementation Scheme 1, wherein the antibody is a monoclonal antibody.

4. The antibody as described in Implementation Scheme 1, wherein the antibody is a humanized antibody.

5. The antibody as described in Implementation Scheme 1, wherein the antibody is an scFv antibody.

6. The antibody as described in any one of embodiments 1 to 5, wherein the antibody or its antigen-binding fragment comprises the V _L peptide as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 79 or 86, or any variant thereof.

7. The antibody as described in any one of embodiments 1 to 6, wherein the antibody or its antigen-binding fragment comprises the V _H peptide as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 80 or 83, or any variant thereof.

8. An antibody or an antigen-binding fragment thereof, wherein the antibody or antibody fragment comprises: (i) a heavy chain variable region comprising heavy chain CDR1, CDR2 and CDR3 sequences, wherein the heavy chain CDR1 sequence has an amino acid sequence of SEQ ID NO: 20, 26, 32, 38, 44, 50 or 56; the heavy chain CDR2 has an amino acid sequence of SEQ ID NO: 21, 27, 33, 39, 45, 51 or 57; and the heavy chain CDR3 sequence has an amino acid sequence of SEQ ID NO: 22, 28, 34, 40, 46, 52 or 58. (i) or a variant thereof; and (ii) a light chain variable region comprising light chain CDR1, CDR2 and CDR3 sequences, wherein the light chain CDR1 sequence has an amino acid sequence of SEQ ID NO: 17, 23, 29, 35, 41, 47 or 53; the light chain CDR2 sequence has an amino acid sequence of SEQ ID NO: 18, 24, 30, 36, 42, 48 or 54; and the light chain CDR3 sequence has an amino acid sequence of SEQ ID NO: 19, 25, 31, 37, 43, 49, 55 or 81; or a variant thereof.

9. An antibody or an antigen-binding fragment thereof, wherein the antibody or antibody fragment comprises: (i) 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:20; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:21; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:22; or a variant thereof; and (ii) 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:17; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:18; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:19; or a variant thereof.

10. An antibody or an antigen-binding fragment thereof, wherein the antibody or antibody fragment comprises: (i) 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:26; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:27; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:28; or a variant thereof; and (ii) 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:23; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:24; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:25; or a variant thereof.

11. An antibody or an antigen-binding fragment thereof, wherein the antibody or antibody fragment comprises: (i) 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:32; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:33; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:34; or a variant thereof; and (ii) 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:29; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:30; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:31; or a variant thereof.

12. An antibody or an antigen-binding fragment thereof, wherein the antibody or antibody fragment comprises: (i) 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:38; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:39; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:40; or a variant thereof; and (ii) 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:35; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:36; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:37; or a variant thereof.

13. An antibody or an antigen-binding fragment thereof, wherein the antibody or antibody fragment comprises: (i) 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:44; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:45; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:46; or a variant thereof; and (ii) 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:41; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:42; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:43; or a variant thereof.

14. An antibody or an antigen-binding fragment thereof, wherein the antibody or antibody fragment comprises: (i) 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:50; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:51; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:52; or a variant thereof; and (ii) 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:47; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:48; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:49; or a variant thereof.

15. An antibody or an antigen-binding fragment thereof, wherein the antibody or antibody fragment comprises: (i) 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:56; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:57; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:58; or a variant thereof; and (ii) 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:53; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:54; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:55; or a variant thereof.

16. An antibody or an antigen-binding fragment thereof, wherein the antibody or antibody fragment comprises: (i) 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:62; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:63; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:64; or a variant thereof; and (ii) 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:59; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:60; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:61; or a variant thereof.

17. An antibody or an antigen-binding fragment thereof, wherein the antibody or antibody fragment comprises: (i) 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:38; the heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:39; and the heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:40; or a variant thereof; and (ii) 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:35; the light chain CDR2 sequence has the amino acid sequence of SEQ ID NO:36; and the light chain CDR3 sequence has the amino acid sequence of SEQ ID NO:81; or a variant thereof.

18. The antibody as described in any one of embodiments 6 to 17, wherein the heavy chain variable region and the light chain variable region are not connected by a linker.

19. The antibody as described in any one of embodiments 6 to 17, wherein the heavy chain variable region and the light chain variable region are linked by a peptide linker.

20. The antibody of embodiment 19, wherein the peptide linker comprises the following sequences: (GGGGS) _n (SEQ ID NO:73); (GGGGA) _n (SEQ ID NO:74), or any combination thereof, wherein each n is independently 1 to 5.

21. The antibody as described in any one of embodiments 1 to 20, wherein the antibody comprises the sequence of SEQ ID NO: 65 to 72, 78, 82 or 85, or a variant thereof.

22. The antibody as described in any one of embodiments 1 to 21, wherein the antibody comprises the V _L sequence as set forth therein: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 79 or 86, or a variant thereof.

23. The antibody as described in any one of embodiments 1 to 21, wherein the antibody comprises the V _H sequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 80 or 83, or a variant thereof.

24. The isolated antibody as described in any one of embodiments 1 to 21, wherein the antibody comprises the sequence of SEQ ID NO: 65 to 72, 78, 82 or 85, or a variant thereof.

25. The antibody as described in any one of embodiments 1 to 24, wherein the variant has 1 to 10 substitutions, deletions or insertions.

26. The antibody as described in any one of embodiments 1 to 24, wherein the variant has 1 to 10 conserved substitutions.

27. The antibody as described in any one of embodiments 1 to 26, wherein the variant has at least 85% homology with the sequence of SEQ ID NO: 1 to 72, 78 to 83 or 85 to 86.

28. The antibody as described in any one of embodiments 1 to 26, wherein the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the sequence of SEQ ID NO: 1 to 72, 78 to 83, or 85 to 86.

29. The antibody as described in any one of embodiments 1 to 26, wherein the variant has at least 85% identity with the sequence of SEQ ID NO: 1 to 72, 78 to 83 or 85 to 86.

30. The antibody as described in any one of embodiments 1 to 26, wherein the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 1 to 72, 78 to 83, or 85 to 86.

31. The antibody as described in any one of embodiments 1 to 26, wherein the antibody is an scFv antibody.

32. The antibody as described in any one of embodiments 1 to 26, wherein the antibody is a monoclonal antibody.

33. The antibody as described in any one of embodiments 1 to 26, wherein the antibody is a humanized antibody.

34. The antibody as described in any of the preceding embodiments, wherein the antibody comprises an Fc region.

35. The antibody as described in embodiment 34, wherein the Fc region is as illustrated in SEQ ID NO: 75 to 77 or 84.

36. The antibody as described in any of the preceding embodiments, wherein the Fc region contains a mutation that prolongs the half-life of the antibody when it is attached to the Fc region.

37. The antibody as described in embodiment 36, wherein the Fc region comprises S228P, L235E, M252Y, S254T, T256E, M428L, N434S, L234F, P331S mutations or any combination thereof.

38. The antibody as described in embodiment 36, wherein the Fc region contains M252Y, S254T and T256E mutations.

39. The antibody as described in embodiment 36, wherein the Fc region contains S228P and L235E mutations.

40. The antibody as described in embodiment 36, wherein the Fc region contains L234F, L235E and P331S mutations.

41. The antibody as described in embodiment 36, wherein the Fc region contains the M252Y, S254T, T256E, S228P and L235E mutations.

42. The antibody as described in embodiment 36, wherein the Fc region contains S228P, L235E, M428L and N434S mutations.

43. The antibody as described in embodiment 36, wherein the Fc region contains M428L and N434S mutations.

44. The antibody as described in embodiment 36, wherein the Fc region contains L234F, L235E, P331S, M252Y, S254T and T256E mutations.

45. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any of the preceding embodiments.

46. A carrier comprising a nucleic acid molecule as described in embodiment 45.

47. A cell comprising a nucleic acid molecule as described in embodiment 45 or a vector as described in embodiment 46.

48. A pharmaceutical composition comprising an antibody or a nucleic acid molecule encoding the antibody as described in any one of embodiments 1 to 44.

49. The pharmaceutical composition as described in embodiment 48, wherein the composition is an injectable pharmaceutical composition.

50. A method for treating thyroid-associated eye disease (TAO) or its symptoms or to reduce its severity, the method comprising administering to a subject an antibody or a pharmaceutical composition comprising any one of embodiments 1 to 44.

51. A method for reducing bulging of the eye in a subject with thyroid-associated eye disease (TAO), comprising administering to the subject an antibody or a pharmaceutical composition comprising any one of embodiments 1 to 44.

52. A method of treating a subject with thyroid ophthalmopathy, comprising administering to the subject an antibody as described in any one of embodiments 1 to 44, or a pharmaceutical composition comprising therethe.

53. A method for reducing the clinical activity score (CAS) of thyroid-associated eye disease (TAO) in a subject, comprising administering to the subject an antibody as described in any one of embodiments 1 to 44, or a pharmaceutical composition comprising the antibody.

54. A method for reducing a) bulging eye by at least 2 mm and b) clinical activity score (CAS) in a subject with thyroid-associated eye disease (TAO), comprising administering to the subject an antibody as described in any one of embodiments 1 to 44, or a pharmaceutical composition comprising the antibody.

55. The method as described in any one of embodiments 50 to 54, wherein the bulging eye is reduced by at least 2 mm.

56. The method as described in any one of embodiments 50 to 54, wherein the protruding eye is reduced by at least 3 mm.

57. The method as described in any one of embodiments 50 to 54, wherein the protruding eye is reduced by at least 4 mm.

58. The method as described in any one of embodiments 50 to 54, wherein the subject’s clinical activity score (CAS) is reduced by at least 2 points.

59. The method as described in any one of embodiments 50 to 54, wherein the subject’s clinical activity score (CAS) is reduced to -(1).

60. The method as described in any one of embodiments 50 to 54, wherein the subject’s clinical activity score (CAS) is reduced to zero (0).

61. A method of treating a subject with thyroid-associated ophthalmopathy (TAO) or reducing its severity, comprising administering to the subject an antibody as described in any one of embodiments 1 to 44, or a pharmaceutical composition comprising the antibody, wherein treatment with said antibody (i) reduces bulging of one eye by at least 2 mm; (ii) without deterioration of the other eye (or contralateral eye) by 2 mm or more; and (iii) reduces the subject's CAS to -1 or zero (0).

62. A method for improving the quality of life of a subject suffering from thyroid-associated eye disease (TAO, also known as Graves' eye disease/Graves' orbital disease), comprising administering to the subject an antibody as described in any one of embodiments 1 to 44, or a pharmaceutical composition comprising the antibody.

63. The method of embodiment 62, wherein quality of life is measured by the Graves Eye Disease Quality of Life (GO-QoL) assessment or its visual function or appearance subscale.

64. The method as described in embodiment 63, wherein the treatment results in an improvement in GO-QoL of 8 points or more.

65. The method of embodiment 63, wherein the treatment results in improvement of the functional subscales of GO-QoL.

66. The method of embodiment 63, wherein the treatment improves the appearance subscale of GO-QoL.

67. A method for treating diplopia or reducing the severity of diplopia in a subject suffering from thyroid-associated eye disease (TAO), comprising administering to the subject an antibody as described in any one of embodiments 1 to 44, or a pharmaceutical composition comprising the antibody.

68. The method as described in implementation scheme 67, wherein the superposition is constant superposition.

69. The method as described in implementation scheme 67, wherein the superposition is non-constant superposition.

70. The method as described in implementation scheme 67, wherein diplopia is intermittent diplopia.

71. The method as described in embodiment 67, wherein the improvement in diplopia or the reduction in its severity persists for at least 20 weeks after the cessation of antibody administration.

72. The method as described in embodiment 67, wherein the improvement in diplopia or the reduction in its severity persists for at least 50 weeks after the cessation of antibody administration.

73. The method of any one of embodiments 50 to 72, wherein the antibody is administered as a first dose at a dose of about 1 mg/kg to about 5 mg/kg of antibody.

74. The method of any one of embodiments 50 to 72, wherein the antibody is administered as a first dose at a dose of about 5 mg/kg to about 10 mg/kg of antibody.

75. The method of any one of embodiments 50 to 72, wherein the antibody is administered as a subsequent dose at a dose of about 5 mg/kg to about 20 mg/kg of antibody.

76. The method of any one of embodiments 50 to 72, wherein the antibody is administered in the following amounts: about 10 mg/kg antibody as a first dose; and about 20 mg/kg antibody as a subsequent dose.

77. The method of embodiment 76, wherein the subsequent dose is administered every three weeks for at least 21 weeks.

78. The method as described in any one of embodiments 50 to 77, wherein the antibody or its antigen-binding fragment is a human antibody, a monoclonal antibody, a human monoclonal antibody, a purified antibody, a bispecific antibody, a single-chain antibody, a multispecific antibody, Fab, Fab', F(ab')2, Fv or scFv.

79. The method of any one of embodiments 50 to 78, wherein the antibody or its antigen-binding fragment is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable diluent or excipient or carrier.

80. The method of embodiment 79, wherein the pharmaceutical composition further comprises one or more pharmaceutically active compounds for treating TAO.

81. The method of embodiment 79 or 80, wherein the pharmaceutical composition further comprises a corticosteroid; rituximab or other anti-CD20 antibody; tocilizumab or other anti-IL-6 antibody; or selenium, infliximab or other anti-TNFα antibody or thyroid-stimulating hormone receptor (TSHR) inhibitor.

82. The method as described in any of the preceding embodiments, wherein the antibody or its antigen-binding fragment may be directly applied to the eye, the anterior chamber of the eye, the vitreous chamber of the eye, the suprachoroidal space, or the posterior orbital sinus.

83. The method of embodiment 82, wherein the antibody or its antigen-binding fragment is administered by injection.

84. The method as described in embodiment 83, wherein the injection is an intravitreal injection, an intraorbital injection, a retroorbital injection, a suprachoroidal injection, or an intra-anterior chamber injection.

85. A method for increasing IGF-1R internalization on cells, the method comprising contacting the cells with an antibody or a pharmaceutical composition comprising any one of embodiments 1 to 44.

86. The method of embodiment 85, wherein contact comprises administering to a subject an antibody or a pharmaceutical composition comprising any one of embodiments 1 to 44.

87. The method as described in implementation scheme 86, wherein the subject has thyroid eye disease (TED) or is at risk of it.

88. A method for inhibiting IGF-1-stimulated receptor phosphorylation on cells, the method comprising contacting the cells with an antibody or a pharmaceutical composition comprising any one of embodiments 1 to 44.

89. The method of embodiment 88, wherein contact comprises administering to a subject an antibody or a pharmaceutical composition comprising any one of embodiments 1 to 44.

90. The method as described in implementation scheme 89, wherein the subject has thyroid eye disease (TED) or is at risk of it.

91. The method as described in any one of embodiments 88 to 90, wherein the antibody has an IC50 of less than or equal to about 0.2 nm, 0.15 nm, 0.10 nm, or 0.09 nm.

92. The method of embodiment 91, wherein IC50 is measured in vitro, as provided herein.

93. The method as described in any one of embodiments 88 to 92, wherein the cells are A549 cells or HOCF cells.

94. A method of treating a subject with thyroid ophthalmopathy, the method comprising administering to the subject an antibody or a pharmaceutical composition comprising the antibody as described in any one of embodiments 1 to 44 or as provided elsewhere herein, wherein, at least 1, 2, or 3 weeks after administration, the serum concentration of the antibody in the subject is at least or about 70 μg/ml, 75 μg/ml, 80 μg/ml, 85 μg/ml, 90 μg/ml, 95 μg/ml, 100 μg/ml, or 105 μg/ml.

95. The method as described in embodiment 94, wherein the antibody or pharmaceutical composition is administered intravenously.

96. The method as described in embodiment 94 or 96, wherein the antibody or pharmaceutical composition is administered at a dose of about 20 mg/kg.

97. The method as described in any one of embodiments 94 to 96, wherein the antibody or pharmaceutical composition is administered at least once a week, once every two weeks, once every three weeks, or once every four weeks.

98. A method for inhibiting IGF-1-induced receptor autophosphorylation in cells by at least 95%, 96%, 97%, 98%, 99%, or 100%, said method comprising contacting the cells with an antibody or a pharmaceutical composition comprising the antibody as described in any one of embodiments 1 to 44 or as provided elsewhere herein.

99. The method of embodiment 98, wherein the inhibition of IGF-1-induced receptor autophosphorylation is measured relative to receptor autophosphorylation induced in the absence of the antibody or pharmaceutical composition.

100. The method as described in embodiment 98 or 99, wherein contact comprises administering an antibody or a pharmaceutical composition containing the antibody to a subject.

101. The method as described in implementation scheme 100, wherein the subject has thyroid eye disease (TED) or is at risk of it.

102. A method for inhibiting IGF-1-induced receptor autophosphorylation in a subject of need by at least 95%, 96%, 97%, 98%, 99%, or 100%, said method comprising administering to the subject an antibody or a pharmaceutical composition comprising thereunder as described in any one of embodiments 1 to 44 or as provided elsewhere herein.

103. The method as described in implementation scheme 102, wherein the subject has thyroid eye disease (TED) or is at risk of it.

104. The method as described in any one of embodiments 102 or 103, wherein the antibody or pharmaceutical composition is administered intravenously.

105. The method of any one of embodiments 98 to 104, wherein the antibody comprises a CDR of VRDN-1100.

106. The method as described in any one of embodiments 98 to 104, wherein the antibody comprises a CDR of an antibody containing VRDN-1100 or a CDR of an antibody containing VRDN-2700.

107. An isolated antibody comprising a light chain having the amino acid sequence of SEQ ID NO:3 and a heavy chain having the amino acid sequence of SEQ ID NO:83.

108. An isolated antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO:13 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:14.

109. The isolated antibody as described in embodiment 108, wherein the antibody comprises a light chain having the amino acid sequence of SEQ ID NO:93 and a heavy chain amino acid sequence of SEQ ID NO:92.

110. The isolated antibody as described in embodiment 108, wherein the antibody comprises a light chain having the amino acid sequence of SEQ ID NO:93 and a heavy chain amino acid sequence of SEQ ID NO:94.

111. The isolated antibody as described in embodiment 108, wherein the antibody comprises a light chain having the amino acid sequence of SEQ ID NO:93 and a heavy chain amino acid sequence of SEQ ID NO:95.

112. A pharmaceutical composition comprising an antibody as described in any one of embodiments 107 to 111.

113. A pharmaceutical composition suitable for intravenous administration, comprising an antibody as described in any one of embodiments 107 to 111.

114. A pharmaceutical composition suitable for subcutaneous administration, comprising an antibody as described in any one of embodiments 107 to 111.

115. A method of treating a subject with thyroid ophthalmopathy, the method comprising administering a pharmaceutical composition comprising an antibody as described in any one of embodiments 107 to 111.

116. The method as described in embodiment 115, wherein the pharmaceutical composition is administered intravenously.

117. The method of embodiment 115, wherein the pharmaceutical composition is administered subcutaneously.

118. A method for treating thyroid-associated eye disease (TAO) or its symptoms or to reduce its severity, the method comprising administering to a subject an antibody or a pharmaceutical composition comprising any one of embodiments 107 to 111.

119. A method for reducing bulging of the eye in a subject with thyroid-associated eye disease (TAO), comprising administering to the subject an antibody or a pharmaceutical composition comprising any one of embodiments 107 to 111.

120. A method of treating a subject with thyroid ophthalmopathy, comprising administering to the subject an antibody or a pharmaceutical composition comprising any one of embodiments 107 to 111.

121. A method for reducing the clinical activity score (CAS) of thyroid-associated eye disease (TAO) in a subject, comprising administering to the subject an antibody as described in any one of embodiments 107 to 111, or a pharmaceutical composition comprising the antibody.

122. A method for reducing a) bulging eye by at least 2 mm and b) clinical activity score (CAS) in a subject with thyroid-associated eye disease (TAO), comprising administering to the subject an antibody as described in any one of embodiments 107 to 111, or a pharmaceutical composition comprising the antibody.

123. The method as described in any one of embodiments 118 to 122, wherein the protruding eye is reduced by at least 2 mm.

124. The method as described in any one of embodiments 118 to 122, wherein the protruding eye is reduced by at least 3 mm.

125. The method as described in any one of embodiments 118 to 122, wherein the bulging eye is reduced by at least 4 mm.

126. The method as described in any one of embodiments 118 to 122, wherein the subject’s clinical activity score (CAS) is reduced by at least 2 points.

127. The method as described in any one of embodiments 118 to 122, wherein the subject’s clinical activity score (CAS) is reduced to -(1).

128. The method as described in any one of embodiments 118 to 122, wherein the subject’s clinical activity score (CAS) is reduced to zero (0).

129. A method of treating a subject with thyroid-associated ophthalmopathy (TAO) or reducing its severity, comprising administering to the subject an antibody as described in any one of embodiments 107 to 111, or a pharmaceutical composition comprising the antibody, wherein treatment with the antibody (i) reduces bulging of one eye by at least 2 mm; (ii) without deterioration of the other eye (or contralateral eye) by 2 mm or more; and (iii) reduces the subject's CAS to -1 or zero (0).

130. A method for improving the quality of life of a subject suffering from thyroid-associated eye disease (TAO, also known as Graves' eye disease/Graves' orbital disease), comprising administering to the subject an antibody as described in any one of embodiments 107 to 111, or a pharmaceutical composition comprising the antibody.

131. The method of embodiment 130, wherein quality of life is measured by the Graves Eye Disease Quality of Life (GO-QoL) assessment or its visual function or appearance subscale.

132. The method as described in embodiment 130, wherein the treatment results in an improvement in GO-QoL of 8 points or more.

133. The method of embodiment 130, wherein the treatment results in improvement of the functional subscales of GO-QoL.

134. The method of embodiment 130, wherein the treatment improves the appearance subscale of GO-QoL.

135. A method for treating diplopia or reducing the severity of diplopia in a subject suffering from thyroid-associated eye disease (TAO), comprising administering to the subject an antibody as described in any one of embodiments 107 to 111, or a pharmaceutical composition comprising the antibody.

136. The method as described in embodiment 135, wherein the superposition is constant superposition.

137. The method as described in embodiment 135, wherein the superposition is non-constant superposition.

138. The method as described in implementation scheme 135, wherein diplopia is intermittent diplopia.

139. The method as described in embodiment 135, wherein the improvement in diplopia or the reduction in its severity persists for at least 20 weeks after the cessation of antibody administration.

140. The method as described in embodiment 135, wherein the improvement in diplopia or the reduction in its severity persists for at least 50 weeks after the cessation of antibody administration.

141. The method of any one of embodiments 115 to 140, wherein the antibody is administered as a first dose at a dose of about 1 mg/kg to about 5 mg/kg of antibody.

142. The method of any one of embodiments 115 to 140, wherein the antibody is administered as a first dose at a dose of about 5 mg/kg to about 10 mg/kg of antibody.

143. The method of any one of embodiments 115 to 140, wherein the antibody is administered as a subsequent dose at a dose of about 5 mg/kg to about 20 mg/kg of antibody.

144. The method of any one of embodiments 115 to 140, wherein the antibody is administered in the following amounts: about 10 mg/kg antibody as a first dose; and about 20 mg/kg antibody as a subsequent dose.

145. The method of embodiment 144, wherein the subsequent dose is administered every three weeks for at least 21 weeks.

146. The method of any one of embodiments 115 to 140, wherein the antibody is administered in the form of a pharmaceutical composition comprising a pharmaceutically acceptable diluent, excipient, or carrier.

147. The method of embodiment 146, wherein the pharmaceutical composition further comprises one or more pharmaceutically active compounds for treating TAO.

148. The method of embodiment 146 or 147, wherein the pharmaceutical composition further comprises a corticosteroid; rituximab or other anti-CD20 antibody; tocilizumab or other anti-IL-6 antibody; or selenium, infliximab or other anti-TNFα antibody or thyroid-stimulating hormone receptor (TSHR) inhibitor.

149. A method for increasing IGF-1R internalization on cells, the method comprising contacting the cells with an antibody or a pharmaceutical composition comprising any one of embodiments 107 to 111.

150. The method of embodiment 149, wherein contact comprises administering an antibody or a pharmaceutical composition containing the antibody to a subject.

151. The method as described in implementation scheme 150, wherein the subject has thyroid eye disease (TED) or is at risk of it.

152. A method for inhibiting IGF-1-stimulated receptor phosphorylation on cells, the method comprising contacting the cells with an antibody or a pharmaceutical composition comprising any one of embodiments 107 to 111.

153. The method of embodiment 152, wherein contact comprises administering to a subject an antibody or a pharmaceutical composition comprising any one of embodiments 1 to 44.

154. The method as described in implementation scheme 153, wherein the subject has thyroid eye disease (TED) or is at risk of it.

155. The method as described in embodiment 153 or 154, wherein the antibody has an IC50 of less than or equal to about 0.2 nm, 0.15 nm, 0.10 nm, or 0.09 nm.

156. The method of embodiment 155, wherein IC50 is measured in vitro, as in the assays provided herein.

157. The method as described in any one of embodiments 152 to 157, wherein the cells are A549 cells or HOCF cells.

158. A method of treating a subject with thyroid ophthalmopathy, the method comprising administering to the subject an antibody or a pharmaceutical composition comprising the antibody as described in any one of embodiments 107 to 111, wherein, at least 1, 2, or 3 weeks after administration, the serum concentration of the antibody in the subject is at least or about 70 μg/ml, 75 μg/ml, 80 μg/ml, 85 μg/ml, 90 μg/ml, 95 μg/ml, 100 μg/ml, or 105 μg/ml.

159. The method of embodiment 158, wherein the antibody or pharmaceutical composition is administered intravenously.

160. The method as described in embodiments 158 or 159, wherein the antibody or pharmaceutical composition is administered as a first dose or subsequent dose at a dose of about 1 mg/kg to about 5 mg/kg (mg antibody/kg subject), about 5 mg/kg to about 10 mg/kg antibody, or about 5 mg/kg to about 20 mg/kg.

161. The method of any one of embodiments 158 to 160, wherein the antibody is administered in the following amounts: about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg antibody as a first dose; and about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg/kg antibody as subsequent doses.

162. The method as described in any one of embodiments 158 to 161, wherein the antibody or pharmaceutical composition is administered at least once a week, once every two weeks, once every three weeks, or once every four weeks.

163. A method for inhibiting IGF-1-induced receptor autophosphorylation in a subject of need by at least 95%, 96%, 97%, 98%, 99%, or 100%, said method comprising administering to the subject an antibody or a pharmaceutical composition comprising any one of embodiments 107 to 111.

164. A pharmaceutical composition comprising an antibody for treating a subject with thyroid ophthalmopathy, wherein the antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO:13 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:14.

165. The pharmaceutical composition of embodiment 164, wherein the antibody comprises an Fc region having M428L and N434S substitutions.

166. The pharmaceutical composition of embodiment 164, wherein the antibody comprises an Fc region having M428L, N434S, M252Y, S254T and T256E substitutions.

167. The pharmaceutical composition of embodiment 164, wherein the antibody comprises an Fc region having M252Y, S254T and T256E substitutions.

168. The pharmaceutical composition of embodiment 164, wherein the antibody comprises a light chain having the amino acid sequence of SEQ ID NO:93 and a heavy chain having the amino acid sequence of SEQ ID NO:92.

169. The pharmaceutical composition of embodiment 164, wherein the antibody comprises a light chain having the amino acid sequence of SEQ ID NO:93 and a heavy chain amino acid sequence of SEQ ID NO:94.

170. The pharmaceutical composition of embodiment 164, wherein the antibody comprises a light chain having the amino acid sequence of SEQ ID NO:93 and a heavy chain amino acid sequence of SEQ ID NO:95.

171. A method of treating a subject with thyroid ophthalmopathy, the method comprising administering an antibody-containing pharmaceutical composition as described in any one of embodiments 164 to 170.

172. The method of embodiment 171, wherein the pharmaceutical composition is administered intravenously.

173. The method of embodiment 171, wherein the pharmaceutical composition is administered subcutaneously.

174. A method for treating a subject with thyroid-associated eye disease (TAO) or its symptoms or to reduce its severity, said method comprising administering to the subject a pharmaceutical composition as described in any one of embodiments 164 to 170.

175. A method for reducing bulging of the eye in a subject suffering from thyroid-associated eye disease (TAO), the method comprising administering to the subject a pharmaceutical composition as described in any one of embodiments 164 to 170.

176. A method of treating a subject with thyroid ophthalmopathy, the method comprising administering to the subject a pharmaceutical composition as described in any one of embodiments 2 to 4.

177. A method for reducing the clinical activity score (CAS) of thyroid-associated eye disease (TAO) in a subject, the method comprising administering to the subject a pharmaceutical composition as described in any one of embodiments 164 to 170.

178. A method for reducing a) bulging eye by at least 2 mm and b) lowering the Clinical Activity Score (CAS) of a subject suffering from thyroid-associated eye disease (TAO), the method comprising administering to the subject a pharmaceutical composition as described in any one of embodiments 164 to 170.

179. The method as described in any one of embodiments 174 to 178, wherein the protruding eye is reduced by at least 2 mm.

180. The method as described in any one of embodiments 174 to 178, wherein the protruding eye is reduced by at least 3 mm.

181. The method as described in any one of embodiments 174 to 178, wherein the bulging eye is reduced by at least 4 mm.

182. The method as described in any one of embodiments 174 to 178, wherein the subject’s clinical activity score (CAS) is reduced by at least 2 points.

183. The method as described in any one of embodiments 174 to 178, wherein the subject’s clinical activity score (CAS) is reduced to -(1).

184. The method as described in any one of embodiments 174 to 178, wherein the subject’s clinical activity score (CAS) is reduced to zero (0).

185. A method of treating a subject with thyroid-associated ophthalmopathy (TAO) or reducing its severity, comprising administering to the subject a pharmaceutical composition as described in any one of embodiments 164 to 170, wherein treatment with the antibody (i) reduces bulging of one eye by at least 2 mm; (ii) without deterioration of the other eye (or contralateral eye) by 2 mm or more; and (iii) reduces the subject's CAS to -1 or zero (0).

186. A method for improving the quality of life of a subject suffering from thyroid-associated eye disease (TAO, also known as Graves' eye disease), the method comprising administering to the subject a pharmaceutical composition as described in any one of embodiments 164 to 170.

187. The method of embodiment 186, wherein quality of life is measured by the Graves Eye Disease Quality of Life (GO-QoL) assessment or its visual function or appearance subscale.

188. The method as described in embodiment 186, wherein the treatment results in an improvement in GO-QoL of 8 points or more.

189. The method of embodiment 186, wherein the treatment results in improvement of the functional subscales of GO-QoL.

190. The method as described in embodiment 186, wherein the treatment results in an improvement in the appearance subscale of GO-QoL.

191. A method for treating diplopia or reducing the severity of thyroid-associated eye disease (TAO) in a subject, the method comprising administering to the subject a pharmaceutical composition as described in any one of embodiments 164 to 170.

192. The method as described in embodiment 191, wherein the superposition is constant superposition.

193. The method as described in embodiment 191, wherein the superposition is non-constant superposition.

194. The method as described in embodiment 191, wherein diplopia is intermittent diplopia.

195. The method as described in embodiment 191, wherein the improvement in diplopia or the reduction in its severity persists for at least 20 weeks after the cessation of antibody administration.

196. The method as described in embodiment 191, wherein the improvement in diplopia or the reduction in its severity persists for at least 50 weeks after the cessation of antibody administration.

197. The method of any one of embodiments 171 to 196, wherein the pharmaceutical composition is administered as a first dose at a dose of about 1 mg/kg to about 5 mg/kg, about 5 mg/kg to about 10 mg/kg, about 10 mg/kg to about 20 mg/kg, about 20 mg/kg to about 30 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, or about 30 mg/kg of antibody.

198. The method of any one of embodiments 171 to 196, wherein the pharmaceutical composition is administered as a first dose at a dose of about 10 mg/kg to about 20 mg/kg of antibody.

199. The method of any one of embodiments 171 to 196, wherein the pharmaceutical composition is administered as a subsequent dose at a dose of about 1 mg/kg to about 10 mg/kg, about 2 mg/kg to about 5 mg/kg, or about 5 mg/kg to about 20 mg/kg of antibody.

200. The method of any one of embodiments 171 to 196, wherein the pharmaceutical composition is administered in the following amounts: about 10 mg/kg antibody as a first dose; and about 20 mg/kg antibody as a subsequent dose.

201. The method of embodiment 200, wherein the subsequent doses are administered every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight weeks for at least 21 to 52 weeks or longer.

202. A method for increasing IGF-1R internalization on cells, the method comprising contacting the cells with a pharmaceutical composition as described in any one of embodiments 164 to 170.

203. The method of embodiment 202, wherein contact comprises administering to a subject the pharmaceutical composition of any one of embodiments 164 to 170.

204. The method as described in embodiment 203, wherein the subject has thyroid eye disease (TED) or is at risk of it.

205. A method for inhibiting IGF-1-stimulated receptor phosphorylation on cells, the method comprising contacting the cells with a pharmaceutical composition as described in any one of embodiments 164 to 170.

206. The method of embodiment 205, wherein contact comprises administering to a subject the pharmaceutical composition of any one of embodiments 164 to 170.

207. The method as described in embodiment 206, wherein the subject has thyroid eye disease (TED) or is at risk of it.

208. The method of any one of embodiments 205 to 207, wherein the antibody has an IC50 of less than or equal to about 0.2 nm, 0.15 nm, 0.10 nm, or 0.09 nm.

209. A method of treating a subject with thyroid ophthalmopathy, the method comprising administering to the subject a pharmaceutical composition as described in any one of embodiments 164 to 170, wherein, at least 1, 2, or 3 weeks after administration, the serum concentration of the antibody in the subject is at least or about 10 μg/ml or 20 μg/ml or 50 μg/ml, 70 μg/ml, 75 μg/ml, 80 μg/ml, 85 μg/ml, 90 μg/ml, 95 μg/ml, 100 μg/ml, or 105 μg/ml.

210. The method of embodiment 209, wherein the pharmaceutical composition is administered intravenously or subcutaneously.

211. An isolated antibody comprising a light chain having the amino acid sequence of SEQ ID NO:3 and a heavy chain having the amino acid sequence of SEQ ID NO:83.

212. An isolated antibody comprising a variable light chain comprising the sequence of SEQ ID NO:98 and a variable heavy chain comprising the sequence of SEQ ID NO:99, and an Fc region comprising the mutations of M252Y, S254T and T256E.

213. An isolated antibody comprising a variable light chain comprising the sequence of SEQ ID NO:98 and a variable heavy chain comprising the sequence of SEQ ID NO:99, and an Fc region comprising M428L and N434S mutations.

214. A pharmaceutical composition comprising an antibody as described in any one of embodiments 211 to 213.

215. A pharmaceutical composition suitable for intravenous administration, comprising an antibody as described in any one of embodiments 211 to 213.

216. A pharmaceutical composition suitable for subcutaneous administration, comprising an antibody as described in any one of embodiments 211 to 213.

217. A method of treating a subject with thyroid ophthalmopathy, the method comprising administering a pharmaceutical composition comprising an antibody as described in any one of embodiments 211 to 213.

218. The method of embodiment 217, wherein the pharmaceutical composition is administered intravenously.

219. The method of embodiment 217, wherein the pharmaceutical composition is administered subcutaneously.

220. A method for treating thyroid-associated eye disease (TAO) or its symptoms or to reduce its severity, the method comprising administering to a subject an antibody or a pharmaceutical composition comprising any one of embodiments 211 to 213.

221. A method for reducing bulging of the eye in a subject with thyroid-associated eye disease (TAO), comprising administering to the subject an antibody or a pharmaceutical composition comprising any one of embodiments 211 to 213.

222. A method of treating a subject with thyroid ophthalmopathy, comprising administering to the subject an antibody or a pharmaceutical composition comprising any one of embodiments 211 to 213.

223. A method for reducing the clinical activity score (CAS) of thyroid-associated eye disease (TAO) in a subject, comprising administering to the subject an antibody as described in any one of embodiments 211 to 213, or a pharmaceutical composition comprising the antibody.

224. A method for reducing a) bulging eye by at least 2 mm and b) clinical activity score (CAS) in a subject with thyroid-associated eye disease (TAO), comprising administering to the subject an antibody as described in any one of embodiments 211 to 213, or a pharmaceutical composition comprising the antibody.

225. The method as described in any one of embodiments 220 to 224, wherein the protruding eye is reduced by at least 2 mm.

226. The method as described in any one of embodiments 220 to 224, wherein the protruding eye is reduced by at least 3 mm.

227. The method as described in any one of embodiments 220 to 224, wherein the bulging eye is reduced by at least 4 mm.

228. The method as described in any one of embodiments 220 to 224, wherein the subject’s clinical activity score (CAS) is reduced by at least 2 points.

229. The method as described in any one of embodiments 220 to 224, wherein the subject’s clinical activity score (CAS) is reduced to -(1).

230. The method as described in any one of embodiments 220 to 224, wherein the subject’s clinical activity score (CAS) is reduced to zero (0).

231. A method of treating a subject with thyroid-associated ophthalmopathy (TAO) or reducing its severity, comprising administering to the subject an antibody as described in any one of embodiments 1 to 3, or a pharmaceutical composition comprising the antibody, wherein treatment with said antibody (i) reduces bulging of one eye by at least 2 mm; (ii) without deterioration of the other eye (or contralateral eye) by 2 mm or more; and (iii) reduces the subject's CAS to -1 or zero (0).

232. A method for improving the quality of life of a subject suffering from thyroid-associated eye disease (TAO, also known as Graves' eye disease/Graves' orbital disease), comprising administering to the subject an antibody as described in any one of embodiments 211 to 213, or a pharmaceutical composition comprising the antibody.

233. The method as described in embodiment 232, wherein quality of life is measured by the Graves Eye Disease Quality of Life (GO-QoL) assessment or its visual function or appearance subscale.

234. The method as described in embodiment 232, wherein the treatment results in an improvement in GO-QoL of 8 points or more.

235. The method of embodiment 232, wherein the treatment results in improvement of the functional subscales of GO-QoL.

236. The method of embodiment 232, wherein the treatment improves the appearance subscale of GO-QoL.

237. A method for treating diplopia or reducing the severity of thyroid-associated eye disease (TAO) in a subject, comprising administering to the subject an antibody as described in any one of embodiments 211 to 213, or a pharmaceutical composition comprising the antibody.

238. The method as described in implementation scheme 237, wherein the superposition is constant superposition.

239. The method as described in implementation scheme 237, wherein the superposition is non-constant superposition.

240. The method as described in embodiment 237, wherein diplopia is intermittent diplopia.

241. The method as described in embodiment 237, wherein the improvement in diplopia or the reduction in its severity persists for at least 20 weeks after the cessation of antibody administration.

242. The method as described in embodiment 237, wherein the improvement in diplopia or the reduction in its severity persists for at least 50 weeks after the cessation of antibody administration.

243. The method of any one of embodiments 217 to 242, wherein the antibody is administered as a first dose at a dose of about 1 mg/kg to about 5 mg/kg, about 5 mg/kg to about 10 mg/kg, about 10 mg/kg to about 20 mg/kg, about 20 mg/kg to about 30 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, or about 30 mg/kg of antibody.

244. The method of any one of embodiments 217 to 242, wherein the antibody is administered as a first dose at a dose of about 10 mg/kg to about 20 mg/kg of antibody.

245. The method of any one of embodiments 217 to 242, wherein the antibody is administered as a subsequent dose at a dose of about 1 mg/kg to about 10 mg/kg, about 2 mg/kg to about 5 mg/kg, or about 5 mg/kg to about 20 mg/kg of antibody.

246. The method of any one of embodiments 217 to 242, wherein the antibody is administered in the following amounts: about 10 mg/kg antibody as a first dose; and about 20 mg/kg antibody as a subsequent dose.

247. The method of embodiment 246, wherein the subsequent doses are administered every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight weeks for at least 21 to 52 weeks or longer.

248. A method for increasing IGF-1R internalization on cells, the method comprising contacting the cells with an antibody or a pharmaceutical composition comprising any one of embodiments 211 to 213.

249. The method of embodiment 248, wherein contact comprises administering an antibody or a pharmaceutical composition comprising the antibody to a subject.

250. The method as described in implementation scheme 249, wherein the subject has thyroid eye disease (TED) or is at risk of it.

251. A method for inhibiting IGF-1-stimulated receptor phosphorylation on cells, the method comprising contacting the cells with an antibody or a pharmaceutical composition comprising any one of embodiments 211 to 213.

252. The method of embodiment 251, wherein contact comprises administering to a subject an antibody or a pharmaceutical composition comprising any one of embodiments 211 to 213.

253. The method as described in implementation scheme 252, wherein the subject has thyroid eye disease (TED) or is at risk of it.

254. The method as described in any one of embodiments 251 to 253, wherein the antibody has an IC50 of less than or equal to about 0.2 nm, 0.15 nm, 0.10 nm, or 0.09 nm.

255. A method of treating a subject with thyroid ophthalmopathy, the method comprising administering to the subject an antibody or a pharmaceutical composition comprising any one of embodiments 211 to 213, wherein, at least 1, 2, or 3 weeks after administration, the serum concentration of the antibody in the subject is at least or about 10 μg/ml or 20 μg/ml or 50 μg/ml, 70 μg/ml, 75 μg/ml, 80 μg/ml, 85 μg/ml, 90 μg/ml, 95 μg/ml, 100 μg/ml, or 105 μg/ml.

256. The method of embodiment 255, wherein the antibody or the pharmaceutical composition is administered intravenously or subcutaneously.

The subject matter is now described with reference to the following embodiments. These embodiments are provided for illustrative purposes only, and the claims should in no way be construed as limiting to these embodiments, but rather as covering any and all variations that become apparent from the teachings provided herein. Those skilled in the art will readily recognize that a variety of non-critical parameters can be changed or modified to produce substantially similar results.

Example

Example 1: IGF-1R antibody blocks IGF-1 stimulation.

Blockage of IGF-1 stimulation was measured by hyaluronic acid secretion in the presence of IGF-1R antibodies VRDN-2700, VRDN-03100, VRDN-02100, VRDN-02200, VRDN-02300, VRDN-02400, VRDN-02500, VRDN-01100, VRDN-02600, and VRDN-02301, all of which are disclosed herein. Immunoglobulins were purified from serum of patients with Graves' eye disease (GO) and their ability to directly activate TSHR and/or IGF-1R, as well as TSHR/IGF-1R crosstalk in primary cultures of GO fibroblasts, was tested. Cells were treated with M22 or GO-Ig antibodies, with or without IGF-1R inhibitory antibodies (such as those provided herein), including (but not limited to) VRDN-2700, VRDN-03100, VRDN-02100, VRDN-02200, VRDN-02300, VRDN-02400, VRDN-02500, VRDN-01100, VRDN-02600, and VRDN-02301, all disclosed herein. Hyaluronic acid (HA) secretion is a major biological response to GO fibroblast stimulation. IGF-1R autophosphorylation was used as a measure of direct IGF-1R activation. TSHR activation was determined by the production of cyclic AMP (cAMP). IGF-1R antibodies as disclosed herein were found to effectively block HA secretion, thus blocking IGF stimulation.

Example 2: Treatment of patients with thyroid eye disease and clinical evaluation of IGF-1R antibody in thyroid eye disease.

Subjects were administered infusions of IGF-1R inhibitory antibodies (such as those described herein), including (but not limited to) VRDN-2700, VRDN-03100, VRDN-02100, VRDN-02200, VRDN-02300, VRDN-02400, VRDN-02500, VRDN-01100, VRDN-02600, and VRDN-02301, all disclosed herein. The number of infusions was individualized for each subject and based on investigator clinical judgment. Day 1 follow-up occurred within 14 days of the final follow-up of the previous trial. The follow-up window was ±1 day for weeks 1 and 4, and ±3 days for weeks 3, 6, 9, 12, 15, 18, 21, and 24. Follow-up was only for subjects who were non-responsive to exophthalmos in the previous trial; subjects who relapsed in the previous trial were excluded from the follow-up period. The follow-up period is ±7 days.

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

Subjects who were nonresponsive to bulging eyes were assigned to participate in the 6-month follow-up period of this extended study; subjects who relapsed in the introductory study and were retreated in this extended study were not included in the follow-up period.

Efficacy assessments were performed on both eyes at each assessment time point. The “study eye” (i.e., the more severely affected eye) was kept the same as the eye identified at baseline (Day 1) of the previous study. Efficacy was assessed on both eyes, but the primary outcome measure was assessed using the study eye.

Efficacy was assessed using exophthalmos (measured using Hertel instruments as an assessment of exophthalmos as a measure of clinical severity to ensure consistency of measurement), CAS (7-item scale), diplopia (measured as a portion of the clinical severity measure), and clinical severity measures (including assessment of motor limitation).

Use the GO-QoL questionnaire to assess quality of life.

Safety was assessed through monitoring of adverse events (AEs) and concomitant drug use, immunogenicity testing, physical and ophthalmological examinations, vital signs, clinical safety laboratory assessments (complete blood count, chemistry (including thyroid examination and HbA1C), and urinalysis), pregnancy testing (if applicable), and electrocardiogram (ECG). The study was also monitored by the Data Safety Monitoring Board (DSMB).

To ensure consistency of measurements, the Hertel ectropion meter was used for assessments, and the same Hertel instrument and the same observer were used for each assessment throughout the full duration of the study (unless unavoidable). Additionally, the same intercanthal distance (ICD) was used in all cases.

Proptosis was measured in each eye on day 1 and at weeks 6, 12, 18, and 24 (or premature discontinuation (PW)) during treatment and at months 7, 9, and 12 (or PW) during follow-up. Measurements of the clinical severity of proptosis, eCRF, were recorded.

As presented in this article, antibodies have been found to be effective in treating thyroid eye disease and improving quality of life.

Example 3: Antibodies that increase pK

Crab-eating macaques were administered an antibody at a dose of 10 mg/kg containing the CDR of VRDN-2700 with a YTE mutation in the Fc domain. Samples were collected at 0.5 h, 2 h, 8 h and on days 1, 3, 7, 10, 14, 21, and 28 for pharmacokinetic analysis by ELISA. Tiltumumab was also administered IV at 10 mg/kg as a comparator. The results shown in Figure 1 indicate that the antibody had a significantly higher pharmacokinetic profile compared to tiltumumab.

This result suggests that antibodies containing a CDR of VRDN-2700 may be administered at lower doses, even when administered subcutaneously, compared to tetumumab. These results were not previously predicted.

Example 4:

VRDN-1100 is an antibody antagonist targeting the insulin-like growth factor-1 receptor (IGF-1R) that is under development for the treatment of thyroid ophthalmopathy (TED). TED is driven by an autoantibody that stimulates the thyroid-stimulating hormone receptor (TSHR) and crosstalk between TSHR and IGF-1R. TED is characterized by the recruitment of fibroblasts expressing both IGF-1R and TSHR into the orbital tissue, where fibroblasts mediate hyaluronic acid deposition and the expansion of orbital muscle and fat.¹ IGF-1R antagonism was found to reverse this orbital tissue expansion and stably alleviate symptoms in TED patients.²

VRDN-1100 is a humanized monoclonal antibody targeting IGF-1R. The IGF-1R binding and antagonist characteristics of VRDN-1100 were analyzed.

method

Surface plasmon resonance (SPR): Anti-Fc capture antibody was immobilized, and the recombinant IGF-1R extracellular domain (ECD) was used as the analyte. The association and dissociation rate constants (ka and kd, respectively), as well as the equilibrium dissociation constant KD, were derived by global fitting of the data with a single-site model.

Epitope binning: VRDN-1100 is immobilized on the chip surface via amine coupling and used to capture IGF-1R-ECD, after which tetamumab is allowed to flow on the chip.

Cell binding: A549 human lung adenocarcinoma cells or primary human ocular choroidal fibroblasts (HOCF) were co-cultured with different concentrations of VRDN-1100 or tetumumab. A single-dose 50 nM IgG1 isotype control was used as a negative control. Unbound antibodies were removed by washing, and cells were then co-cultured with Alexa Fluor 488-goat anti-human antibody and cell-impermeable dye to gate live cells. Median fluorescence intensity (MFI) of live cells was measured by flow cytometry, and data were analyzed using FlowJo software. A nonlinear regression model was used to fit dose curves; log(agonist) versus response-variable slope (four parameters).

Internalization: Cells were incubated with different concentrations of the antibody of interest at 4°C and 37°C for 60 minutes each. Cells were then washed three times and incubated with FITC-labeled goat anti-human Fc secondary antibody at 4°C for 30 minutes. MFI of live cells was measured by flow cytometry, and data were analyzed using FlowJo software.

Cell surface marker expression: HOCF cells were cultured together with 10 μg/mL of directly labeled antibody or IgG isotype control. Median fluorescence intensity (MFI) was measured by flow cytometry and data were analyzed using FlowJo software.

Antagonistic effect: Serum-starved A549 or HOCF cells were pre-incubated with different concentrations of test antibody at 37°C for one hour, followed by stimulation at 37°C for 7 minutes with the addition of 100 ng/mL (A549) or 200 ng/mL (HOCF) IGF-1. Phosphorylated IGF-1R (pIGF1R) of the bioreplicas was measured using the R&D Systems pIGF-1R ELISA according to the manufacturer's protocol, and the pIGF-1R concentration was normalized relative to the lowest test antibody concentration. A nonlinear regression model was used to fit the dose curve; log(inhibitor) versus response - variable slope (four parameters).

result

VRDN-1100 binds to IGF-1R with sub-nanomolar affinity. Figure 2A shows that the increasing concentration of IGF-1R-ECD bound to either anti-FC-captured VRDN-1100 or tetumumab reveals a gradual increase in SPR signal, allowing for a global fit to the binding model. Following IGF-1R clearance, VRDN-1100 exhibits a more sustained binding interaction. Figure 2B shows that IGF-1R-ECD is robustly bound to immobilized VRDN-1100. Tetumumab did not show binding to the IGF-1R:VRDN-1100 complex, indicating that tetumumab and VRDN-1100 have overlapping epitopes. The data are also illustrated in the table shown in Figure 2.

VRDN-1100 binds to IGF-1R on A549 cells with high affinity. As shown in Figure 3, the binding of VRDN-1100 to A549 cells was assessed by flow cytometry and a similar binding distribution to tetumumab was found at three different concentrations. Also shown in Figure 3, the dose-response curve for VRDN-1100 shows an EC50 of 0.1 nM. As shown in Figure 3, VRDN-1100, VRDN-2700 with M252Y, S254T, and T256E mutations in the Fc domain, and tetumumab exhibit similar binding at temperatures that block IGF-1R receptor internalization. Figure D shows that VRDN-1100, VRDN-2700 with M252Y, S254T, and T256E mutations in the Fc domain, and tetumumab induced similar internalization levels (approximately 50%), as measured by the decrease in membrane IGF-1R receptor levels at 37°C compared to 4°C. In the bar chart of Figure 3, the leftmost bar chart is the isotype control, the second bar chart from the left is the teltolimumab set, the second bar chart from the right is the VRDN-1100 set, and the rightmost bar chart is the VRDN-2700 set.

HOCF serves as an in vitro model of TED pathology.

CD34+ and Thy-1+ orbital fibroblasts are involved in extracellular matrix deposition and pathogenic fibrosis in TED5. As shown in Figure 4, HOFCs express (Figure A) IGF-1R and (Figure B) TSHR, as well as (Figure C) CD34 and Thy-1, which confirms their ability to serve as an in vitro model system for IGF-1R function in TED.

VRDN-1100 binds to IGF-1R on HOCF cells with high affinity.

Figure 5 illustrates the binding of VRDN-1100 to HOCF cells, as assessed by flow cytometry, and shows binding at three different concentrations that is largely similar to that of tetumumab. The dose-response curve in the lower right corner of Figure 5 confirms that VRDN-1100 has an EC50 of 0.4 nM.

VRDN-1100 is a sub-nanomolar IGF-1R antagonist. VRDN-1100 potently inhibits IGF-1-stimulated receptor phosphorylation on A549 cells (IC50 = 0.09 nM) and HOCF cells (IC50 = 0.09 nM), as shown in Figures A and B of Figure 6.

These results confirm the epitope overlap between VRDN-1100 and tetumumab on IGF-1R. VRDN-1100 binds to IGF-1R on cells at sub-nanomolar EC50, promotes IGF-1R internalization, and inhibits IGF-1R phosphorylation at sub-nanomolar IC50. Therefore, VRDN-1100 binds to, antagonizes, and internalizes IGF-1R at sub-nanomolar concentrations, suggesting that VRDN-1100 should be potentially effective in potently inhibiting the pathophysiology driving TED.

Example 4. VRDN-2700, with M252Y, S254T, and T256E mutations in its Fc domain, is a novel anti-IGF-1R antibody as described herein, with a half-life extension modification incorporated into its Fc region, and is indicated for the treatment of thyroid eye disease (TED). Pharmacokinetic (PK) parameters of VRDN-2700 with these Fc mutations were measured in cynomolgus monkeys relative to the commercially available IGF-1R antibody tetumumab, and a PK model was constructed to plan possible human dosing regimens.

Tetanus eye disease (TED) is the most common autoimmune condition associated with Graves' disease and hyperthyroidism, but it can also occur in patients with normal or hypothyroidism. The ocular manifestations in TED are driven by thyroid-stimulating hormone receptor (TSHR) agonist autoantibodies and crosstalk between TSHR and IGF-1R. Pathological remodeling of the orbit and periorbital tissues produces various presentations, which may include dry eye, increased tearing, local irritation, eyelid retraction and eventual ptosis, diplopia, and optic nerve compression, accompanied by subsequent visual loss.

The underlying pathology of TED is the activation of an intraorbital inflammatory cascade primarily due to the recruitment of fibroblasts and immune cells. Overexpression of IGF-1R has been observed in the orbit of TED patients, and it is hypothesized that IGF-1R inhibitory antibodies can disrupt IGF-1R and TSHR crosstalk and attenuate the inflammatory cascade. Indeed, IGF-1R antagonism has been shown to reliably alleviate many inflammatory symptoms affecting TED patients.

VRDN-2700 is a monoclonal antibody that inhibits IGF-1-mediated signaling at sub-nanomolar titers via IGF-1R and incorporates clinically validated, extended-half-life Fc modifications (M252Y, S254T, and T256E). Compared to conventional IgG therapeutic antibodies, this antibody has been found to have a more favorable PK profile, potentially offering a less burdensome treatment modality for patients.

VRDN-2700 with the Fc mutation was administered to cynomolgus monkeys via intravenous (IV) infusion at 2, 10, and 50 mg/kg over 30 min, and via subcutaneous (SC) injection at 2 and 10 mg/kg. Tiltumumab was similarly administered via IV infusion at 10 mg/kg over 30 min. Serum levels of VRDN-2700 and tiltumumab were measured using a human IgG-specific ELISA assay. Data were analyzed using the WinNonlin non-compartmental model. A semi-mechanistic model of target-mediated drug disposition was constructed using available human and cynomolgus monkey data. Data are described below.

The tables and diagrams in Figure 7 provide a more favorable PK overview.

The table shows the PK parameters +/- SD. Evidence of target-mediated drug disposal (TMDD) was observed at 2 mg/kg, but not at doses of 10 and 50 mg/kg, consistent with reports of TMDD saturation of tetumumab and other IGF-1R antibodies at higher doses.

VRDN-2700 half-life extension modification prolongs exposure.

At equivalent doses, SC-administered VRDN-2700 with the YTE mutation had greater exposure than intravenously infused tetumumab and reached approximately twice the half-life of tetumumab in the NHP. The bioavailability (F) of SC-administered VRDN-2700 using the preliminary discovery phase formulation was estimated at 62%. Parameter estimates +/- SD are shown in Figure 8.

Model simulations predict that administering VRDN-2700 at 10 mg/kg every 3 weeks or 20 mg/kg every 6 weeks will produce a Cmin >100 ug/mL, similar to the approved tetumumab regimen (10 mg/kg first dose followed by seven 20 mg/kg doses q3w). The 10 mg/kg q3w regimen will have a lower Cmax value. Longer dosing intervals will increase patient convenience and reduce treatment costs, while lower doses and Cmax values may potentially reduce toxicity. Furthermore, the model predicts that a fixed weekly subcutaneous dose of 300 mg VRDN-2700 will achieve a steady-state Cmin of approximately 130 ug/mL, allowing for home self-administration. If a lower Cmin value is effective, a fixed weekly subcutaneous dose of 300 mg VRDN-2700 is predicted to achieve a steady-state Cmin level of approximately 50 ug/mL. In conclusion, the predicted extended half-life of VRDN-2700 provides patients with a wider range of options for more suitable dosing intervals and routes of administration.

Example 5: VRDN2700 Properties During antibody evaluation, the expression of VRDN-2700 was compared with other antibodies possessing mutations in the Fc domain (such as the L/S mutation described herein). Unexpectedly, the antibody with the YTE mutation in the Fc domain (VRDN2700) exhibited approximately 80% higher yield than a similar antibody with the L/S mutation. This was surprising and unexpected, as other antibodies targeting IGF-1R with either the YTE or LS mutations tested showed similar expression regardless of the Fc mutation. The YTE form has fewer low-molecular-weight species compared to the LS form. Therefore, it is indicated that the YTE antibody has fewer impurities and produces a more homogeneous composition, offering an advantage over the antibody with the LS mutation. This was also unpredictable, as another antibody evaluated showed the opposite effect on such species. Furthermore, during purification, the LS mutant was found to form more aggregates than VRDN-2700 upon purification on a cation exchange column. The aggregation of LS mutants would cause numerous manufacturing problems, which have not been observed with VRDN-2700. Therefore, this difference in Fc mutants against this antibody may not have been predicted or anticipated and produces a large number of unexpected advantages for the antibody referred to herein as VRDN-2700.

The extended half-life of VRDN-2700 (YTE) confirms that, compared to conventional therapeutic IgG antibodies, it can be used for convenient SC injection or as an IV infusion, requiring less and/or less frequent treatment, and has superior properties compared to other Fc mutant types (with the same variable region) of the same antibody.

Example 6: VRDN-1100 with YTE or YTE/C22S mutations binds to IGF-1R and inhibits IGF-1R autophosphorylation. The binding of VRDN-1100 with an Fc YTE mutation in the heavy chain (SEQ ID NO: 94) or both C22S and Fc YTE mutations in the heavy chain (SEQ ID NO: 95) to IGF-1R was evaluated in a cell-based binding assay (A549 cells). The light chain has the sequence of SEQ ID NO: 93. The YTE Fc mutant form of VRDN1100 was found to bind to A549 cells with an EC50 of 0.30 nm, while the C22S and Fc YTE mutants had an EC50 of 0.36 nm. The ability of the antibody to inhibit IGF-1R autophosphorylation was also evaluated. Only the YTE mutant had an IC50 of 0.40 nm, and the C22S plus YTE mutant had an IC50 of 0.37 nm. Therefore, it was found that the antibody could both bind to IGF-1R and inhibit its autophosphorylation.

Example 7: VRDN-1100 with a C22S mutation binds to IGF-1R. The binding of a mutant of VRDN-1100 with a C22S mutation in the heavy chain (SEQ ID NO:96) and a VL containing the sequence of SEQ ID NO:97 to IGF-1R was evaluated in a surface plasmon resonance assay. Using this assay, the antibody was found to bind to IGF-1R at pH 7.4 with 1.04 × ^10⁵ _k⁻¹ (1/Ms), 2.18 × ^10⁻⁵ _k⁻¹ (1/s), and 2.10 × ^10⁻¹⁰ _k⁻¹ (M).

Each of the embodiments and implementations provided herein demonstrates that the antibodies provided herein can be used to treat TED and its related symptoms.

All references cited herein are incorporated by reference as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent specifically and individually indicated to be incorporated by reference. The applicant follows 37 C.F.R. §1.57(b)(1) that this incorporated reference refers to each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent, each of which is expressly identified in accordance with 37 C.F.R. §1.57(b)(2), even if such reference does not closely approximate the specific expression incorporated by reference. The inclusion of specific expressions (if any) within this specification does not in any way weaken this general expression incorporated by reference. The citation of references herein is not intended as an admission that the references are relevant background art, nor does it constitute any admission of the content or dates of such publications or documents.

The scope of the embodiments of this invention is not limited to the specific embodiments described herein. In fact, those skilled in the art will readily recognize various modifications other than those described herein based on the foregoing description. These modifications are intended to fall within the scope of the embodiments and any appended claims.

This specification is intended to enable those skilled in the art to practice the embodiments. Various modifications other than those shown and described herein will be readily apparent to those skilled in the art from the foregoing specification, and such modifications fall within the scope of this disclosure and any appended claims.

Claims (19)

1. Use of a pharmaceutical composition comprising an antibody in the preparation of a medicament for treating a subject with thyroid ophthalmopathy, wherein the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 13, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 14. 2. The use as claimed in claim 1, wherein the antibody comprises an Fc region, and the amino acid sequence of the Fc region is SEQ ID NO: 89 or SEQ ID NO: 90. 3. The use as described in claim 1, wherein the amino acid sequence of the light chain of the antibody is SEQ ID NO: 93, and the amino acid sequence of the heavy chain is SEQ ID NO: 92. 4. The use as described in claim 1, wherein the amino acid sequence of the light chain of the antibody is SEQ ID NO: 93, and the amino acid sequence of the heavy chain is SEQ ID NO: 94. 5. The use as described in claim 1, wherein the amino acid sequence of the light chain of the antibody is SEQ ID NO: 93, and the amino acid sequence of the heavy chain is SEQ ID NO: 95. 6. The use as described in any one of claims 1-5, wherein the pharmaceutical composition is formulated for intravenous administration. 7. The use as described in any one of claims 1-5, wherein the pharmaceutical composition is formulated for subcutaneous administration. 8. The use as described in any one of claims 1-5, wherein the drug is suitable for reducing bulging of the subject's eye and/or reducing the subject's clinical activity score (CAS). 9. The use as claimed in claim 8, wherein the drug is suitable for reducing the subject's a) bulging eye by at least 2 mm and b) clinical activity score (CAS). 10. The use as claimed in claim 8, wherein the protruding eye is reduced by at least 2 mm. 11. The use as claimed in claim 8, wherein the protruding eye is reduced by at least 3 mm. 12. The use as claimed in claim 8, wherein the subject's Clinical Activity Score (CAS) is reduced by at least 2 points. 13. The use as described in claim 8, wherein the subject's Clinical Activity Score (CAS) is reduced to one. 14. The use as described in claim 8, wherein the subject's Clinical Activity Score (CAS) is reduced to zero. 15. The use of the drug as described in any one of claims 1-5, wherein the drug (i) reduces bulging of one eye by at least 2 mm; (ii) without causing deterioration of the other eye (or contralateral eye) by 2 mm or more; and (iii) reduces the subject's CAS to one or zero. 16. The use as described in any one of claims 1-5, wherein the drug is suitable for treating diplopia in the subject or reducing its severity. 17. The use according to any one of claims 1-5, wherein the drug is formulated to be administered with the antibody at a dose of 10 mg/kg as a first dose. 18. The use according to any one of claims 1-5, wherein the drug is formulated to be administered with the antibody as a subsequent dose at a dose of 10 mg/kg of antibody. 19. The use as described in claim 17, wherein the drug is formulated for administration of the antibody at subsequent doses every three weeks.