Therapeutic Anti-IGF-1r and Anti-TSHR bispecific antibody and use thereof

Abstract

Provided are a therapeutic anti-IGF-1R and anti-TSHR bispecific antibody and a use thereof. Specifically disclosed are a single-domain antibody against IGF-1R, a bispecific antibody against IGF-1R and TSHR, a nucleic acid encoding the single-domain antibody or the bispecific antibody, a vector comprising the nucleic acid, and a host cell comprising the nucleic acid or the vector. Also provided are a pharmaceutical composition and conjugate or immune fusion comprising the antibody, and a treatment method using the antibody.

Description

A therapeutic bispecific antibody against IGF-1R and TSHR and its application

[0001] This application claims priority to Chinese Patent Application No. 2024105516265, filed May 6, 2024, entitled "A Therapeutic Bispecific Antibody Against IGF-1R and TSHR and Its Application Thereof," and Chinese Patent Application No. 2025105262348, filed April 24, 2025, entitled "A Therapeutic Bispecific Antibody Against IGF-1R and TSHR and Its Application Thereof." The contents of both applications are incorporated herein by reference. Technical Field

[0002] This invention relates to the fields of biomedical or biopharmaceutical technology, and more specifically to anti-IGF-1R antibodies, particularly bispecific antibodies against IGF-1R and TSHR, and their medical uses. Background Technology

[0003] Graves' disease (GD) is a refractory autoimmune disease characterized by goiter and hyperactivity of the thyroid gland, tachycardia, and ocular abnormalities. The most prominent ocular abnormality is known as Graves' ophthalmopathy, also called thyroid-associated ophthalmopathy (TAO) or thyroid eye disease (TED). It is a complex organ-specific autoimmune disease with the highest incidence rate among orbital diseases. TED can occur at any stage of hyperthyroidism and has complex clinical manifestations, including unilateral or bilateral eyelid retraction, proptosis, diplopia, restrictive strabismus, and exposure corneal lesions, severely impacting patients' quality of life. Depending on the severity, treatment options include intravenous corticosteroid pulse therapy, orbital radiotherapy, immunosuppressants, and biologics. Ocular corrective surgery may be considered in some cases.

[0004] The humoral immune response to thyroid ophthalmopathy (TED) begins with the abnormal recognition of the autoantigen thyroid stimulating hormone receptor (TSHR). Activated B cells then produce TSHR antibodies (TRAb). Activated TRAb binds to TSHR, promoting the secretion of inflammatory and chemokine-like factors by orbital fibroblasts, increasing hyaluronic acid (HA) production and fat formation, ultimately leading to remodeling of orbital connective tissues, such as thickening of extraocular muscles and hyperplasia of orbital fat. Insulin-like growth factor 1 receptor (IGF-1R) is another possible TED autoantigen, widely expressed in normal human tissues and highly expressed in thyroid ophthalmopathy. Activation of IGF-1R can stimulate cell proliferation, survival, transformation, metastasis, and angiogenesis, while inhibition of IGF-1R can alleviate and improve symptoms of thyroid ophthalmopathy. IGF-IR may form a physical and functional complex with TSHR, which can cause downstream signal transduction pathways to bind to each other through the interaction of G protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK), ultimately leading to a large amount of hyaluronic acid secretion.

[0005] In 2020, the FDA approved Teprotumumab (brand name TEPEZZATM) for the treatment of TED. It targets IGF-1R and is currently the only approved drug for the treatment of Graves' disease. However, the treatment time is long and significant side effects such as hearing loss and hyperglycemia have been observed in clinical practice.

[0006] Several IGF-1R-targeting drugs are currently under development. For example, the drugs under development by Viridian and Acelyrin in the United States are antibodies obtained by immunizing mice with IGF-1R protein, and have been developed for tumor-related indications, completing phase I-III clinical trials, and are now being explored for the treatment of thyroid eye diseases. US patent application US20230084477A1 discloses an antibody targeting IGF-1R, VRDN-5000, which is a more effective IGF-1 to IGF1R binding inhibitor compared to teprotumumab.

[0007] In addition, antibody drugs targeting the thyroid-stimulating hormone receptor (TSHR) have entered the clinical stage. For example, the TSHR antibodies developed by RSR in the UK are derived from patients' TSHR autoantibodies, including stimulatory TSHR autoantibodies (such as M22) and inhibitory TSHR autoantibodies (such as K1-70). Among them, the phase I clinical trial of K1-70 showed that K1-70 can improve the clinical symptoms of TED to some extent, such as shortening the proptosis.

[0008] Currently, there is still a lack of drugs for treating TED. Single IGF-1R antagonists and single TSHR antagonists have shown partial efficacy; for example, existing data indicate that single anti-IGF-1R antibodies cannot completely inhibit HA production induced by the stimulating TSHR autoantibody M22. Therefore, developing bispecific antibodies targeting TSHR and IGF-1R holds promise for providing new treatment options for TED. Summary of the Invention

[0009] Given that the TSHR and IGF-1R signaling pathways are directly or indirectly involved in the pathogenesis of TED, the inventors developed a single-domain antibody targeting IGF-1R and designed a bispecific antibody molecule targeting both IGF-1R and TSHR. This bispecific antibody molecule blocks IGF-1R and TSHR by simultaneously binding to them, inhibiting downstream signaling pathways and their potential interactions. Simultaneous targeting of IGF-1R and TSHR has the potential to reduce the toxicity of high doses of single-drug therapy, such as hyperglycemia or hearing impairment known to be associated with IGF-1 targets, as seen with teprotumumab.

[0010] In a first aspect, the present invention provides an antigen-binding protein that specifically binds to IGF-1R, said antigen-binding protein comprising a VHH domain, wherein

[0011] (i) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:4; or

[0012] (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:8; or

[0013] (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:12; or

[0014] (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:16; or

[0015] (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:20; or

[0016] (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:24; or

[0017] (vii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:25; or

[0018] (viii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:26; or

[0019] (ix) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:27; or

[0020] (x) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:28.

[0021] In some embodiments, (i) the VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO: 1-3; or (ii) the VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO: 5-7; or (iii) the VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO: 9-11; or (iv) the VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO: 13-15; or (v) the VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO: 17-19; or (vi) the VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO: 1-3. The amino acid sequences shown in NO:21-23 are heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3.

[0022] In some embodiments, (i) the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; or (ii) the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; or (iii) the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; or (iv) the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4. NO:16 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20; or (v) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20; or (vi) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:24; or (vii) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:24; or (vii) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20. NO:25 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:25; or (viii) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:26; or (ix) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:27; or (x) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:25; NO:28 has an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0023] In some embodiments, (i) the VHH comprises the amino acid sequence shown in SEQ ID NO:4; or (ii) the VHH comprises the amino acid sequence shown in SEQ ID NO:8; or (iii) the VHH comprises the amino acid sequence shown in SEQ ID NO:12; or (iv) the VHH comprises the amino acid sequence shown in SEQ ID NO:16; or (v) the VHH comprises the amino acid sequence shown in SEQ ID NO:20; or (vi) the VHH comprises the amino acid sequence shown in SEQ ID NO:24; or (vii) the VHH comprises the amino acid sequence shown in SEQ ID NO:25; or (viii) the VHH comprises the amino acid sequence shown in SEQ ID NO:26; or (ix) the VHH comprises the amino acid sequence shown in SEQ ID NO:27; or (x) the VHH comprises the amino acid sequence shown in SEQ ID NO:28.

[0024] In some embodiments of the antigen-binding protein of the present invention, the antigen-binding protein is an antibody.

[0025] In some implementations, the antibody is a monoclonal antibody.

[0026] In some implementations, the antibody is a single-domain antibody or a heavy-chain antibody.

[0027] In some implementations, the antibody is a camel antibody, a humanized antibody, or a chimeric antibody.

[0028] In some implementations, the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0029] In some embodiments, the antibody is a bispecific antibody, further comprising a second antigen-binding region that binds to a second antigen. In some embodiments, the second antigen is an autoantigen. In a preferred embodiment, the second antigen is a TSHR.

[0030] In a second aspect, the present invention provides a bispecific antibody comprising a first antigen-binding region that binds to IGF-1R and a second antigen-binding region that binds to TSHR.

[0031] In some embodiments, the first antigen-binding region includes a VHH domain, wherein,

[0032] (i) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:4; or

[0033] (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:8; or

[0034] (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:12; or

[0035] (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:16; or

[0036] (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:20; or

[0037] (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:24; or

[0038] (vii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:25; or

[0039] (viii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:26; or

[0040] (ix) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:27; or

[0041] (x) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:28.

[0042] In some embodiments, the first antigen-binding region includes a VHH domain, wherein,

[0043] (i) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:1-3; or

[0044] (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:5-7; or

[0045] (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO: 9-11; or

[0046] (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:13-15, respectively; or

[0047] (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:17-19; or

[0048] (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:21-23, respectively.

[0049] In some embodiments, (i) the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; or (ii) the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; or (iii) the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; or (iv) the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16; or (v) the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4. NO:20 has an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20; or (vi) the VHH contains an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:24; or (vii) the VHH contains an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:25; or (viii) the VHH contains an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:26; or (ix) the VHH contains an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20. NO:27 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity; or (x) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:28.

[0050] In some embodiments, (i) the VHH comprises the amino acid sequence shown in SEQ ID NO:4; or (ii) the VHH comprises the amino acid sequence shown in SEQ ID NO:8; or (iii) the VHH comprises the amino acid sequence shown in SEQ ID NO:12; or (iv) the VHH comprises the amino acid sequence shown in SEQ ID NO:16; or (v) the VHH comprises the amino acid sequence shown in SEQ ID NO:20; or (vi) the VHH comprises the amino acid sequence shown in SEQ ID NO:24; or (vii) the VHH comprises the amino acid sequence shown in SEQ ID NO:25; or (viii) the VHH comprises the amino acid sequence shown in SEQ ID NO:26; or (ix) the VHH comprises the amino acid sequence shown in SEQ ID NO:27; or (x) the VHH comprises the amino acid sequence shown in SEQ ID NO:28.

[0051] In some embodiments, the second antigen-binding region comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequence shown in SEQ ID NO:36; and the VH comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequence shown in SEQ ID NO:32.

[0052] In some embodiments, the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO:33-35, and the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:29-31, respectively.

[0053] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:36, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:32.

[0054] In some embodiments, the VL contains an amino acid sequence as shown in SEQ ID NO:36 and the VH contains an amino acid sequence as shown in SEQ ID NO:32.

[0055] In some embodiments, in the bispecific antibody, the VHH of the first antigen-binding region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:9-11, respectively; the VL of the second antigen-binding region comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in any one of SEQ ID NO:33-35, respectively; and the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in any one of SEQ ID NO:29-31, respectively.

[0056] In some embodiments, in the bispecific antibody, the VHH of the first antigen-binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:27; the VL of the second antigen-binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:36; and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:32.

[0057] In some embodiments, in the bispecific antibody, the VHH of the first antigen-binding region comprises the amino acid sequence of SEQ ID NO:27; the VL of the second antigen-binding region comprises the amino acid sequence of SEQ ID NO:36 and the VH comprises the amino acid sequence of SEQ ID NO:32.

[0058] In some implementations, the binding valence ratio of the first antigen-binding region and the second antigen-binding region is 1:1, 2:1 or 1:2, preferably 1:1.

[0059] In some embodiments of the bispecific antibody of the present invention, the bispecific antibody comprises (i) a heavy chain comprising, from the N-terminus to the C-terminus, the VH, heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3); and (ii) a light chain comprising, from the N-terminus to the C-terminus, the VL and light chain constant region (CL); wherein the VHH is optionally attached to the N-terminus or C-terminus of the heavy chain or the light chain via a linker.

[0060] In some embodiments, (i) the heavy chain from N end to C end comprises: VHH-connector-VH-CH1-CH2-CH3; and the light chain from N end to C end comprises: VL-CL; or (ii) the heavy chain from N end to C end comprises: VH-CH1-CH2-CH3-connector-VHH; and the light chain from N end to C end comprises: VL-CL; or (iii) the heavy chain from N end to C end comprises: VH-CH1-CH2-CH3; and the light chain from N end to C end comprises: VL-CL-connector-VHH; or (iv) the heavy chain from N end to C end comprises: VH-CH1-CH2-CH3; and the light chain from N end to C end comprises: VHH-connector-VL-CL.

[0061] In some embodiments, the linker comprises an amino acid sequence selected from (G4S)n, where n is an integer selected from 1 to 5.

[0062] In some embodiments, the adapter comprises an amino acid sequence such as any one of SEQ ID NO:47-49.

[0063] In some embodiments of the bispecific antibody of the present invention, (i) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:37; or

[0064] (ii) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:39; or

[0065] (iii) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:40; or

[0066] (iv) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:41; or

[0067] (v) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:43, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:42.

[0068] In some embodiments of the bispecific antibody of the present invention, (i) the light chain comprises the amino acid sequence of SEQ ID NO:38 and the heavy chain comprises the amino acid sequence of SEQ ID NO:37; or (ii) the light chain comprises the amino acid sequence of SEQ ID NO:38 and the heavy chain comprises the amino acid sequence of SEQ ID NO:39; or (iii) the light chain comprises the amino acid sequence of SEQ ID NO:38 and the heavy chain comprises the amino acid sequence of SEQ ID NO:40; or (iv) the light chain comprises the amino acid sequence of SEQ ID NO:38 and the heavy chain comprises the amino acid sequence of SEQ ID NO:41; or (v) the light chain comprises the amino acid sequence of SEQ ID NO:43 and the heavy chain comprises the amino acid sequence of SEQ ID NO:42.

[0069] In a third aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein according to the first aspect of the present invention or a bispecific antibody according to the second aspect of the present invention.

[0070] In a fourth aspect, the present invention provides a carrier comprising nucleic acids according to a third aspect of the present invention.

[0071] In a fifth aspect, the present invention provides a host cell comprising a nucleic acid according to a third aspect of the present invention or a vector according to a fourth aspect of the present invention.

[0072] In a sixth aspect, the present invention provides a pharmaceutical composition comprising (i) an antibody or an antigen-binding fragment thereof according to the first aspect of the present invention, or a bispecific antibody according to the second aspect of the present invention; and (ii) a pharmaceutically acceptable carrier or excipient.

[0073] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent, optionally selected from antibodies, chemotherapeutic agents, and small molecule drugs.

[0074] In a seventh aspect, the present invention provides conjugates or immunofusions comprising an antigen-binding protein according to a first aspect of the present invention, or a bispecific antibody according to a second aspect of the present invention, and a chemical moiety thereto.

[0075] In some embodiments, the chemical component is selected from therapeutic agents, detectable components, and immunostimulatory molecules.

[0076] In an eighth aspect, the present invention provides a chimeric antigen receptor comprising an antigen-binding protein according to a first aspect of the present invention, or a bispecific antibody according to a second aspect of the present invention.

[0077] In a ninth aspect, the present invention provides genetically modified cells comprising a chimeric antigen receptor according to an eighth aspect of the present invention.

[0078] In a tenth aspect, the present invention provides a method for treating IGF-1R and / or TSHR-mediated diseases in a subject, comprising administering to the subject an effective amount of an antigen-binding protein according to a first aspect of the invention, a bispecific antibody according to a second aspect of the invention, a pharmaceutical composition according to a sixth aspect of the invention, a conjugate or immunofusion according to a seventh aspect of the invention, a chimeric antigen receptor according to an eighth aspect of the invention, or a genetically modified cell according to a ninth aspect of the invention. The disease is an autoimmune disease, such as Graves' disease or thyroid eye disease (TED). In a preferred embodiment, the disease is thyroid eye disease (TED).

[0079] In some embodiments, the method further includes administering a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.

[0080] In an eleventh aspect, the present invention provides the use of an antigen-binding protein according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, a conjugate or immune fusion compound according to the seventh aspect of the present invention, or a chimeric antigen receptor according to the eighth aspect of the present invention, or a genetically modified cell according to the ninth aspect of the present invention in the preparation of a medicament for treating IGF-1R and / or TSHR-mediated diseases; wherein the disease is an autoimmune disease, such as Graves' disease or thyroid eye disease (TED); preferably, the disease is thyroid eye disease (TED).

[0081] In a twelfth aspect, the present invention provides the use of an antigen-binding protein according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, a conjugate or immune fusion compound according to the seventh aspect of the present invention, or a chimeric antigen receptor according to the eighth aspect of the present invention, or a genetically modified cell according to the ninth aspect of the present invention in the treatment of IGF-1R and / or TSHR-mediated diseases; wherein said diseases are autoimmune diseases, such as Graves' disease or thyroid eye disease (TED); preferably, said diseases are thyroid eye disease (TED). Attached Figure Description

[0082] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail below. A brief description of the drawings is as follows:

[0083] Figure 1. ELISA binding curves of anti-IGF-1R antibody and IGF-1R antigen.

[0084] Figure 2. ELISA curves showing the inhibition of IGF-1 binding to IGF-1R by anti-IGF-1R antibody.

[0085] Figure 3. FACS binding curves of the anti-IGF-1R antibody D09 mutant with MCF-7 cells.

[0086] Figure 4. Configuration diagram of anti-IGF-1R and TSHR bispecific antibodies.

[0087] Figure 5. FACS binding curves of anti-IGF-1R / TSHR bispecific antibody with MCF-7 cells.

[0088] Figure 6. FACS binding curves of anti-IGF-1R / TSHR bispecific antibody with HEK293-TSHR sc26 cells.

[0089] Figure 7. FACS curves of the anti-IGF-1R / TSHR bispecific antibody inhibiting the binding of IGF-1 to MCF-7.

[0090] Figure 8. FACS curves of the inhibition of M22 binding to HEK293-TSHR sc26 by the anti-IGF-1R / TSHR bispecific antibody.

[0091] Figure 9. FACS curves of candidate bispecific antibody molecules V-7 and V-10 inhibiting the binding of IGF-1 to cellular IGF-1R.

[0092] Figure 10. FACS curves of candidate bispecific antibodies V-7 and V-10 inhibiting the binding of M22 to cellular TSHR.

[0093] Figure 11 ELISA curves of candidate bispecific antibodies V-7 and V-10 inhibiting IGF-1R phosphorylation induced by IGF-1.

[0094] Figure 12 ELISA curves of candidate bispecific antibodies V-7 and V-10 inhibiting M22-induced cAMP production.

[0095] Figure 13 shows the detection results of the candidate bispecific antibody molecules V-7 and V-10 inhibiting M22-induced hyaluronic acid production.

[0096] Figure 14 shows the efficacy of candidate bispecific antibody molecules V-7 and V-10 in mice with thyroid-associated eye disease (TED). Figure 14A shows the changes in body weight of mice in the control group, model group, model group + V-7 group, and model group + V-10 group; Figure 14B shows the appearance of eyeball protrusion and eyelid edema in the control group, model group, model group + V-7 group, and model group + V-10 group; Figure 14C shows the H&E staining results of thyroid tissue sections in the control group, model group, model group + V-7 group, and model group + V-10 group; Figures 14D and 14E show the H&E staining results of orbital tissue in the control group, model group, model group + V-7 group, and model group + V-10 group; Figures 14F and 14G show the Masson staining results of orbital tissue in the control group, model group, model group + V-7 group, and model group + V-10 group. Detailed Implementation

[0097] The above-described features and advantages of the present invention, as well as their additional features and advantages, will become more clearly understood below by taking into account the accompanying drawings and the detailed description of the following embodiments.

[0098] The embodiments described herein with reference to the accompanying drawings are illustrative, exemplary, and intended for a general understanding of the invention. The embodiments should not be construed as limiting the scope of the invention. Identical or similar elements and elements having the same or similar functions are represented by the same reference numerals throughout the specification.

[0099] Terminology Definition

[0100] Unless otherwise stated or defined, all terms used have the common meaning in the art as is clear to a person skilled in the art. For example, refer to standard manuals such as Leuenberger, H.G., Nagel, B. and Klbl, H. eds., "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., eds., "Current protocols in molecular biology", Green Publishing and Wiley InterScience, New York (1987); Roitt et al., "Immunology" (6th Ed.), Mosby / Elsevier, Edinburgh (2001); and Janeway et al., "Immunobiology" (6th Ed.), Garland Science Publishing / Churchill Livingstone, New York. York (2005), and the general background technology cited above.

[0101] As used herein, "antigen" in a broad sense refers to a molecule or part of a molecule that can be bound by an antibody, and additionally, can induce an animal to produce antibodies that can bind to the epitopes of that antigen. An antigen may have one epitope or more than one epitope.

[0102] As used herein, "autoantigen" refers to antigenic substances that, due to changes in the structure and composition of one's own tissue cells caused by biological, physical, or chemical factors, elicit an immune response from the body's immune system against these tissue cell components. The release of occult antigens can generate an immune response. Changes in self-components may produce autoantigens. For example, denatured IgG can stimulate the body to produce anti-denatured IgG antibodies (rheumatoid factor), causing rheumatoid arthritis. Clinically, the use of certain drugs can alter the antigenicity of blood cell surfaces, causing autoimmune hemolytic anemia or granulocytopenia. Cross-reactions triggered by common antigens can generate an immune response. Some bacteria and viruses have similar antigenic determinants to certain tissue cells in normal human bodies; autoantibodies and sensitized lymphocytes generated against these bacterial or viral antigenic determinants can cross-react with self-tissue cells, causing autoimmune diseases. Autoantigens can exist in the host at levels different from those present in healthy or disease-free patients (e.g., elevated or decreased). TSHR (thyroid-stimulating hormone receptor) in this invention is a type of "autoantigen."

[0103] As used herein, the term "epitope" refers to the site on an antigen where an antigen-binding protein binds. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed in the ternary folding of one or more proteins. Epitopes formed from consecutive amino acids (also known as linear epitopes) are generally preserved upon exposure to denaturing solvents, while epitopes formed from ternary folds (also known as conformational epitopes) are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least two, more commonly at least five, or eight to ten amino acids in a unique spatial conformation. Epitopes define the minimum binding site for antigen-binding proteins and are therefore specific targets of these proteins.

[0104] As used herein, “antigen-binding protein” generally refers to a protein that contains a portion that binds to an antigen. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments, immunoconjugates or immunofusions, antibody derivatives, antibody analogs, fusion proteins, T-cell receptors (TCRs) or chimeric antigen receptors (CARs), as long as they exhibit the desired antigen-binding activity.

[0105] As used herein, the term "antibody" generally refers to an immunoglobulin that can specifically bind to a corresponding antigen, possessing the ability to specifically bind to a particular antigen. The antibody may be secreted by immune cells and includes, but is not limited to, full-length antibodies comprising two light chains and two heavy chains, and / or heavy chain antibodies. The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may be a monospecific antibody, a bispecific antibody, or a multispecific antibody. The antibody may be a murine antibody, a camelid antibody, a humanized antibody, a fully human antibody (fully human antibody), or a chimeric antibody. The antibody may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. In this document, "antibody" may refer to an IgG form of antibody targeting TSHR or a heavy chain antibody targeting IGF-1R.

[0106] A full-length antibody consists of two light chains and two heavy chains. Each heavy chain comprises a variable region (or domain) (abbreviated as VH) and a constant region. The constant region consists of three domains: CH1, CH2, and CH3. Each light chain comprises a variable region (or domain) (abbreviated as VL) and a constant region. The constant region consists of one domain: CL. The variable regions of both the antibody heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical complement activation pathway). The heavy chain of this type of immunoglobulin can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (crystallizable fragment). The Fd region contains the VH and CH1 domains and binds to the light chain to form the Fab (antigen-binding fragment). The Fc fragment is responsible for immunoglobulin effector functions, including complement binding and binding to homologous Fc receptors on effector cells. The Fc region of an immunoglobulin generally contains two constant domains, the CH2 and CH3 domains, and optionally includes a CH4 domain. Hinge regions, found in the IgG, IgA, and IgD immunoglobulin classes, act as flexible spacers, allowing the Fab moiety to move freely in space relative to the Fc region. Hinge domains are structurally diverse, varying in sequence and length across immunoglobulin classes and subclasses.

[0107] As used herein, the term "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies in the cluster are identical, except for a small number of possible natural mutations. Monoclonal antibodies typically exhibit high specificity against a single antigenic site. Moreover, unlike conventional polyclonal antibody formulations (which usually have different antibodies targeting different determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used according to the invention can be prepared in hybridoma cells or by recombinant DNA methods.

[0108] As used herein, the “light chain variable region” (VL) or “heavy chain variable region” (VH) consists of a “framework” region separated by three “complementarity-determining regions” or “CDRs”. The framework region is used to align the CDRs that specifically bind to the antigenic epitopes. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL and VH domains contain the following framework (FR) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; and the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0109] As used herein, “heavy chain antibody” refers to an antibody that lacks a light chain, which is a conventional antibody. The term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen-binding domain and an Fc domain, such as VHH-CH2-CH3 or VHH-CH1-CH2-CH3 from the N-terminus to the C-terminus; functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NARs and functional fragments of homodimers comprising one variable domain (V-NAR) and five C-like constant domains (C-NAR); and soluble single-domain antibodies (sUniDabs™).

[0110] In this article, the antibody-related terms “valence,” “binding valence,” or “valence number” refer to the total number of antigen-binding sites in an antibody molecule, or the number of antigen-binding sites with the same antigen-binding specificity.

[0111] In this text, "single specificity" refers to the ability to bind to only one epitope. In contrast, the term "multispecificity" refers to the ability to bind to multiple different epitopes. Accordingly, "bispecificity" refers to the ability to bind to two different epitopes; and "triple specificity" refers to the ability to bind to three different epitopes. The different epitopes can be different epitopes on different antigens or different epitopes on the same antigen.

[0112] In the context of this invention, the term "bispecific antibody" should be understood as an antibody having two distinct antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a single target. As used herein, the term "bispecific antibody" should be understood in its broadest sense, including full-length bispecific antibodies and their antigen-binding fragments. Bispecific antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Bispecific antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

[0113] As used herein, the term “antigen-binding fragment” includes, but is not limited to, Fab, Fab', F(ab)2, Fv fragment, F(ab')2, crossFab, scFv, di-scFv, sdAb (single-domain antibody, including nanobodies), or diabody. Unless otherwise stated herein or explicitly contradicted by the context, the term “antigen-binding protein” as used herein is equivalent to “antigen-binding protein or antigen-binding fragment thereof,” and the term “antibody” as used herein is equivalent to “antibody or antigen-binding fragment thereof.”

[0114] In this context, "sdAb" refers to an antibody peptide fragment capable of specifically recognizing and binding to a target antigen through a single variable domain, such as a single heavy chain variable domain or a single light chain variable domain, without pairing with additional immunoglobulin variable domains. For a single-domain antibody, its structure can be considered to consist of four frame regions ("FR") and three complementarity-determining regions ("CDR"), arranged in the following order from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single-domain antibodies can include fully human sequences, humanized sequences, sequences optimized in other ways, or chimeric immunoglobulin sequences. Single-domain antibodies can be used alone in isolation or as part of a larger protein to perform antigen-binding functions. sdAbs can originate from the VH / VL domains of conventional antibodies (such as humanized single-domain antibodies) or can include other single-domain antibody types such as camelid VHH and shark VNAR. "Nanobodies" are the variable regions (VHH) of natural heavy chain antibodies (HCAbs) from camel-like animals (such as camels and alpacas). Nanobodies are a type of single-domain antibody.

[0115] As used herein, the term "VHH" generally refers to the variable region of a heavy chain antibody that is naturally lacking the antibody light chain. The term "VHH" is used to distinguish between the heavy chain variable domain (VH) and the light chain variable domain region (VL) present in conventional four-chain antibodies. The terms "VHH" and "VHH domain" are used interchangeably to refer to a heavy chain variable domain derived from a heavy chain antibody lacking the light chain. VHHs can be derived from antibodies produced in camelid species (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides camelids may also produce naturally lacking heavy chain antibodies, and such VHHs are also within the scope of this invention.

[0116] The VHH domain and other structural characteristics and functional properties of peptides containing it can be summarized as follows:

[0117] The VHH domain (which is naturally "designed" to functionally bind to antigens in the absence of and without interaction with light chain variable domains) can be used as a single, relatively small functional antigen-binding structural unit, domain, or peptide. This distinguishes the VHH domain from the VH and VL domains of conventional 4-chain antibodies, which are generally not suitable on their own for practical applications as a single antigen-binding protein or a single variable domain of an immunoglobulin, but need to be combined in one or another form to provide a functional antigen-binding unit (e.g., in the form of a conventional antibody fragment such as a Fab fragment; or in the form of an scFv consisting of VH domains covalently linked to a VL domain).

[0118] Due to these unique properties, using VHH domains offers many significant advantages over using conventional VH and VL domains, scFv, or conventional antibody fragments (e.g., Fab- or F(ab')2- fragments): only a single domain is required to bind antigens with high affinity and selectivity, thus eliminating the need for two separate domains or ensuring that these two domains are in appropriate spatial conformation and configuration (e.g., scFv generally requires specially designed linkers); VHH domains can be expressed from a single gene without post-translational folding or modification; VHH domains can be easily modified into multivalent and multispecific formats (formatted); VHH domains are highly soluble and do not exhibit aggregation tendency; VHH domains are resistant to heat, pH, and protein. The enzyme and other denaturants or conditions are highly stable, and therefore no freezing equipment is needed during preparation, storage, or transportation, thus saving costs, time, and the environment; the VHH domain is easy to prepare and relatively inexpensive, even at the scale required for production; the VHH domain is relatively small compared to conventional 4-chain antibodies and their antigen-binding fragments (approximately 15 kDa or 1 / 10 the size of conventional IgG), thus exhibiting higher tissue permeability and allowing for higher doses compared to conventional 4-chain antibodies and their antigen-binding fragments; the VHH domain can exhibit so-called cavity-binding properties (especially due to its elongated CDR3 loop compared to conventional VH domains), thereby reaching targets and epitopes that are inaccessible to conventional 4-chain antibodies and their antigen-binding fragments.

[0119] Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185–195; Li et al., J Biol Chem., 287(2012)13713–13721; Deffar et al., African Journal of Biotechnology Vol.8(12),pp.2645-2652,17June,2009 and WO94 / 04678.

[0120] The VHH domain derived from the Camelidae family can be "humanized" (also referred to herein as "sequence optimization," which, in addition to humanization, may also encompass other modifications to the sequence by one or more mutations that provide improved VHH properties, such as removal of potential post-translational modification sites) by replacing one or more amino acid residues in the original VHH sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a conventional human 4-chain antibody. The humanized VHH domain may contain one or more fully human framework regions. Humanization can be accomplished using protein surface amino acid resurfacing methods and / or CDR grafting to a universal framework, for example, as exemplified in the examples.

[0121] Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region and CDR, it means the numbering position according to the Kabat numbering system.

[0122] The amino acid assignments of the VL, VH, and VHH domains in this application are in accordance with any conventional definition of a CDR. These conventional definitions are known in the art. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); the complex of the Chothia Kabat CDR, wherein CDR-H1 is a complex of the Chothia CDR and the Kabat CDR; the AbM definition used by the antibody modeling software Oxford Molecular; and the CONTACT definition by Martin et al. (world wide web bioinfo.org.uk / abs).

[0123] In the technical solutions of this invention, the amino acid residues in the variable domain sequence can be determined using Kabat definitions, Chothia definitions, Combined definitions that include both Kabat and Chothia definitions, AbM definitions, CONTACT definitions, etc., as detailed in the table below. Those skilled in the art should understand that, unless otherwise specified, the “CDR” and “complementarity-determining region” of an antibody or its region (e.g., the variable region) should be understood to encompass the complementarity-determining region defined by any of the known schemes described above. Although in a preferred embodiment, the amino acid sequence of the CDR is based on the AbM definition rule, the amino acid sequences corresponding to other CDR definition rules should also be within the scope of protection of this invention.

[0124] However, it should be noted that, as is known in the art regarding VH and VHH domains, the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, in general, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence.

[0125] For example, a CDR may include "extended CDRs", such as: 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2) and 89-97 or 89-96 (LCDR3) in VL; 26-35 (HCDR1), 50-65 or 49-65 (HCDR2) and 93-102, 94-102 or 95-102 (HCDR3) in VH.

[0126] The total number of amino acid residues in the VHH domain will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0127] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or cohesion. Affinity, expressed as the equilibrium constant (KD) for antigen-antibody dissociation, is a measure of the strength of binding between an antigenic determinant and the antigen-binding site of the antibody: the smaller the KD value, the stronger the binding between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1 / KD. Cohesion is a measure of the strength of binding between an antibody and its associated antigen. Cohesion involves the affinity between the antigenic determinant and the antigen-binding site of the antibody, as well as the number of associated binding sites present on the antibody. Typically, antibodies will bind antigens with the following dissociation constant (KD): 10 -5 M to 10 -12 M or smaller, and preferably 10 -7 M to 10 -12 M or smaller, and more preferably 10 -8 M to 10 -12 M, and / or binds to the antigen with the following binding affinity: at least 10 7 M -1 Preferably at least 10 8 M -1 More preferably at least 10 9 M -1 such as at least 10 10 M -1 It is generally considered that anything greater than 10... -4 The KD value of M indicates nonspecific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays, as well as various variants known in the art.

[0128] In this application, the term "KD" generally refers to the equilibrium dissociation constant, which is the ratio of the dissociation rate constant (kdis, also known as "off-rate" (koff) or "kd") to the binding rate constant (kon, also known as "on-rate" or "ka"). The binding rate constant (kon), dissociation rate constant (kdis), and equilibrium dissociation constant (KD) can be used to represent the binding affinity of an antigen-binding protein (e.g., an antibody) to an antigen. Methods for determining the binding and dissociation rate constants are well known in the art, including but not limited to biomembrane interference (BLI), radioimmunoassay (RIA), equilibrium dialysis, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), co-immunoprecipitation (Co-IP), and protein chip technology. The affinity of a particular protein-protein interaction may vary depending on the conditions measured (e.g., salt concentration, pH).

[0129] As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same positions when aligned. For example, "the amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence is % identical to SEQ ID NO: Y and is described as having X% of the residues in the amino acid sequence being identical to the residues in the sequence disclosed in SEQ ID NO: Y.

[0130] Such calculations are typically performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0131] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody from a laboratory animal such as a rodent ("parental antibody") and the constant region is derived from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in human individuals compared to parental antibodies (e.g., mouse-derived, alpaca-derived).

[0132] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., an alpaca antibody) are replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions, or modifications of amino acids within the CDR region are also permissible, as long as they retain the antibody's ability to bind to a specific antigen. Humanized antibodies may optionally contain at least a portion of the constant region of human immunoglobulins. "Humanized antibodies" retain antigen specificity similar to the original antibody. The "humanized" form of a non-human (e.g., alpaca) antibody may minimally contain a chimeric antibody with a sequence derived from a non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (receptor antibody) may be replaced with CDR region residues from a non-human species (donor antibody) (such as an alpaca, mouse, rat, rabbit, or non-human primate) having the desired properties, affinity, and / or capabilities. In some cases, FR region residues in a human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications not found in receptor antibodies or in donor antibodies. These modifications can be made to further improve antibody performance, such as binding affinity.

[0133] In this application, the term "fully human antibody" generally refers to an antibody expressed by transferring a human antibody-encoding gene into a genetically engineered animal lacking an antibody gene. All parts of the antibody (including the variable and constant regions) are encoded by human-derived genes. Fully human antibodies can significantly reduce the immune side effects caused by heterologous antibodies in humans. Methods for obtaining fully human antibodies in this field include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-peptide technology.

[0134] In this application, the term "subject" generally refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). The term "primate" generally refers to monkey and ape species, and includes monkey species such as those from the genera *Macaca* (e.g., cynomolgus monkeys and rhesus monkeys) and baboons (e.g., pig-tailed baboons), as well as marmosets (species from the genus *Callithrix*), squirrel monkeys (species from the genus *Saimiri*) and tamarins (species from the genus *Saguinus*), and ape species such as chimpanzees (*Pan troglodytes*), and also includes *Homo sapiens*.

[0135] In this application, the term "nucleic acid" generally refers to any length of isolated nucleotide, deoxyribonucleotide, or ribonucleotide or analogue thereof, isolated from its natural environment or synthesized artificially.

[0136] In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers inserted nucleic acid molecules into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having a variety of the functions described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0137] In this application, the term "host cell" generally refers to an individual cell, cell line, or cell culture that may contain or already contains a plasmid or vector including the nucleic acid molecules described in this application, or that is capable of expressing the antibodies or antigen-binding fragments described in this application. The cell may include progeny of a single host cell. Due to natural, accidental, or intentional mutations, progeny cells may not necessarily be morphologically or genomically identical to the original parent cell, but they must be capable of expressing the antibodies or antigen-binding fragments described in this application. The cell can be obtained by in vitro transfection of cells using the vector described in this application. The cell can be a prokaryotic cell (e.g., *Escherichia coli*) or a eukaryotic cell (e.g., yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells). In some cases, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell. In this application, the term "recombinant cell" generally refers to a cell in which a recombinant expression vector has been introduced. The recombinant host cell includes not only a specific type of cell but also the progeny of these cells.

[0138] In this application, the term "pharmaceutical composition" generally refers to a formulation which is present in a form that allows the biological activity of the active ingredient to be effective and does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition will be administered.

[0139] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations employed. Typically, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers may include buffers, antioxidants, low molecular weight (less than about 10 residues) peptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming anti-charge ions such as sodium; and / or nonionic surfactants.

[0140] In this application, the terms "conjugate" or "immunofusion" generally refer to a molecule formed by the conjugation or fusion of an antigen-binding protein with one or more other chemical moieties. These other chemical moieties can be protein-like molecules, such as peptides, polypeptides, or proteins, where the antigen-binding protein fuses with a protein-like molecule to form an "immunofusion"; or non-protein-like molecules, such as chemical toxins. In cases involving multiple other chemical moieties, these moieties may be the same or different from each other.

[0141] In this application, the term "chimeric antigen receptor" ("CAR") refers to an engineered receptor that includes an extracellular antigen-binding domain (which is typically derived from a monoclonal antibody or a fragment thereof, such as scFv), a transmembrane domain, a co-stimulatory domain, and an intracellular signal transduction domain.

[0142] "IGF-1R" or "IGF1R" refers to the receptor for insulin-like growth factor 1 (IGF-1, formerly known as somatomedin C). IGF-1R also binds to and is activated by insulin-like growth factor 2 (IGF-2). IGF1-R is a receptor tyrosine kinase that autophosphorylates upon activation by IGF-1 or IGF-2. IGF1-R is a TED autoantigen, widely expressed in normal human tissues and highly expressed in thyroid eye diseases. Activation of IGF-1R stimulates cell proliferation, survival, transformation, metastasis, and angiogenesis, while inhibition of IGF-1R can alleviate and improve symptoms of thyroid eye diseases. The complete amino acid sequence of human IGF-1R has the Swiss-Prot accession number P08069 (IGF1R_HUMAN).

[0143] TSHR is a G protein-coupled receptor composed of three domains: a leucine-rich repeat domain (LRD), a cleavage domain (CD), and a transmembrane domain (TMD) (Nunez Miguel R, et al 2004. Thyroid 14:991-1011). Thyroid function is regulated by TSH secreted by the pituitary gland (Szkudlinski MW, et al, 2002. Physiological Reviews 82:473-502). TSH binds to TSHR on the surface of thyroid cells, and this is the first step in initiating the TSHR signaling cascade. The binding of TSH to TSHR leads to stimulation of the formation and release of thyroid hormones: thyroxine (T4) and triiodothyronine (T3). Feedback mechanisms involving circulating T4 and T3 levels and the levels of thyrotropin-releasing hormone (TRH) secreted by the hypothalamus control TSH release, which in turn controls thyroid stimulation and serum thyroid hormone levels (Szkudlinski MW, et al, 2002). There are two main types of TSHR autoantibodies (TRAbs): stimulatory and blocking. Thyroid-stimulating autoantibodies bind to TSHR and mimic the effects of TSH, stimulating the thyroid gland to produce high levels of T4 and T3; these autoantibodies are also described as TRAbs with stimulatory or TSH-activating activity (Rees Smith B, et al, 2007. Thyroid 17:923-938). This feedback control mechanism of thyroid function is no longer effective in the presence of thyroid-stimulating autoantibodies, and patients exhibit clinical symptoms of hyperthyroidism, characterized by excessive thyroid hormones and their metabolites in the serum. This condition is known as Graves' disease. The complete amino acid sequence of human TSHR has the Swiss-Prot accession number P16473 (TSHR_HUMAN).

[0144] As used herein, the terms “treatment,” “therapy,” “treatment,” etc., refer to the administration of a drug or the performance of a procedure for the purpose of achieving an effect. These effects may be preventative in the complete or partial prevention of a disease or its symptoms, and / or therapeutic in the partial or complete cure of a disease and / or its symptoms. As used herein, “treatment” may include the treatment of diseases or conditions in mammals, particularly humans (e.g., diseases mediated by IGF-1R and / or TSHR), and includes: (a) preventing the onset of a disease or its symptoms in subjects who may be susceptible to the disease (e.g., including diseases that may be related to or caused by the primary disease) but have not yet been diagnosed with the disease; (b) suppressing the disease, i.e., halting its development; and (c) alleviating the disease, i.e., causing its remission. Treatment may refer to any indicator of success in the treatment or improvement or prevention of a disease, including any objective or subjective parameter such as a reduction in symptoms; remission; elimination or making the disease or its symptoms more tolerable for the patient; slowing the rate of deterioration or decline; or weakening the final stage of deterioration. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician’s examination. Therefore, the term "treatment" includes the administration of the antibodies or compositions or conjugates or immunofusions disclosed herein to prevent or delay, alleviate, or prevent or inhibit the development of symptoms associated with a disease or condition (e.g., IGF-1R and / or TSHR-mediated diseases).

[0145] As used in this article, the term "effective dose" refers to a dose administered to a subject that is sufficient to treat the disease.

[0146] As used herein, the singular forms “a,” “an,” and “the” include plural objects unless the context clearly indicates otherwise. Thus, for example, references to “an antibody” include multiple antibodies, and in some embodiments, references to “an antibody” include multiple antibodies, and so on.

[0147] Unless otherwise stated or defined, the terms “comprising,” “including,” “containing,” and “having” should be understood to mean including the said element or step or group of elements or steps, but not excluding any other element or step or group of elements or steps.

[0148] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0149] Antigen-binding protein that binds to IGF-1R

[0150] In a first aspect, the present invention provides an antigen-binding protein that specifically binds to IGF-1R, said antigen-binding protein comprising a VHH domain, wherein

[0151] (i) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:4; or

[0152] (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:8; or

[0153] (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:12; or

[0154] (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:16; or

[0155] (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:20; or

[0156] (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:24; or

[0157] (vii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:25; or

[0158] (viii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:26; or

[0159] (ix) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:27; or

[0160] (x) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:28.

[0161] In some implementations, the CDR sequence is defined according to the AbM definition rule. In some implementations, the CDR sequence is defined according to the Kabat definition rule. In some implementations, the CDR sequence is defined according to the Chothia definition rule. In some implementations, the CDR sequence is defined according to the Combined definition rule. In some implementations, the CDR sequence is defined according to the CONTACT definition rule.

[0162] When the CDR sequence is defined according to the Abm definition rules, (i) the VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having the amino acid sequences shown in SEQ ID NO:1 (GFDFSNYYMS), SEQ ID NO:2 (SVNAAGDLTR), and SEQ ID NO:3 (RRRLAYARYGNDYDY), respectively; or (ii) the VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having the amino acid sequences shown in SEQ ID NO:5 (GFDFSNYYMY), SEQ ID NO:6 (SVNPTGDMTR), and SEQ ID NO:7 (RRRLAYARTGNDYDY), respectively; or (iii) the VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having the amino acid sequences shown in SEQ ID NO:9 (GFIFSNYYMS), SEQ ID NO:10 (SVNPAGDMTR), and SEQ ID NO:3 (RRRLAYARYGNDYDY), respectively. Heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having the amino acid sequences shown in SEQ ID NO:11 (RRRLAYSRTGNDYDY); or (iv) the VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having the amino acid sequences shown in SEQ ID NO:13 (GFTLDELHIG), SEQ ID NO:14 (CIAGRAGLSYYTY), and SEQ ID NO:15 (VASYFRQCRLSHAEYAH); or (v) the VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having the amino acid sequences shown in SEQ ID NO:17 (GSTFSLYDMG), SEQ ID NO:18 (GNSWRGGTTY), and SEQ ID NO:19 (GGDYSRKNYEGNY); or (vi) the VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having the amino acid sequences shown in SEQ ID NO:21 (GFSLDYYAIG), SEQ ID NO:22 (CITNPGGRIN), and SEQ ID NO:19 (GFSLDYYAIG), respectively. The heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are shown in NO:23(QRSMQSNYCPRAPGSQYDY).

[0163] In some embodiments, (i) the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; or (ii) the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; or (iii) the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; or (iv) the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4. NO:16 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20; or (v) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20; or (vi) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:24; or (vii) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:24; or (vii) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20. NO:25 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:25; or (viii) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:26; or (ix) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:27; or (x) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:25; NO:28 has an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0164] In some embodiments, VHH includes a functional variant formed by inserting, deleting and / or substituting one or more amino acids in an amino acid sequence such as SEQ ID NO:4, 8, 12, 16, 20, 24, 25, 26, 27 and 28, provided that the functional variant retains the ability to bind IGF-1R.

[0165] The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.

[0166] In the context of functional variants, the number of inserted, deleted, and / or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parental amino acid sequence, more preferably not more than 35%, more preferably 1% to 33%, and even more preferably 5% to 30%, more preferably 10% to 25%, and even more preferably 15% to 20%. For example, the number of inserted, deleted, and / or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and most preferably 1 to 2. In preferred embodiments, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0167] In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Such a conservative substitution is preferably a substitution in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0168] The particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0169] In some embodiments, (i) the VHH comprises the amino acid sequence shown in SEQ ID NO:4; or (ii) the VHH comprises the amino acid sequence shown in SEQ ID NO:8; or (iii) the VHH comprises the amino acid sequence shown in SEQ ID NO:12; or (iv) the VHH comprises the amino acid sequence shown in SEQ ID NO:16; or (v) the VHH comprises the amino acid sequence shown in SEQ ID NO:20; or (vi) the VHH comprises the amino acid sequence shown in SEQ ID NO:24; or (vii) the VHH comprises the amino acid sequence shown in SEQ ID NO:25; or (viii) the VHH comprises the amino acid sequence shown in SEQ ID NO:26; or (ix) the VHH comprises the amino acid sequence shown in SEQ ID NO:27; or (x) the VHH comprises the amino acid sequence shown in SEQ ID NO:28.

[0170] In some embodiments of the antigen-binding protein of the present invention, the antigen-binding protein is an antibody.

[0171] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a heavy chain antibody. In some embodiments, the antibody is a humanized chimeric antibody.

[0172] The antibodies disclosed in this article may be complete antibodies or their antigen-binding fragments.

[0173] In some embodiments, the antibody is a heavy chain antibody (HCAb). In some embodiments, the antigen-binding fragment is an sdAb (single-domain antibody).

[0174] In one embodiment, the heavy chain antibody comprises a VHH domain, the domain being composed of frames 1, CDR1, 2, CDR2, 3, CDR3, and 4. In another embodiment, the heavy chain antibody comprises a VHH domain, at least a portion of a hinge region, and CH2 and CH3 domains. In yet another embodiment, the heavy chain antibody comprises a VHH domain, at least a portion of a hinge region, and a CH2 domain. In yet another embodiment, the heavy chain antibody comprises a VHH domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. Heavy chain antibodies may be in dimer form, wherein two heavy chains are linked by disulfide bonds, or otherwise covalently or non-covalently attached to each other. Heavy chain antibodies may belong to IgG subclasses, such as camel IgG2 and IgG3. Non-limiting examples of heavy chain antibodies are described, for example, in WO2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0175] In some implementations, the antigen-binding protein is a camelid antibody, a humanized antibody, or a chimeric antibody.

[0176] In some implementations, the antigen-binding protein is a bispecific antibody or a multispecific antibody.

[0177] In some embodiments, the antigen-binding protein is a bispecific antibody, which further includes a second antigen-binding region that binds to a second antigen. In some embodiments, the second antigen is an autoantigen.

[0178] In a preferred embodiment, the second antigen is TSHR, and the second antigen-binding region comprises VL and VH, wherein VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequence shown in SEQ ID NO:36; and VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence shown in SEQ ID NO:32. Preferably, VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO:33-35, and VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO:29-31.

[0179] In some embodiments, the second antigen-binding region comprises VL and VH, wherein VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:36, and VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:32.

[0180] In some embodiments, VL comprises a functional variant of the amino acid sequence shown in SEQ ID NO:36, formed by inserting, deleting, and / or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to TSHR. In some embodiments, VH comprises a functional variant of the amino acid sequence shown in SEQ ID NO:32, formed by inserting, deleting, and / or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to TSHR.

[0181] In some embodiments, the number of inserted, deleted, and / or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parental amino acid sequence, more preferably not more than 35%, even more preferably 1% to 33%, and even more preferably 5% to 30%, more preferably 10% to 25%, and even more preferably 15% to 20%. For example, the number of inserted, deleted, and / or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and most preferably 1 to 2. In preferred embodiments, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0182] In some implementations, insertions, deletions, and / or substitutions can be performed in frame (FR) regions, such as FR1, FR2, FR3, and / or FR4.

[0183] In some implementations, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

[0184] In a preferred embodiment, the second antigen-binding region comprises VL and VH, wherein VL comprises the amino acid sequence shown in SEQ ID NO:36, and VH comprises the amino acid sequence shown in SEQ ID NO:32.

[0185] In some embodiments, the adapter comprises an amino acid sequence selected from (G4S)n, where n is an integer selected from 1-5. In some embodiments, the adapter comprises an amino acid sequence such as any one of SEQ ID NO:47-49.

[0186] In some embodiments, (i) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:37; or

[0187] (ii) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:39; or

[0188] (iii) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:40; or

[0189] (iv) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:41; or

[0190] (v) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:43, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:42.

[0191] In a preferred embodiment, (i) the light chain comprises the sequence shown in SEQ ID NO:38 and the heavy chain comprises the sequence shown in SEQ ID NO:37; or (ii) the light chain comprises the sequence shown in SEQ ID NO:38 and the heavy chain comprises the sequence shown in SEQ ID NO:39; or (iii) the light chain comprises the sequence shown in SEQ ID NO:38 and the heavy chain comprises the sequence shown in SEQ ID NO:40; or (iv) the light chain comprises the sequence shown in SEQ ID NO:38 and the heavy chain comprises the sequence shown in SEQ ID NO:41; or (v) the light chain comprises the sequence shown in SEQ ID NO:43 and the heavy chain comprises the sequence shown in SEQ ID NO:42.

[0192] Bispecific antibodies

[0193] Secondly, the present invention provides a bispecific antibody comprising a first antigen-binding region for binding IGF-1R and a second antigen-binding region for binding TSHR, wherein the first antigen-binding region comprises a VHH domain (e.g., the antigen-binding protein specifically binding IGF-1R disclosed in the first aspect of the present invention), and the second antigen-binding region comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein,

[0194] (i) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:4; or

[0195] (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:8; or

[0196] (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:12; or

[0197] (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:16; or

[0198] (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:20; or

[0199] (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:24; or

[0200] (vii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:25; or

[0201] (viii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:26; or

[0202] (ix) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:27; or

[0203] (x) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:28.

[0204] In some implementations, in the first antigen-binding region:

[0205] (i) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:1-3; or

[0206] (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:5-7; or

[0207] (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO: 9-11; or

[0208] (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:13-15, respectively; or

[0209] (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:17-19; or

[0210] (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having the amino acid sequences shown in SEQ ID NO:21-23, respectively; and the VL in the second antigen-binding region comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequences shown in SEQ ID NO:36; and the VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequences shown in SEQ ID NO:32; preferably, the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO:33-35, respectively, and the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO:29-31, respectively.

[0211] In some embodiments, the first antigen-binding region comprises: (i) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; or (ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; or (iii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; or (iv) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; NO:16 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20; or (v) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20; or (vi) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:24; or (vii) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:25; or (viii) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:25. NO:26 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:27; or (ix) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:28; or (x) the VHH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:28.

[0212] In some embodiments, the VL in the second antigen-binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:36, and the VH in the second antigen-binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:32.

[0213] In some embodiments, VHH comprises a functional variant of the amino acid sequence shown in any one of SEQ ID NO:4, 8, 12, 16, 20, 24, 25, 26, 27 and 28, which is formed by inserting, deleting and / or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind IGF-1R.

[0214] In some embodiments, VL comprises a functional variant of the amino acid sequence shown in SEQ ID NO:36, formed by inserting, deleting, and / or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind TSHR. In some embodiments, VH comprises a functional variant of the amino acid sequence shown in SEQ ID NO:32, formed by inserting, deleting, and / or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind TSHR.

[0215] The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.

[0216] In the content of the functional variant, the number of inserted, deleted, and / or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parental amino acid sequence, more preferably not more than 35%, even more preferably 1% to 33%, more preferably 5% to 30%, even more preferably 10% to 25%, and even more preferably 15% to 20%. For example, the number of inserted, deleted, and / or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and most preferably 1 to 2. In a preferred embodiment, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0217] In some implementations, insertions, deletions, and / or substitutions can be performed in frame (FR) regions, such as FR1, FR2, FR3, and / or FR4.

[0218] In some implementations, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

[0219] In some embodiments, (i) the VHH comprises the amino acid sequence shown in SEQ ID NO:4; or (ii) the VHH comprises the amino acid sequence shown in SEQ ID NO:8; or (iii) the VHH comprises the amino acid sequence shown in SEQ ID NO:12; or (iv) the VHH comprises the amino acid sequence shown in SEQ ID NO:16; or (v) the VHH comprises the amino acid sequence shown in SEQ ID NO:20; or (vi) the VHH comprises the amino acid sequence shown in SEQ ID NO:24; or (vii) the VHH comprises the amino acid sequence shown in SEQ ID NO:25; or (viii) the VHH comprises the amino acid sequence shown in SEQ ID NO:26; or (ix) the VHH comprises the amino acid sequence shown in SEQ ID NO:27; or (x) the VHH comprises the amino acid sequence shown in SEQ ID NO:28.

[0220] In some embodiments, the VL contains an amino acid sequence as shown in SEQ ID NO:36 and the VH contains an amino acid sequence as shown in SEQ ID NO:32.

[0221] In some implementations, the binding valence ratio of the first antigen-binding region and the second antigen-binding region in the bispecific antibody is 1:1, 2:1 or 1:2, preferably 1:1.

[0222] In some embodiments of the bispecific antibody of the present invention, the bispecific antibody comprises (i) a heavy chain comprising, from the N-terminus to the C-terminus, the VH, heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3); and (ii) a light chain comprising, from the N-terminus to the C-terminus, the VL and light chain constant region (CL); wherein the VHH is optionally attached to the N-terminus or C-terminus of the heavy chain or the light chain via a linker.

[0223] In some embodiments, (i) the heavy chain from N end to C end comprises: VHH-connector-VH-CH1-CH2-CH3; and the light chain from N end to C end comprises: VL-CL; or (ii) the heavy chain from N end to C end comprises: VH-CH1-CH2-CH3-connector-VHH; and the light chain from N end to C end comprises: VL-CL; or (iii) the heavy chain from N end to C end comprises: VH-CH1-CH2-CH3; and the light chain from N end to C end comprises: VL-CL-connector-VHH; or (iv) the heavy chain from N end to C end comprises: VH-CH1-CH2-CH3; and the light chain from N end to C end comprises: VHH-connector-VL-CL.

[0224] In some embodiments, the linker can be any flexible linker. In some embodiments, the linker comprises an amino acid sequence selected from (G4S)n, where n is an integer selected from 1-5. In some embodiments, the linker may comprise the amino acid sequence (GGGGS)2 (SEQ ID NO:47). In some embodiments, the linker may comprise the amino acid sequence (GGGGS)3 (SEQ ID NO:48). In some embodiments, the linker may comprise the amino acid sequence (GGGGS)4 (SEQ ID NO:49).

[0225] The bispecific antibodies disclosed in this article may contain Fc regions of CH2 and CH3 containing the antibody.

[0226] The Fc region can be any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and may contain one or more mutations or modifications. In one embodiment, the Fc region is an IgG1 isotype or derived therefrom, optionally having one or more mutations or modifications. In another embodiment, the Fc region is an IgG4 isotype or derived therefrom, optionally having one or more mutations or modifications. In one embodiment, the Fc region is human IgG1 Fc.

[0227] In one embodiment, the Fc region has an extended half-life. An increase in the circulating half-life of an antibody, antibody Fc region conjugate, or immunofusion can be achieved by increased binding to nascent Fc receptors and results in improved efficacy, reduced dosage or administration frequency, or improved target delivery. In one embodiment, the Fc region having altered binding affinity for FcRn is an Fc region having amino acid alterations at one or more of the following amino acid positions: 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439, and / or 447. In a preferred embodiment, the antibody of the present invention comprises YTE mutations (M252Y, S254T, T256E).

[0228] In one implementation, the Fc region has reduced effector function, such as reduced binding to ADCC, ADCP, CDC and / or Clq, FcγRI, FcγRII, or FcγRIIIA. For example, the Fc region may be an IgG1 isotype or a non-IgG1 type, such as IgG2, IgG3, or IgG4, which has been mutated to reduce or even eliminate its ability to mediate effector function. Such mutations have been described, for example, in Dall'Acqua WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.; 75(24):12161-12168 (2001). For example, compared to the wild-type sequence, the Fc region may contain an amino acid sequence with one or more of the following amino acid substitutions: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G. In a preferred embodiment, the antibody of the present invention comprises LALA mutations (L234A and L235A).

[0229] In one embodiment, the Fc region contains a mutation that removes the Asn-linked glycosylation receptor site or is otherwise manipulated to alter the glycosylation properties. For example, in the IgG1 Fc region, the N297Q mutation can be used to remove the Asn-linked glycosylation site. Therefore, in a specific embodiment, the Fc region contains an IgG1 sequence with the N297Q mutation.

[0230] In a further embodiment, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, for example by adding a compound to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, it can be used... The method described by Natsume et al. (1999) Nature Biotech 17:176 optimizes ADCC. In another implementation, the Fc region is engineered to enhance complement activation, as described by Natsume et al. (2009) Cancer Sci. 100:2411.

[0231] In some implementations, the Fc region contains modifications or mutations that can inhibit Fc homodimerization, such as mortis mutations. Detailed descriptions of the mortis concept can be found, for example, in U.S. Patent Nos. 5,731,168 and 7,186,076; and Ridgway et al., Protein Engineering, Design and Selection, 1996, 9(7):617–621; Atwell et al., J Mol Biol, 1997, 270(1):26-35; Merchant et al., Nat Biotechnol, 1998, 16:677-681; and Carter, J. Immunological Methods, 2001, 24(1-2):7-15. In short, a pestle can be generated at the CH3 domain interface of the first IgG Fc chain by replacing a smaller amino acid side chain with a larger one (e.g., T366W); and a mortar can be generated at a tandem position at the CH3 interface of the second IgG Fc chain by replacing a larger one with a smaller amino acid side chain (e.g., Y407V). Exemplary amino acid substitutions in the CH3 domain forming the pestle include, but are not limited to, T366W, T366Y, or F405W substitutions. Exemplary amino acid substitutions in the CH3 domain forming the mortar include, but are not limited to, T366S, L368A, F405A, Y407A, Y407T, and Y407V substitutions.

[0232] In some embodiments of the bispecific antibody of the present invention, (i) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:37; or

[0233] (ii) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:39; or

[0234] (iii) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:40; or

[0235] (iv) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:41; or

[0236] (v) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:43, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:42.

[0237] In some embodiments, the light chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO:38 or 43, formed by inserting, deleting, and / or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to IGF-1R and TSHR. In some embodiments, the heavy chain comprises a functional variant of the amino acid sequence shown in any one of SEQ ID NO:37, 39, 40, 41, and 42, formed by inserting, deleting, and / or substituting one or more amino acids therein, the precursor being a functional variant that retains the ability to bind to IGF-1R and TSHR.

[0238] The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.

[0239] In some embodiments, the number of inserted, deleted, and / or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parental amino acid sequence, more preferably not more than 35%, more preferably 1% to 33%, and even more preferably 5% to 30%, more preferably 10% to 25%, and even more preferably 15% to 20%. For example, the number of inserted, deleted, and / or substituted amino acids can be 1 to 50, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. In preferred embodiments, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0240] In some implementations, insertions, deletions, and / or substitutions may be performed in frame (FR) regions, such as FR1, FR2, FR3, and / or FR4; and / or constant regions, such as CL, CH1, CH2, and / or CH3.

[0241] In some implementations, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

[0242] In a preferred embodiment, the light chain comprises the amino acid sequence shown in SEQ ID NO:38; and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:37. In a preferred embodiment, the light chain comprises the amino acid sequence shown in SEQ ID NO:38; and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:39. In a preferred embodiment, the light chain comprises the amino acid sequence shown in SEQ ID NO:38; and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:40. In a preferred embodiment, the light chain comprises the amino acid sequence shown in SEQ ID NO:38; and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:41. In a preferred embodiment, the light chain comprises the amino acid sequence shown in SEQ ID NO:43; and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:42.

[0243] Nucleic acid

[0244] This disclosure provides nucleic acids containing nucleotide sequences encoding antigen-binding proteins or bispecific antibodies disclosed herein.

[0245] The term "nucleic acid" includes single-stranded and double-stranded nucleotide polymers. Nucleic acids can be ribonucleotides or deoxyribonucleotides or modified forms of any type of nucleotide. These modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and nucleotide bond modifications such as thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, phenylthiophosphates, aniline phosphates, and aminophosphates.

[0246] For example, the present invention provides a nucleic acid molecule encoding any of the VHH sequences disclosed herein. The present invention also provides a nucleic acid molecule having at least 90%, at least 95%, at least 98%, or at least 99% identity with a nucleic acid encoding any of the VHH sequences disclosed herein.

[0247] For example, the present invention provides a nucleic acid molecule encoding any of the VH sequences disclosed herein. The present invention also provides a nucleic acid molecule having at least 90%, at least 95%, at least 98%, or at least 99% identity with a nucleic acid encoding any of the VH sequences disclosed herein.

[0248] For example, the present invention provides nucleic acid molecules encoding any of the VL sequences disclosed herein. The present invention also provides nucleic acid molecules having at least 90%, at least 95%, at least 98%, or at least 99% identity with nucleic acids encoding any of the VL sequences disclosed herein.

[0249] For example, the present invention provides a nucleic acid molecule encoding a VHH sequence comprising a CDR sequence of any VHH sequence disclosed herein. The present invention also provides a nucleic acid molecule encoding a VHH sequence comprising a CDR sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity with a CDR sequence of any VHH sequence disclosed herein.

[0250] For example, the present invention provides a nucleic acid molecule encoding a VH sequence comprising a CDR sequence of any VH sequence disclosed herein. The present invention also provides a nucleic acid molecule encoding a VH sequence comprising a CDR sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity with a CDR sequence of any VH sequence disclosed herein.

[0251] For example, the present invention provides a nucleic acid molecule encoding a VL sequence comprising a CDR sequence of any VL sequence disclosed herein. The present invention also provides a nucleic acid molecule encoding a VL sequence comprising a CDR sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity with a CDR sequence of any VL sequence disclosed herein.

[0252] In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the present invention provides ribonucleic acid (RNA) comprising a nucleotide sequence encoding an antibody disclosed herein. In some embodiments, the present invention provides deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an antibody disclosed herein.

[0253] In some embodiments, deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a vector or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of a cell.

[0254] In some embodiments, ribonucleic acid (RNA) can be introduced into human cells in vivo. In some embodiments, the ribonucleic acid (RNA) of the present invention is contained in a vector or delivery agent.

[0255] carrier

[0256] This disclosure provides vectors containing the nucleic acids disclosed herein.

[0257] In some embodiments, the vector is an expression vector capable of expressing a polypeptide containing an antibody, such as VHH, VH, or VL. For example, the present invention provides an expression vector comprising any of the above-described nucleic acid molecules.

[0258] Any vector may be used in this disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0259] The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression, or both, such as plasmids and viruses. For example, vectors can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors that can be used in this disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors that can be used in this disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0260] Recombinant expression vectors can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain the functionality of the replication system in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, COLEL, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0261] For example, the vector could be an adenoviral vector containing a nucleotide sequence encoding an antibody disclosed herein. The vector could be administered to a subject, then enter the subject's cells, thereby integrating the nucleotide sequence encoding the antibody disclosed herein into the cell's genome, and subsequently the cells express the antibody disclosed herein.

[0262] host cells

[0263] This disclosure provides host cells containing the nucleic acids or vectors disclosed herein.

[0264] Any cell can be used as the host cell for the nucleic acids or vectors disclosed herein. In some embodiments, the cell may be a prokaryotic cell, fungal cell, yeast cell, or higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescens. The bacteria include *Bacillus subtilis* and *Shigella*; *Bacillus* species, such as *B. subtilis* and *B. licheniformis*; *Pseudomonas* species, such as *P. aeruginosa*; and *Streptomyces*. In some embodiments, the cells are human cells. In some embodiments, the cells are immune cells. In some embodiments, the host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0265] The host cells of the present invention are prepared by introducing the vectors or nucleic acids disclosed herein in vitro or ex vivo. The host cells of the present invention can be administered to a subject, and the host cells express the antibodies disclosed herein in vivo.

[0266] This invention provides host cells in which any of the aforementioned vectors have been introduced. This invention also provides a method for preparing the antibodies of this invention, the method comprising a) culturing the host cells of the fifth aspect of this invention under conditions suitable for antibody production; and b) obtaining the antibodies from the culture.

[0267] Pharmaceutical Composition

[0268] This disclosure provides pharmaceutical compositions comprising the antigen-binding protein disclosed herein, or the bispecific antibody disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0269] The antibodies or antigen-binding fragments or agents thereof (also referred to herein as “active compounds”) of the present invention, and their derivatives, fragments, analogs, and homologues, may be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise antibodies or antigen-binding fragments or agents thereof, along with a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solution, dextran solution, and 5% human serum albumin. Liposomes and non-aqueous media such as non-volatile oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Additional active compounds may also be incorporated into the composition.

[0270] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.

[0271] Conjugates or immune fusion compounds

[0272] This disclosure provides conjugates or immunofusions comprising the antigen-binding protein or the bispecific antibody disclosed herein, and a chemical moiety conjugated or fused thereto.

[0273] In the context of this disclosure, a "conjugate" or "immunofusion" is an antigen-binding protein covalently linked to a chemical moiety. The chemical moiety can be, for example, a drug, toxin, therapeutic agent, detectable marker, protein, nucleic acid, lipid, nanoparticle, carbohydrate, or recombinant virus. Antibody conjugates or antibody fusion proteins are commonly referred to as "immunoconjugates" or "immunofusions." When a conjugate or immunofusion contains an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is commonly referred to as an "antibody-drug conjugate" or "ADC."

[0274] The terms "fusion" or "linkage" can refer to the formation of a single, continuous polypeptide molecule from two polypeptides. The terms "conjugation" or "linkage" can refer to the linking of a small, non-protein molecule to a protein to form a biomolecule. In one embodiment, an antibody is linked to a chemical moiety. In another embodiment, the antibody linked to the chemical moiety is further linked to a lipid or other molecule to a protein or peptide to increase its half-life in vivo. Linkage can be performed chemically or recombinantly. In one embodiment, the linking is chemical, wherein a reaction between the antibody moiety and the chemical moiety produces a covalent bond formed between the two molecules to form a single molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the chemical moiety.

[0275] The chemical moiety can be linked to the antibody of the present invention using any number of methods known to those skilled in the art. Covalent and non-covalent attachment methods can be used. The procedure for attaching the chemical moiety to the antibody varies depending on the chemical structure of the chemical moiety. Peptides typically contain multiple functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) moieties, which can be used to react with suitable functional groups on the antibody to result in the binding of the chemical moiety. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. Derivatization can involve attaching any of a number of known linker molecules. The linker can be any molecule used to link the antibody to the chemical moiety. The linker is capable of forming a covalent bond with both the antibody and the chemical moiety. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In the case where the antibody and chemical moiety are peptides, the linker can be linked via its side groups to the constituent amino acid (e.g., via a disulfide bond to cysteine) or to the α-carbon amino and carboxyl groups of the terminal amino acid.

[0276] In some cases, when an immunoconjugate or immunofusion reaches its target site, it is desirable to release a chemical moiety from the antibody. Therefore, in these cases, the immunoconjugate or immunofusion will contain a cleavable linker near the target site.

[0277] Enzymatic activity or conditions experienced by immune conjugates or immune fusions within or near target cells may induce linker cleavage to release the chemical portion from the antibody.

[0278] Given the numerous methods reported for attaching various radiodiagnostic compounds, radiotherapy compounds, markers (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, those skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other peptide.

[0279] In some embodiments of the conjugates or immunofusions disclosed in this invention, the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.

[0280] Chimeric antigen receptor

[0281] This disclosure provides chimeric antigen receptors comprising the antigen-binding proteins disclosed herein, or the bispecific antibodies disclosed herein. This disclosure also provides genetically modified cells comprising the chimeric antigen receptors disclosed herein.

[0282] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule engineered to contain an antigen-binding domain that targets a specific antigen, and upon binding to that antigen, activates immune cells (such as T cells or NK cells, including naive T cells, central memory T cells, effector memory T cells, or combinations thereof) to attack and destroy cells carrying that antigen. When these antigens are present on tumor cells, CAR-expressing immune cells can target and kill the tumor cells.

[0283] A classic chimeric antigen receptor (CAR) is a chimeric type I transmembrane protein that links an extracellular antigen-binding domain to an intracellular signaling domain. The antigen-binding domain is typically an antigen-binding fragment derived from a monoclonal antibody (mAb) (e.g., scFv), but it can be based on other forms containing antibody-like antigen-binding sites or on a native ligand derived from the antigen. A hinge domain is usually required to separate the antigen-binding domain from the membrane and allow for its proper orientation. A common hinge domain used is the Fc of IgG1. Depending on the antigen, more compact spacer regions may be suitable, such as the stem from CD8α, and even just the IgG1 hinge alone. The transmembrane domain anchors the protein within the cell membrane and connects the hinge domain to the intracellular domain (intramocyte domain).

[0284] According to at least one non-limiting viewpoint, at least three “generations” of CAR molecules have existed. In first-generation CARs, they are designed with an intracellular domain having an intracellular portion of the γ chain derived from FcεR1 or the intracellular portion of CD3ζ. Therefore, these first-generation CARs deliver an immune signal¹ sufficient to trigger T cell killing of homologous target cells, but cannot fully activate T cell proliferation and survival. To overcome this limitation, second-generation CARs have been constructed with a complex intracellular domain resulting from the fusion of the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3ζ, thus enabling the simultaneous delivery of activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is the CD28 costimulatory domain. This provides the most potent costimulatory signal—i.e., an immune signal²—that triggers T cell proliferation. Several CARs have also been described that include intracellular domains of the TNF receptor family, such as the closely associated OX40 and 41BB, which deliver survival signals. Even more potent third-generation CARs have now been described, possessing intracellular domains capable of delivering activation, proliferation, and survival signals.

[0285] Therefore, a CAR typically comprises: (i) an antigen-binding domain; (ii) a hinge domain; (iii) a transmembrane domain; and (iv) an intracellular domain that includes a signal transduction domain and one or more co-stimulatory domains.

[0286] An "antigen-binding domain" refers to the portion of a chimeric antigen receptor that recognizes an antigen. In a classic CAR, the antigen-binding domain comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody. CARs have also been generated using domain-specific antibodies (dAbs), VHH antigen-binding domains, or antigen-binding domains derived from natural ligands of the antigen. In this application, the antigen-binding domain may be the antibody or its antigen-binding fragment described herein.

[0287] The "hinge domain" positions the antigen-binding domain away from the effector cell surface to facilitate proper cell / cell contact, antigen binding, and activation. CARs optionally include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can contain the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0288] As used herein, a “transmembrane domain” (TM domain) refers to a portion of a CAR that optionally fuses an extracellular binding portion with an intracellular portion (e.g., a co-stimulatory domain and an intracellular signaling domain) via a hinge domain, and anchors the CAR to the plasma membrane of an immune effector cell. A transmembrane domain is typically a hydrophobic region of the CAR that crosses the cell membrane. A TM domain can be a transmembrane region or fragment of a transmembrane protein (e.g., a type I transmembrane protein or other transmembrane proteins), an artificial hydrophobic sequence, or a combination thereof.

[0289] The intracellular domain (intracytoplasmic domain) is the signaling portion of a chimeric antigen receptor. It contains a signal transduction domain and one or more co-stimulatory domains. Upon antigen recognition, the receptor clusters native CD45 and CD148 are expelled from the synapse, and signals are transduced into the cell, thereby activating one or more immune cell effector functions (e.g., innate immune cell effector functions). The most commonly used intracellular domain component is the CD3ζ intracellular domain component containing three ITAMs. After antigen binding, it transmits an activation signal to T cells. CD3ζ may not provide a completely sufficient activation signal, and additional co-stimulatory signal transduction may be required. Co-stimulatory signals promote T cell proliferation and survival.

[0290] "Intracellular signal transduction domain" refers to a portion of the CAR polypeptide that participates in transducing information about the binding of an effective CAR to a target antigen into immune effector cells to trigger effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors into CAR-bound target cells or other cellular responses triggered after the antigen binds to the extracellular CAR domain.

[0291] The "co-stimulatory domain" refers to the intracellular signal transduction domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide the second signal required for effective activation and function of T lymphocytes when binding to antigens.

[0292] Treatment methods and uses

[0293] This disclosure provides a method for treating IGF-1R and / or TSHR-mediated disease in a subject, comprising administering to the subject an effective amount of the antigen-binding protein disclosed herein, the bispecific antibody disclosed herein, the pharmaceutical composition disclosed herein, the conjugate or immunofusion disclosed herein, or the chimeric antigen receptor disclosed herein.

[0294] In some embodiments, the IGF-1R and / or TSHR-mediated diseases are autoimmune diseases. In some embodiments, the IGF-1R and / or TSHR-mediated diseases are cancers, such as hematologic malignancies or solid tumors. In a preferred embodiment, the IGF-1R and / or TSHR-mediated diseases are Graves' disease or thyroid eye disease (TED).

[0295] In some implementation methods, the dose administered to the subject may vary depending on the implementation method, the drug used, the method of administration, and the site of treatment and the subject. However, the dose should be sufficient to provide a therapeutic response. Clinicians can determine the effective amount to administer to a person or other subject to treat a medical condition. The precise amount required for effective treatment may depend on many factors, such as antibody activity and the route of administration.

[0296] The dosage of the antibodies, compositions, conjugates, or immunofusions described herein may be administered to mammals in a single dose or in a series of sub-dose over an appropriate period of time, such as daily, bi-weekly, weekly, bi-weekly, bi-weekly, bi-monthly, semi-annually, or annually as needed. Dosage units containing an effective amount of the antibody, composition, conjugate, or immunofusion may be administered as a single daily dose, or the total daily dose may be administered as needed in two, three, four, or more daily sub-dose administrations.

[0297] The appropriate route of administration can be chosen by the physician. Administration routes may include parenteral administration, such as by injection, nasal administration, pulmonary administration, or percutaneous administration. Systemic or local administration may be performed via intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. In some embodiments, antibodies, compositions or conjugates, or immunofusions are selected for parenteral delivery, inhalation, or delivery via the digestive tract, such as oral administration. The dosage and method of administration can vary depending on the subject's weight, age, condition, etc., and can be appropriately selected.

[0298] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or forms. As used herein, the terms “combined” or “in combination” administration should be understood as the delivery of two (or more) different treatments to a subject during the course of the subject’s illness, such that the effects of the treatments on the patient overlap at some point in time. In some embodiments, the delivery of one treatment is still in progress when the second treatment begins, thus creating an overlap in administration. This is sometimes referred to herein as “simultaneous” or “simultaneous delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective due to combined administration. For example, fewer doses of the second treatment may be observed when combined with the first treatment compared to when the second treatment is administered alone without the first treatment, or the second treatment may alleviate symptoms to a greater extent; or a similar situation may exist with the first treatment. In some embodiments, combined administration results in a reduction in symptoms or other parameters related to the illness that is greater than that observed in the absence of the other treatment. The effects of the two treatments may be partially additive, completely additive, or greater than additive. Combination therapy allows the effect of the first treatment to still be detectable when the second treatment is administered.

[0299] In some embodiments, the method further includes administering a second therapeutic agent to the subject. In some embodiments, the antibodies, compositions, conjugates, or immunofusions or chimeric antigen receptors disclosed herein are administered before, substantially simultaneously with, or after the administration of the second therapeutic agent.

[0300] This disclosure provides the use of the antigen-binding proteins, bispecific antibodies, pharmaceutical compositions, conjugates or immunofusions, or chimeric antigen receptors disclosed herein in the preparation of medicaments for treating IGF-1R and / or TSHR-mediated diseases in subjects.

[0301] This disclosure also provides antigen-binding proteins disclosed herein, bispecific antibodies disclosed herein, pharmaceutical compositions disclosed herein, conjugates or immunofusions disclosed herein, or chimeric antigen receptors disclosed herein for the treatment of subjects with IGF-1R and / or TSHR-mediated diseases.

[0302] In embodiments of the uses disclosed herein, the IGF-1R and / or TSHR-mediated diseases are autoimmune diseases. In some embodiments, the IGF-1R and / or TSHR-mediated diseases are cancers, such as hematologic malignancies or solid tumors. In preferred embodiments, the IGF-1R and / or TSHR-mediated diseases are Graves' disease or thyroid eye disease (TED).

[0303] In some embodiments, the antigen-binding protein disclosed herein, the bispecific antibody disclosed herein, the pharmaceutical composition disclosed herein, the conjugate or immunofusion compound disclosed herein, or the chimeric antigen receptor disclosed herein is combined with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.

[0304] Reagent test kit

[0305] This disclosure provides pharmaceutical packaging or kits comprising one or more containers containing one or more components of the pharmaceutical compositions described herein, such as antibodies or antigen-binding fragments disclosed herein.

[0306] In a specific embodiment, the kit includes a first container containing the antibody disclosed herein. In another specific embodiment, the kit includes a first container which is a vial containing the antibody as a lyophilized sterile powder under vacuum, and the kit also includes a second container containing a pharmaceutically acceptable fluid.

[0307] In a specific embodiment, this document provides an injection device containing antibodies. In a specific embodiment, the injection device contains antibodies in a sterile solution. In a specific embodiment, the injection device is a syringe.

[0308] In one embodiment, the kit includes instructional material disclosing how to use the antibodies of this disclosure. The instructional material may be in written, electronic (e.g., computer floppy disk or optical disc) or visual (e.g., video file). The kit may also include additional components to facilitate the application for which the kit is designed. Thus, for example, the kit may additionally contain tools for detecting labels (e.g., enzyme substrates for enzymatic labeling, filter sets for detecting fluorescent labels, suitable secondary labels such as secondary antibodies, etc.). The kit may also include buffers and other reagents conventionally used to perform a particular method. Such kits and suitable contents are well known to those skilled in the art.

[0309] The present invention also provides the following embodiments I-1 to I-32:

[0310] Implementation Scheme I-1. An antigen-binding protein or antigen-binding fragment thereof that specifically binds to IGF-1R, wherein the antigen-binding protein or antigen-binding fragment thereof comprises a single-chain variable region (VHH) of a single-domain antibody, wherein

[0311] (i) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:4; or

[0312] (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:8; or

[0313] (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:12; or

[0314] (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:16; or

[0315] (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:20; or

[0316] (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:24; or

[0317] (vii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:25; or

[0318] (viii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:26; or

[0319] (ix) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:27; or

[0320] (x) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:28.

[0321] Implementation Scheme I-2. The antigen-binding protein or its antigen-binding fragment as described in Implementation Scheme I-1, wherein...

[0322] (i) The VHH comprises heavy chains CDR 1-3 having amino acid sequences as shown in SEQ ID NO: 1-3; or

[0323] (ii) The VHH comprises heavy chain CDRs 1-3, each having an amino acid sequence as shown in SEQ ID NO: 5-7; or

[0324] (iii) The VHH comprises heavy chain CDRs 1-3, each having an amino acid sequence as shown in SEQ ID NO: 9-11; or

[0325] (iv) The VHH comprises heavy chain CDRs 1-3, each having an amino acid sequence as shown in SEQ ID NO: 13-15; or

[0326] (v) The VHH comprises heavy chain CDRs 1-3, each having an amino acid sequence as shown in SEQ ID NO: 17-19; or

[0327] (vi) The VHH comprises heavy chain CDR 1-3 having amino acid sequences as shown in SEQ ID NO:21-23.

[0328] Implementation Scheme I-3. The antigen-binding protein or its antigen-binding fragment as described in Implementation Scheme I-1 or Implementation Scheme I-2, wherein...

[0329] (i) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; or

[0330] (ii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; or

[0331] (iii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; or

[0332] (iv) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16; or

[0333] (v) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20; or

[0334] (vi) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:24; or

[0335] (vii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:25; or

[0336] (viii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:26; or

[0337] (ix) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:27; or

[0338] (x) The VHH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:28.

[0339] Implementation Scheme I-4. An antigen-binding protein or an antigen-binding fragment thereof according to any one of Implementation Schemes I-1 to I-3, wherein the antigen-binding protein is an antibody.

[0340] Implementation Scheme I-5. The antigen-binding protein or its antigen-binding fragment as described in Implementation Scheme I-4, wherein the antibody is a monoclonal antibody.

[0341] Implementation Scheme I-6. An antigen-binding protein or antigen-binding fragment thereof as described in Implementation Scheme I-4 or Implementation Scheme I-5, wherein the antibody is a single-domain antibody or a heavy chain antibody.

[0342] Implementation Scheme I-7. An antigen-binding protein or antigen-binding fragment thereof according to any one of Implementation Schemes I-4 to I-6, wherein the antibody is a camelid antibody, a humanized antibody or a chimeric antibody.

[0343] Implementation Scheme I-8. The antigen-binding protein or its antigen-binding fragment according to Implementation Scheme I-4, wherein the antibody is a bispecific antibody or a multispecific antibody.

[0344] Implementation Scheme I-9. An antigen-binding protein or antigen-binding fragment thereof according to Implementation Scheme I-8, wherein the antibody is a bispecific antibody and further comprises a second antigen-binding region that binds to a second antigen.

[0345] Implementation Scheme I-10. The antigen-binding protein or its antigen-binding fragment according to Implementation Scheme I-9, wherein the second antigen is an autoantigen, preferably TSHR.

[0346] Implementation Scheme I-11. A bispecific antibody or its antigen-binding fragment, comprising a first antigen-binding region for binding IGF-1R and a second antigen-binding region for binding TSHR, wherein the first antigen-binding region comprises a single-chain variable region (VHH) of a single-domain antibody, wherein

[0347] (i) The VHH comprises heavy chains CDR 1-3 having amino acid sequences as shown in SEQ ID NO: 1-3; or

[0348] (ii) The VHH comprises heavy chain CDRs 1-3, each having an amino acid sequence as shown in SEQ ID NO: 5-7; or

[0349] (iii) The VHH comprises heavy chain CDRs 1-3, each having an amino acid sequence as shown in SEQ ID NO: 9-11; or

[0350] (iv) The VHH comprises heavy chain CDRs 1-3, each having an amino acid sequence as shown in SEQ ID NO: 13-15; or

[0351] (v) The VHH comprises heavy chain CDRs 1-3, each having an amino acid sequence as shown in SEQ ID NO: 17-19; or

[0352] (vi) The VHH comprises heavy chain CDR 1-3 having amino acid sequences as shown in SEQ ID NO:21-23.

[0353] Implementation Scheme I-12. The bispecific antibody or its antigen-binding fragment according to Implementation Scheme I-11, wherein...

[0354] (i) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; or

[0355] (ii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; or

[0356] (iii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; or

[0357] (iv) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16; or

[0358] (v) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20; or

[0359] (vi) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:24; or

[0360] (vii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:25; or

[0361] (viii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:26; or

[0362] (ix) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:27; or

[0363] (x) The VHH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:28.

[0364] Implementation Scheme I-13. A bispecific antibody or antigen-binding fragment thereof according to Implementation Scheme I-11 or Implementation Scheme I-12, wherein the second antigen-binding region comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises LCDR 1-3 having amino acid sequences as shown in SEQ ID NO:33-35, and the VH comprises HCDR 1-3 having amino acid sequences as shown in SEQ ID NO:29-31.

[0365] Implementation Scheme I-14. The bispecific antibody or its antigen-binding fragment according to Implementation Scheme I-13, wherein the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:36 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:32.

[0366] Implementation Scheme I-15. A bispecific antibody or antigen-binding fragment thereof according to any one of Implementation Schemes I-11 to I-14, wherein the bispecific antibody comprises:

[0367] (i) a heavy chain, which from the N-end to the C-end includes the VH, heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3); and

[0368] (ii) A light chain that includes the VL and the light chain constant region (CL) from the N end to the C end;

[0369] The VHH is connected to the N or C end of the heavy chain or the light chain via an optional connector.

[0370] Implementation Scheme I-16. The bispecific antibody or its antigen-binding fragment according to Implementation Scheme I-15, wherein:

[0371] (i) The heavy chain from N-end to C-end comprises: VHH-connector-VH-CH1-CH2-CH3; and the light chain from N-end to C-end comprises: VL-CL; or

[0372] (ii) The heavy chain from N-end to C-end comprises: VH-CH1-CH2-CH3-connector-VHH; and the light chain from N-end to C-end comprises: VL-CL; or

[0373] (iii) The heavy chain from the N-end to the C-end comprises: VH-CH1-CH2-CH3; and the light chain from the N-end to the C-end comprises: VL-CL-connector-VHH; or

[0374] (iv) The heavy chain from the N end to the C end comprises: VH-CH1-CH2-CH3; and the light chain from the N end to the C end comprises: VHH-connector-VL-CL.

[0375] Implementation Scheme I-17. The bispecific antibody or antigen-binding fragment thereof according to Implementation Scheme I-16, wherein the linker comprises an amino acid sequence selected from (G4S)n, where n is an integer selected from 1 to 5.

[0376] Implementation Scheme I-18. The bispecific antibody or antigen-binding fragment thereof according to Implementation Scheme I-17, wherein the adapter comprises an amino acid sequence such as any one of SEQ ID NO:47-49.

[0377] Implementation Scheme I-19. A bispecific antibody or antigen-binding fragment thereof according to any one of Implementation Schemes I-15 to I-18, wherein

[0378] (i) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:37; or

[0379] (ii) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:39; or

[0380] (iii) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:40; or

[0381] (iv) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:41; or

[0382] (v) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:43, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:42.

[0383] Implementation Scheme I-20. Nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein or an antigen-binding fragment thereof according to any one of Implementation Schemes I-1 to I-10, or a bispecific antibody or an antigen-binding fragment thereof according to any one of Implementation Schemes I-11 to I-19.

[0384] Implementation scheme I-21. A vector comprising the nucleic acid described in implementation scheme I-20.

[0385] Implementation Scheme I-22. A host cell comprising the nucleic acid described in Implementation Scheme I-20 or the vector described in Implementation Scheme I-21.

[0386] Implementation Scheme I-23. A pharmaceutical composition comprising (i) an antigen-binding protein or an antigen-binding fragment thereof according to any one of Implementation Schemes I-1 to I-10, or a bispecific antibody or an antigen-binding fragment thereof according to any one of Implementation Schemes I-11 to I-19; and (ii) a pharmaceutically acceptable carrier or excipient.

[0387] Implementation Scheme I-24. The pharmaceutical composition according to Implementation Scheme I to Implementation Scheme I-23 further comprises a second therapeutic agent.

[0388] Implementation Scheme I-25. The pharmaceutical composition according to Implementation Scheme I to Implementation Scheme I-24, wherein the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs.

[0389] Implementation Scheme I-26. A conjugate comprising an antigen-binding protein or an antigen-binding fragment thereof according to any one of Implementation Schemes I-1 to I-10, or a bispecific antibody or an antigen-binding fragment thereof according to any one of Implementation Schemes I-11 to I-19, and a chemical moiety conjugated thereto.

[0390] Implementation Scheme I-27. The conjugate according to Implementation Scheme I-26, wherein the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.

[0391] Implementation Scheme I-28. A chimeric antigen receptor (CAR) comprising an antigen-binding protein or an antigen-binding fragment thereof according to any one of Implementation Schemes I-1 to I-10, or a bispecific antibody or an antigen-binding fragment thereof according to any one of Implementation Schemes I-11 to I-19.

[0392] Implementation Scheme I-29. A method for treating a disease in a subject, comprising administering to the subject an effective amount of an antigen-binding protein or an antigen-binding fragment thereof according to any one of Implementation Schemes I-1 to I-10, a bispecific antibody or an antigen-binding fragment thereof according to any one of Implementation Schemes I-11 to I-19, a pharmaceutical composition according to any one of Implementation Schemes I-23 to I-25, a conjugate according to Implementation Scheme I-26 or I-27, or a chimeric antigen receptor according to Implementation Scheme I-28, wherein the disease is an IGF-1R and / or TSHR-mediated disease.

[0393] Implementation Scheme I-30. The method according to Implementation Scheme I-29, wherein the disease is thyroid ophthalmopathy (TED).

[0394] Implementation Scheme I-31. The method according to Implementation Scheme I-30, wherein the method further includes administering a second therapeutic agent.

[0395] Implementation Scheme I-32. The method according to Implementation Scheme I-31, wherein the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs.

[0396] The present invention also provides the following embodiments II-1 to II-33:

[0397] Implementation Scheme II-1. An antigen-binding protein that specifically binds to IGF-1R, wherein the antigen-binding protein comprises a VHH domain, wherein

[0398] (i) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:4; or

[0399] (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:8; or

[0400] (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:12; or

[0401] (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:16; or

[0402] (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:20; or

[0403] (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:24; or

[0404] (vii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:25; or

[0405] (viii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:26; or

[0406] (ix) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:27; or

[0407] (x) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:28.

[0408] Implementation Scheme II-2. The antigen-binding protein according to Implementation Scheme II-1, wherein

[0409] (i) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:1-3; or

[0410] (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:5-7; or

[0411] (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO: 9-11; or

[0412] (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:13-15, respectively; or

[0413] (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:17-19; or

[0414] (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:21-23, respectively.

[0415] Implementation Scheme II-3. The antigen-binding protein according to Implementation Scheme II-1 or Implementation Scheme II-2, wherein

[0416] (i) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; or

[0417] (ii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; or

[0418] (iii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; or

[0419] (iv) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16; or

[0420] (v) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20; or

[0421] (vi) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:24; or

[0422] (vii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:25; or

[0423] (viii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:26; or

[0424] (ix) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:27; or

[0425] (x) The VHH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:28.

[0426] Implementation scheme II-4. An antigen-binding protein according to any one of implementation schemes II-1 to II-3, wherein the antigen-binding protein is an antibody.

[0427] Implementation scheme II-5. The antigen-binding protein according to implementation scheme II-4, wherein the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0428] Implementation scheme II-6. An antigen-binding protein according to any one of implementation schemes II-4 to II-5, wherein the antibody further comprises a second antigen-binding region that binds to a second antigen.

[0429] Implementation Scheme II-7. The antigen-binding protein according to Implementation Scheme II-6, wherein the second antigen is an autoantigen, preferably TSHR.

[0430] Implementation Scheme II-8. A bispecific antibody comprising a first antigen-binding region that binds to IGF-1R and a second antigen-binding region that binds to TSHR, wherein the first antigen-binding region comprises a VHH domain, wherein

[0431] (i) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:4; or

[0432] (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:8; or

[0433] (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:12; or

[0434] (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:16; or

[0435] (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:20; or

[0436] (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:24; or

[0437] (vii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:25; or

[0438] (viii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:26; or

[0439] (ix) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:27; or

[0440] (x) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:28.

[0441] Implementation Scheme II-9. The bispecific antibody according to Implementation Scheme II-8, wherein (i) the VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:1-3, respectively; or

[0442] (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:5-7; or

[0443] (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO: 9-11; or

[0444] (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:13-15, respectively; or

[0445] (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:17-19; or

[0446] (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:21-23, respectively.

[0447] Implementation Scheme II-10. The bispecific antibody according to Implementation Scheme II-9, wherein

[0448] (i) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; or

[0449] (ii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; or

[0450] (iii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; or

[0451] (iv) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16; or

[0452] (v) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20; or

[0453] (vi) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:24; or

[0454] (vii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:25; or

[0455] (viii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:26; or

[0456] (ix) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:27; or

[0457] (x) The VHH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:28.

[0458] Implementation Scheme II-11. A bispecific antibody according to any one of Implementation Schemes II-8 to II-10, wherein the second antigen-binding region comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequence shown in SEQ ID NO:36; and the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence shown in SEQ ID NO:32; preferably, the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequence shown in any one of SEQ ID NO:33-35, and the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence shown in any one of SEQ ID NO:29-31.

[0459] Implementation Scheme II-12. The bispecific antibody according to Implementation Scheme II-11, wherein the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:36 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:32.

[0460] Implementation Scheme II-13. The bispecific antibody according to any one of Implementation Schemes II-8 to II-12, wherein the VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:9-11; the VL comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in any one of SEQ ID NO:33-35; and the VH comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in any one of SEQ ID NO:29-31.

[0461] Implementation Scheme II-14. The bispecific antibody according to Implementation Scheme II-13, wherein the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:27; the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:36; and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:32.

[0462] Implementation Scheme II-15. The bispecific antibody according to any one of Implementation Scheme II- to Implementation Scheme II-14, wherein the binding valence ratio of the first antigen-binding region and the second antigen-binding region is 1:1, 2:1 or 1:2, preferably 1:1.

[0463] Implementation Scheme II-16. The bispecific antibody according to any one of Implementation Schemes II-8 to II-15, wherein the bispecific antibody comprises:

[0464] (i) a heavy chain, which from the N-end to the C-end includes the VH, heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3); and

[0465] (ii) A light chain that includes the VL and the light chain constant region (CL) from the N end to the C end;

[0466] The VHH is connected to the N or C end of the heavy chain or the light chain via an optional connector.

[0467] Implementation Scheme II-17. The bispecific antibody according to Implementation Scheme II-16, wherein:

[0468] (i) The heavy chain from N-end to C-end comprises: VHH-connector-VH-CH1-CH2-CH3; and the light chain from N-end to C-end comprises: VL-CL; or

[0469] (ii) The heavy chain from N-end to C-end comprises: VH-CH1-CH2-CH3-connector-VHH; and the light chain from N-end to C-end comprises: VL-CL; or

[0470] (iii) The heavy chain from the N-end to the C-end comprises: VH-CH1-CH2-CH3; and the light chain from the N-end to the C-end comprises: VL-CL-connector-VHH; or

[0471] (iv) The heavy chain from the N end to the C end comprises: VH-CH1-CH2-CH3; and the light chain from the N end to the C end comprises: VHH-connector-VL-CL.

[0472] Implementation Scheme II-18. The bispecific antibody according to Implementation Scheme II-17, wherein the linker comprises an amino acid sequence selected from (G4S)n, where n is an integer selected from 1 to 5.

[0473] Implementation Scheme II-19. The bispecific antibody according to Implementation Scheme II-18, wherein the adapter comprises an amino acid sequence such as any one of SEQ ID NO:47-49.

[0474] Implementation Scheme II-20. A bispecific antibody according to any one of Implementation Schemes II-8 to II-19, wherein

[0475] (i) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:37; or

[0476] (ii) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:39; or

[0477] (iii) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:40; or

[0478] (iv) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:41; or

[0479] (v) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:43, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:42.

[0480] Implementation scheme II-21. Nucleic acid, comprising a nucleotide sequence encoding an antigen-binding protein according to any one of implementation schemes II-1 to II-7 or a bispecific antibody according to any one of implementation schemes II-8 to II-20.

[0481] Implementation scheme II-22. A vector comprising the nucleic acid as described in implementation scheme II-21.

[0482] Implementation scheme II-23. A host cell comprising the nucleic acid described in implementation scheme II-21 or the vector described in implementation scheme II-22.

[0483] Implementation Scheme II-24. A pharmaceutical composition comprising (i) an antigen-binding protein according to any one of Implementation Schemes II-1 to II-7, or a bispecific antibody according to any one of Implementation Schemes II-8 to II-20; and (ii) a pharmaceutically acceptable carrier or excipient.

[0484] Implementation Scheme II-25. The pharmaceutical composition according to Implementation Scheme II-24 further comprises a second therapeutic agent.

[0485] Implementation Scheme II-26. The pharmaceutical composition according to Implementation Scheme II-25, wherein the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.

[0486] Implementation Scheme II-27. A conjugate or immunofusion compound comprising an antigen-binding protein according to any one of Implementation Schemes II-1 to II-7, or a bispecific antibody according to any one of Implementation Schemes II-8 to II-20, and a chemical moiety thereto.

[0487] Implementation scheme II-28. The conjugate or immunofusion compound according to implementation scheme II-27, wherein the chemical portion is selected from therapeutic agents, detectable portions and immunostimulatory molecules.

[0488] Implementation scheme II-29. Chimeric antigen receptor (CAR) comprising an antigen-binding protein according to any one of implementation schemes II-1 to II-7, or a bispecific antibody according to any one of implementation schemes II-8 to II-20.

[0489] Implementation Scheme II-30. A method for treating a disease in a subject, comprising administering to the subject an effective amount of an antigen-binding protein according to any one of Implementation Schemes II-1 to II-7, a bispecific antibody according to any one of Implementation Schemes II-8 to II-20, a pharmaceutical composition according to any one of Implementation Schemes II-24 to II-26, a conjugate or immunofusion according to Implementation Scheme II-27 or II-28, or a chimeric antigen receptor according to Implementation Scheme II-29, wherein the disease is an IGF-1R and / or TSHR-mediated disease.

[0490] Implementation scheme II-31. The method according to implementation scheme II-30, wherein the disease is an autoimmune disease, such as Graves' disease or thyroid eye disease (TED).

[0491] Implementation scheme II-32. The method according to implementation scheme II-31, wherein the method further includes administering a second therapeutic agent.

[0492] Implementation scheme II-33. The method according to implementation scheme II-32, wherein the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs.

[0493] Example

[0494] Construction of overexpression engineered cell lines: The full-length genes of human TSHR and human IGF-1R were inserted into the lentiviral expression vector pLVX-puro (Clontech-632164), respectively. Lentiviral viruses were packaged using 293T cells and used to infect HEK293 cells. Cells were cultured in medium containing 1 μg / mL puromycin and 400 μg / mL genimycin under G418 selection pressure to obtain polyclonal cell lines. Through limiting dilution, HEK293-TSHR (also referred to as HEK293-TSHR sc26 cells in this paper) overexpressing TSHR and HEK293-TSHR-IGF-1R monoclonal stable expression cell lines with dual overexpression of TSHR and IGF-1R were obtained. The CHOS-IGF-1R overexpression stable cell line was constructed in a similar manner.

[0495] Reference antibody

[0496] The following antibodies were used as reference antibodies in this article:

[0497] The anti-IGF-1R antibody Teprotumumab has heavy chain (HC) and light chain (LC) sequences of SEQ ID NO:50 and SEQ ID NO:51, respectively.

[0498] The anti-IGF-1R antibody VRDN-5000 (also referred to herein as VRDN-001 or VR1.1) has sequences derived from SEQ ID NO:10 and SEQ ID NO:11 as described in U.S. Patent Application US20230084477.

[0499] The anti-TSHR reference antibody has variable region sequences derived from SEQ ID NO:51 and SEQ ID NO:69 described in US Patent US10428153B2, and its heavy chain (HC) and light chain (LC) sequences are SEQ ID NO:44 and SEQ ID NO:38 described in this application, respectively.

[0500] The anti-TSHR antibody M22 has variable region sequences derived from SEQ ID NO:1 and SEQ ID NO:6 described in US Patent US8110664B2, and its heavy chain (HC) and light chain (LC) sequences are SEQ ID NO:45 and SEQ ID NO:46 described in this application, respectively.

[0501] Example 1: Development of IGF-1R-targeting antibody

[0502] 1.1 Development of Single-Domain Antibodies

[0503] The extracellular fragment sequence (Glu31–Asn932) of human IGF-IR (P08069-1) was retrieved from the UniProt database. After synthesizing a codon-optimized nucleotide sequence, the sequence was cloned and expressed to obtain the recombinant IGF-IR protein. Alpaca were immunized with the recombinant protein, peripheral blood was collected, PBMCs were isolated, RNA was extracted, and reverse transcribed into cDNA. Two rounds of PCR amplification were performed using the cDNA as a template. The amplification products were analyzed by agarose gel electrophoresis, and the VHH fragment with a molecular weight of approximately 400 bp was isolated. The VHH PCR product was recovered using a gel recovery kit (Qiagen, CAT#:28706). A single-domain antibody yeast display library was constructed using the VHH PCR product and the yeast display vector pDisplay (iCareab). Subsequently, the yeast display library was sorted using magnetic beads, and the sorted yeast clones were analyzed by flow cytometry. Genomic DNA was extracted from positive clones, and antibody sequences were obtained by PCR and sequenced. Based on the sequencing results, the obtained VHH antibody sequences were constructed into vectors and subcloned into the expression vector pcDNA3.4-hIgG1-Fc. The constructed expression vectors were transiently transfected into 293F cells, and the supernatant of the expressed antibodies was used for FACS detection. The expressed antibodies were incubated for 3 × 10⁶ cells. 5 CHO and CHO-IGF-IR cells were thoroughly mixed and incubated at room temperature for 1 hour. After centrifugation at 800×g for 5 minutes at room temperature, the supernatant containing antibodies was removed, and the cells were washed three times with PBS. 100 μl of PE-labeled anti-human IgG (eBioscience, Cat#:12-4998-82) (1:500 dilution) was added, thoroughly mixed, and incubated at room temperature in the dark for 30 minutes; after centrifugation at 800×g for 5 minutes at room temperature, the supernatant containing the secondary antibody (PE-Streptavidin) was removed, and the cells were washed three times with PBS; the cells were resuspended in 500 μl of PBS and analyzed by flow cytometry. The results showed that the six selected antibody clones, C10, H05, D09, F09, G10, and G09, could bind to IGF-IR. The amino acid sequences of these antibodies are shown in Table 1 (where CDR is defined as Abm).

[0504] Table 1. Sequences of anti-IGF-1R single-domain antibodies

[0505] 1.2 Antibody Function Validation

[0506] 1.2.1 ELISA Combination Experiment

[0507] IGF-1R antigen (AcroBiosystems-IGR-H5229) was diluted to 0.5-2.0 μg / mL with PBS, 100 μl / well was applied to each well, and incubated overnight at 2-8°C or for 2 hours at 33-37°C. The plate was washed three times with 0.05% PBST, and 250 μl / well of blocking buffer (1% BSA or 8% skim milk powder in PBS) was added. The plate was then incubated at room temperature or 33-37°C for at least 1 hour. The sample to be tested was diluted to a suitable concentration with blocking buffer. The plate was washed three times with 0.05% PBST, and 100 μl / well of the diluted sample was added. The plate was then incubated at room temperature or 33-37°C for approximately 1 hour. Wash the plate three times with 0.05% PBST using a plate washer. Add 100 μl / well of secondary antibody-HRP (Novus, CAT#NBP1-75095) diluted to the target dilution and incubate at room temperature or 33-37°C for approximately 0.5 to 1 hour. Wash the plate three times with 0.05% PBST using a plate washer. Add 100 μl / well of TMB and observe the color change. When the color reaches the appropriate level, immediately add 50-100 μl / well of stop solution. Read the OD450-OD650 values. Use reference antibodies Teprotumumab and VR1.1 as controls.

[0508] The results of the detection of the binding between anti-IGF-1R antibodies and IGF-1R antigen are shown in Figure 1 and Table 2. The results show that antibodies C10, D09, F09, G10, G09, and H05 all exhibited strong binding activity.

[0509] Table 2. Results of anti-IGF-1R antibody binding to IGF-1R ELISA

[0510] 1.2.2 ELISA Inhibition Binding Assay

[0511] The ELISA procedure was similar to that in Example 1.2.1, except that the test sample was serially diluted with blocking buffer and then mixed with biotin-labeled IGF-1 (Acrobiosystems-IG1-H82Q6). Reference antibodies Teprotumumab and VR1.1 were used as controls.

[0512] The detection results of the inhibition of IGF-1 binding to IGF-1R antigen by anti-IGF-1R antibodies are shown in Figure 2 and Table 3. The results indicate that antibodies C10, D09, F09, G10, G09, and H05 all exhibited strong inhibitory activity.

[0513] Table 3. Results of ELISA detection of anti-IGF-1R antibody blocking IGF-1 binding with IGF-1R

[0514] Example 2: Sequence optimization and characterization of anti-IGF-1R antibody D09

[0515] 2.1 Sequence optimization of anti-IGF-1R antibody D09

[0516] The anti-IGF-1R VHH sequence D09 was selected and its sequence was optimized.

[0517] The original VHH sequence was humanized using the "best-matching method". The amino acid sequences of the VHH framework region were compared and analyzed using a human germline V gene database to select the best germline sequence. The best-matching human CDR sequence was replaced with the VHH CDR sequence to generate a humanized VHH sequence, and one or more amino acid mutations were introduced to form multiple humanized mutant sequences. The humanized sequences were reverse-translated and synthesized by Genewiz (Shanghai, China). They were then constructed into the pcDNA 3.4 expression vector and expressed as humanized VHH with a C-terminal fusion of human IgG1 sequence, thereby obtaining the VHH antibody protein. Finally, humanized anti-IGF-1R antibody D09 mutants znD09.m1, znD09.m2, znD09.m3, and znD09.m4 were obtained, and their amino acid sequences are shown in Table 4 (where CDR is defined by Abm).

[0518] Table 4. Humanized IGF-1R antibody sequences

[0519] 2.2 In vitro characterization of the humanized anti-IGF-1R antibody D09 mutant

[0520] FACS combined with experiment

[0521] MCF-7 (IGF-1R positive, Chinese Academy of Sciences Type Culture Collection, TCHu 74) was cultured at 1-5 × 10⁻⁶ cells per well. 5Seed cells at a density of 100 μL / well in 96-well plates and centrifuge at 300 g for 5 minutes at 4°C. Add the test sample diluted to the appropriate concentration and incubate at 2-8°C for approximately 1 hour. Centrifuge at 4°C, remove the supernatant, wash twice with 200 μL / well FACS buffer (PBS containing 1-2% FBS), and centrifuge at 4°C. Add 100 μL / well of flow cytometry secondary antibody diluted to the target dilution, resuspend the cells, and incubate at 2-8°C in the dark for approximately 0.5 h or 1 h. Wash twice with 200 μL / well FACS buffer, resuspend the cells with 60-100 μL / well FACS buffer, and perform flow cytometry analysis. Measure the mean fluorescence index (MFI) of the cells using a Beckman Coulter flow cytometer. The binding results of the mutants of anti-IGF-1R antibody D09, znD09.m1, znD09.m2, znD09.m3, and znD09.m4, to MCF-7 cells are shown in Figure 3 and Table 5. The results show that the mutants znD09.m1, znD09.m2, znD09.m3, and znD09.m4 exhibited similar binding activity in MCF-7 cells, indicating that the humanized antibody molecules retained the binding activity of the parent D09.

[0522] Table 5. FACS binding detection results of anti-IGF-1R antibody D09 mutant with MCF-7.

[0523] Example 3: Generation of anti-IGF-1R / TSHR bispecific antibody

[0524] 3.1 Bispecific antibody molecule design

[0525] The following bispecific antibody molecule was designed: it is a symmetrical double-stranded form, composed of four pairwise symmetrical polypeptide chains. The light chain, from N-terminus to C-terminus, contains: (VHH)n-VL-CL-(VHH)n; the heavy chain, from N-terminus to C-terminus, contains: (VHH)n-VH-CH1-CH2-CH3-(VHH)n. Each n is independently 0 or 1. Five bispecific antibody structures were designed (designated V-7, V-8, V-9, V-10, and V-11), and their specific structures are shown in Figure 4.

[0526] Due to the dimerization of the Fc region of immunoglobulins, polypeptide chains can associate to form homodimers, thereby producing bispecific binding molecules in double-stranded form.

[0527] 3.2 Construction of double-stranded bispecific antibody molecules

[0528] Bispecific antibodies were constructed using anti-IGF-1R VHH and anti-TSHR IgG antibody sequences as components, according to the antibody structures shown in Table 6 and the antibody sequences shown in Table 7. The VHH amino acid sequence was derived from znD09.m3. VL is the light chain variable region of the anti-TSHR IgG antibody, and VH is the heavy chain variable region of the anti-TSHR IgG antibody.

[0529] In addition, half-life extension mutations and Fc function silencing mutations were introduced into the Fc region, specifically YTE mutations (M252Y, S254T, T256E) and LALA mutations (L234A and L235A). These antibody elements were constructed into the V-7, V-8, V-9, V-10, and V-11 antibody structures described above, and their structural and sequence information are shown in Table 6 (where the symbol "-" indicates that the two domains are connected by a linker or directly) and Table 7, respectively.

[0530] Table 6. Fc dimer form of bispecific antibodies

[0531] Table 7. Bispecific antibody sequences (with CDR Abm definition)

[0532] The heavy / light chain of the Fc dimer double-stranded antibody described above was constructed into the pcDNA 3.4 expression vector and transfected into HEK293F cells. Cells were cultured for 3 days, and the culture supernatant was collected and loaded into a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibody was eluted with acetate-sodium acetate solution (pH 3.5) and immediately neutralized with 2M Tris. Antibody concentration was measured using Nano Drop. Protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and the protein was then stored at -80°C.

[0533] 3.3. In vitro characterization of anti-IGF-1R / TSHR bispecific antibody

[0534] 3.3.1 FACS binding assay of anti-IGF-1R / TSHR bispecific antibody

[0535] The FACS assay for the anti-IGF-1R / TSHR bispecific antibody was performed according to the method described in Example 2.2. The reference antibody Teprotumumab and an anti-TSHR reference antibody containing LALA and YTE mutations in the Fc region (referred to as anti-TSHR reference antibody (LALA-YTE)) were used as controls. Simultaneously, bivalent antibodies formed by fusing the C-terminus of D09m3 with human IgG Fc containing or without LALA and YTE mutations (referred to as D09m3-Fc (LALA-YTE) and D09m3-Fc, respectively) were used as controls.

[0536] The results of detecting the binding of anti-IGF-1R / TSHR bispecific antibodies to MCF-7 and HEK293-TSHR sc26 cells are shown in Figures 5 and 6 and Table 8. The results showed that antibodies V-7, V-8, V-9, V-10, and V-11 all exhibited binding activity on both MCF-7 and HEK293-TSHR sc26 cells. V-7, V-8, and V-9 showed similar binding activity, exhibiting higher binding on MCF-7 compared to monoclonal antibodies D09m3-Fc (LALA-YTE), D09m3-Fc, and the reference antibody Teprotumumab, and also showing higher binding on HEK293-TSHR sc26 compared to the anti-TSHR reference antibody (LALA-YTE).

[0537] Table 8. Detection results of binding of anti-IGF-1R / TSHR bispecific antibodies to IGF-1R and TSHR via FACS.

[0538] 3.3.2 FACS inhibition binding assay of anti-IGF-1R / TSHR bispecific antibody

[0539] The FACS experimental procedure is similar to that in Example 2.2, except that the appropriate concentration of the test sample is mixed with biotin-labeled IGF-1 antigen or M22 before being added to a 96-well plate. Reference antibodies Teprotumumab, anti-TSHR reference antibody (LALA-YTE), and D09m3-Fc are used as controls.

[0540] Figure 7 shows the detection results of the anti-IGF-1R / TSHR bispecific antibody inhibiting the binding of IGF-1 to MCF-7, and Figure 8 shows the detection results of the anti-TSHR antibody M22 inhibiting the binding of HEK293-TSHR sc26. The results show that antibodies V-7, V-8, V-9, V-10, and V-11 all exhibited inhibitory activity on both MCF-7 and HEK293-TSHR sc26. V-7, V-8, and V-9 showed similar inhibitory activity on MCF-7, all exhibiting stronger and more complete inhibitory activity than the reference antibody Teprotumumab. V-10 and V-11 more completely inhibited the binding of IGF-1 and IGF-1R compared to Teprotumumab, which only showed partial inhibitory activity. The D09m3 antibody also showed stronger and more complete inhibitory activity than the reference antibody Teprotumumab.

[0541] 3.4. In vitro characterization of candidate bispecific antibody molecules

[0542] 3.4.1 FACS Inhibition Experiment of Candidate Bispecific Antibody Molecules V-7 and V-10

[0543] The FACS experimental procedure is similar to that in Example 2.2, except that the appropriate concentration of the test sample is mixed with biotin-labeled IGF-1 antigen or M22 before being added to a 96-well plate. Reference antibodies Teprotumumab, VR1.1, D09m3-Fc (LALA-YTE), and anti-TSHR reference antibody are used as controls.

[0544] The results of detecting the inhibition of IGF-1 binding to MCF-7, HEK293, HEK293-TSHR, and HEK293-TSHR-IGF-1R by candidate bispecific antibodies V-7 and V-10 are shown in Figure 9 and Table 9. The results show that V-7 and V-10 exhibit inhibitory activity on MCF-7, HEK293, HEK293-TSHR, and HEK293-TSHR-IGF-1R. V-7 and V-10 showed significantly better inhibitory activity than the reference antibodies VR1.1 and Teprotumumab on HEK293-TSHR cells, which exhibit low IGF-1R expression and high TSHR expression, indicating that the anti-TSHR terminus of the bispecific antibody can assist in inhibiting IGF-1 binding to IGF-1R. For the D09m3 antibody, it showed more complete inhibitory activity than the reference antibodies Teprotumumab and VR1.1 on both MCF-7 and HEK293-TSHR-IGF-1R cells.

[0545] The results of detecting the inhibition of M22 binding to HEK293-TSHR and HEK293-TSHR-IGF-1R by candidate bispecific antibodies V-7 and V-10 are shown in Figure 10 and Table 10. The results show that both V-7 and V-10 exhibit inhibitory activity against both HEK293-TSHR and HEK293-TSHR-IGF-1R. V-7 and V-10 showed significantly better inhibitory activity than the anti-TSHR reference antibody against HEK293-TSHR-IGF-1R, which exhibits high expression of both IGF-1R and TSHR, indicating that the anti-IGF-1R end of the bispecific antibody can assist in inhibiting M22 binding to TSHR.

[0546] Table 9. Detection results of the inhibition of IGF-1 binding to cellular IGF-1R by candidate bispecific antibody molecules V-7 and V-10.

[0547] Table 10 Results of detection of the inhibition of M22 binding to cellular TSHR by candidate bispecific antibody molecules V-7 and V-10

[0548] 3.4.2 Inhibition of IGF-1R phosphorylation by candidate bispecific antibody molecules V-7 and V-10

[0549] IGF-1R positive 1~2×10 5 A549 cells (TCHu150, Chinese Academy of Sciences Cell Bank) were seeded in 24-well plates and incubated at 37°C in a 5% CO2 incubator with complete cell culture medium containing 10% FBS for 24 hours. The culture medium was removed the following day, and the cells were starved in serum-free medium for another 24 hours. Serially diluted antibodies were added to the serum-free medium, and the cells were incubated at 37°C in a 5% CO2 incubator for 30 minutes. Then, IGF-1 was added at 37°C to a final concentration of 10.5 nM for stimulation. Cells were lysed on ice with 200 μL of lysis buffer (1% NP-40 with a cocktail of protease inhibitors and a cocktail of phosphatase inhibitors). Cell lysates were collected and lysed at 14,000 rpm at 4°C. The supernatant was transferred to fresh tubes, frozen at -80°C, and used for IGF-1R phosphorylation ELISA assays (R&D, DYC1770-2). The reference antibodies Teprotumumab and D09m3-Fc (LALA-YTE) were used as controls.

[0550] The results of the detection of IGF-1R phosphorylation induced by candidate bispecific antibodies V-7 and V-10 are shown in Figure 11 and Table 11. The results show that antibodies V-7, V-10 and monoclonal antibody D09m3 all inhibited IGF-1R phosphorylation more completely than the reference antibody Teprotumumab.

[0551] Table 11 Detection results of the inhibition of IGF-1R phosphorylation by candidate bispecific antibody molecules V-7 and V-10.

[0552] 3.4.3 Inhibition of cyclic adenosine monophosphate (cAMP) by candidate bispecific antibody molecules V-7 and V-10

[0553] 1~3×10 4 HEK293-TSHR or HEK293-TSHR-IGF-1R cells were seeded in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours in complete cell culture medium containing 10% FBS. The next day, the culture medium was removed, and the cells were starved in serum-free cell culture medium for another 16–24 hours. Serially diluted antibody and 1 nM M22 were added to complete cell culture medium containing 0.5% FBS, and the cells were incubated at 37°C in a 5% CO2 incubator for 2 hours, after which the cells were lysed. The supernatant was transferred to fresh tubes and frozen at -80°C for cAMP ELISA (GenScript, A01509). Anti-TSHR reference antibody and a D09.m3 / anti-TSHR reference antibody combination (antibody ratio 1:1) were used as controls.

[0554] The results of detecting the inhibition of M22-induced cAMP production by candidate bispecific antibodies V-7 and V-10 are shown in Figure 12 and Table 12. The results show that both V-7 and V-10 exhibited inhibitory activity against M22-induced cAMP production. In HEK293-TSHR-IGF-1R cells, V-7 and V-10 showed superior inhibitory activity compared to the anti-TSHR reference antibody and the D09.m3 / anti-TSHR reference antibody combination group.

[0555] Table 12. Detection results of the inhibition of M22-induced cAMP production by four candidate bispecific antibody molecules V-7 and V-10.

[0556] Example 4: Experiment on the inhibition of M22-induced hyaluronic acid (HA) production by candidate bispecific antibodies V-7 and V-10.

[0557] 1~3×10 4HEK293-TSHR-IGF-1R cells were seeded in 96-well plates and incubated at 37°C with 5% CO2 in a complete cell culture medium containing 10% FBS for 24 hours. The next day, the medium was removed, and the cells were starved in complete cell culture medium containing 0.5–1% FBS for another 16–24 hours. An appropriate concentration of antibody (to a final concentration of 250 nM) and M22 were added to the complete cell culture medium containing 0.5–1% FBS, and the cells were incubated at 37°C with 5% CO2 for 48–72 hours. The supernatant was transferred to new tubes and frozen at -80°C for hyaluronic acid ELISA (CUSABIO: CSB-E04805h; R&D: DHYAL0). Untreated HEK293 and HEK293-TSHR cells were cultured and subjected to hyaluronic acid ELISA in the same manner.

[0558] The test results are shown in Figure 13. The results show that both V-7 and V-10 exhibited inhibitory activity against hyaluronic acid production, and their inhibitory activity was superior to that of the anti-TSHR reference antibody and Teprotumumab.

[0559] Example 5: Efficacy experiment of candidate bispecific antibody molecules V-7 and V-10 in mice with thyroid-associated eye disease (TED).

[0560] This experiment established a mouse model of thyroid-associated ophthalmopathy (TED) and observed the effects of dual anti-inflammatory molecules V-7 and V-10 on TED mice, including the following steps:

[0561] (1) Construction of a mouse model of thyroid ophthalmopathy (TED)

[0562] The TED animal model was established using an adenovirus-induced autoimmune method. An adenovirus encoding the human thyroid-stimulating hormone receptor A subunit (TSHRA) (Ad-TSHRA) was constructed and injected intramuscularly into the femoral muscle of 6-week-old female Balb / c mice to induce an autoimmune response (viral titer 1×10⁻⁶). 10 The dosage was PFU / ml, 50 μl per injection. The control group received an adenovirus encoding green fluorescent protein (Ad-EGFP) via injection. The drug treatment groups received intraperitoneal administration of IGF-1R / TSHR bispecific antibody drugs V-7 or V-10 (10 mg / kg body weight) weekly, in addition to TED modeling. The induction injection process included an initial induction period and a maintenance induction period, with a total of nine adenovirus injections. The initial induction period was performed with injections every three weeks for three times, and the maintenance induction period was performed with injections every four weeks for six times. Four weeks after the completion of all injection induction, the mice were evaluated.

[0563] (2) Mouse thyroid tissue sampling, section staining

[0564] After euthanizing mice with cervical dislocation, the skin of their necks was cut open, and the subcutaneous tissue was carefully separated. The thyroid tissue was located between the trachea and the seventh cartilage ring, and was completely separated and removed. It was fixed in 4% paraformaldehyde solution for 24 hours, dehydrated, and then embedded in paraffin. Coronal sections were then prepared, stained with hematoxylin and eosin (H&E), and observed and photographed under a microscope to assess the pathological changes in the thyroid gland.

[0565] (3) Obtaining and staining mouse orbital tissue sections

[0566] The skin around the mouse eye was completely separated, and the orbital tissue, along with the eyeball, was removed intact and fixed in 4% paraformaldehyde solution for 24 hours. After dehydration, it was embedded in paraffin, and coronal sections were then prepared along the optic nerve pathway. After staining with hematoxylin and eosin (H&E), the orbital tissue was observed and photographed under a microscope to assess pathological changes. The retrobulbar fat content of the orbit was defined as the ratio of retrobulbar fat area to optic nerve area using Imagej image analysis software, to assess the extent of retrobulbar fat hyperplasia in mice.

[0567] (4) Masson staining of orbital tissue

[0568] After dewaxing the above-mentioned mouse orbital tissue sections with xylene-ethanol, they were stained with prepared Weigert iron hematoxylin staining solution and Masson blue solution, thoroughly washed with distilled water, stained with Ponceau S and fuchsin staining solution and aniline blue staining solution, and washed with weak acid. After dehydration with ethanol-xylene and mounting with neutral resin, the sections were observed and photographed under a microscope. Collagen fibers appeared blue, indicating fibrotic lesions. Image analysis software Imagej was used to quantitatively analyze the area of ​​blue-stained region / total tissue area to assess the degree of extraocular muscle fibrosis.

[0569] The results are analyzed as follows:

[0570] As shown in Figure 14A, the model group experienced continuous weight loss starting from week 22 until the end of the experiment. In contrast, the weight remained stable in the V-7 and V-10 treatment groups, especially in the V-7 group, where weight recovery was significant from week 22. Figure 14B shows that the model group exhibited significant TED symptoms such as exophthalmos and eyelid edema, while the V-7 and V-10 treatment groups showed significant improvement, indicating that the bispecific antibodies V-7 and V-10 have a significant therapeutic effect on TED in terms of ocular appearance. Furthermore, Figure 14C shows that the thyroid gland in the model group exhibited diffuse hyperplasia, follicular epithelial cell proliferation, irregular follicular morphology, and significantly increased blood supply. The V-7 and V-10 treatment groups showed significant improvement compared to the model group.

[0571] In TED (Eye Tissue Degeneration), orbital fat deposition and fibrosis are recognized pathological changes. This invention defines retrobulbar orbital fat content as the ratio of retrobulbar fat area to optic nerve area, i.e., relative fat content. Figures 14D and 14 show that the relative fat content was significantly increased in the model group compared to the control group, while the V-7 and V-10 administration groups showed a significant decrease in relative fat content compared to the model group, indicating that the bispecific antibody of this invention has significant efficacy in inhibiting orbital fat deposition in TED.

[0572] Furthermore, extraocular muscle fibrosis leads to muscle fiber disorder; after Masson staining, collagen fibers appear blue, indicating fibrosis. Quantitative analysis was performed as the ratio of the blue-stained area to the total tissue area. Figures 14F and 14G show that the model group exhibited multiple blue fibrotic lesions, while the V-7 and V-10 treatment groups showed significant improvement.

[0573] In summary, using the method described herein, this invention successfully established a mouse TED pharmacodynamic model. In this model, the V-7 and V-10 administration groups significantly improved the ocular appearance, thyroid pathology, and orbital tissue pathology of TED mice, suggesting that the bispecific antibodies V-7 and V-10 have a significant therapeutic effect on TED.

Claims

1. An antigen-binding protein that specifically binds to IGF-1R, wherein the antigen-binding protein contains a VHH domain, wherein (i) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:4; or (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:8; or (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:12; or (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:16; or (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:20; or (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:24; or (vii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:25; or (viii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:26; or (ix) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:27; or (x) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:

28.

2. The antigen-binding protein according to claim 1, wherein... (i) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:1-3; or (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:5-7; or (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO: 9-11; or (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:13-15, respectively; or (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:17-19; or (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:21-23, respectively.

3. The antigen-binding protein according to claim 1 or 2, wherein... (i) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; or (ii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; or (iii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; or (iv) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16; or (v) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20; or (vi) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:24; or (vii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:25; or (viii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:26; or (ix) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:27; or (x) The VHH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

28.

4. The antigen-binding protein according to any one of claims 1-3, wherein the antigen-binding protein is an antibody.

5. The antigen-binding protein according to claim 4, wherein the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

6. The antigen-binding protein according to any one of claims 4-5, wherein the antibody further comprises a second antigen-binding region that binds to a second antigen.

7. The antigen-binding protein according to claim 6, wherein the second antigen is an autoantigen, preferably TSHR.

8. A bispecific antibody comprising a first antigen-binding region for binding IGF-1R and a second antigen-binding region for binding TSHR, wherein the first antigen-binding region comprises a VHH domain. (i) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:4; or (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:8; or (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:12; or (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:16; or (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:20; or (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:24; or (vii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:25; or (viii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:26; or (ix) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:27; or (x) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:

28.

9. The bispecific antibody according to claim 8, wherein (i) the VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:1-3, respectively; or (ii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:5-7; or (iii) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO: 9-11; or (iv) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:13-15, respectively; or (v) The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each having an amino acid sequence as shown in SEQ ID NO:17-19; or (vi) The VHH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:21-23, respectively.

10. The bispecific antibody according to claim 9, wherein... (i) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; or (ii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; or (iii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; or (iv) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16; or (v) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:20; or (vi) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:24; or (vii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:25; or (viii) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:26; or (ix) The VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:27; or (x) The VHH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

28.

11. The bispecific antibody according to any one of claims 8-10, wherein the second antigen-binding region comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequence shown in SEQ ID NO:36; and the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence shown in SEQ ID NO:32; preferably, the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequence shown in any one of SEQ ID NO:33-35, and the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence shown in any one of SEQ ID NO:29-31.

12. The bispecific antibody of claim 11, wherein the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:36 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

32.

13. The bispecific antibody according to any one of claims 8-12, wherein, The VHH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 having amino acid sequences as shown in SEQ ID NO:9-11, respectively; the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in any one of SEQ ID NO:33-35, respectively; and the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in any one of SEQ ID NO:29-31, respectively.

14. The bispecific antibody of claim 13, wherein the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:27; the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:36; and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

32.

15. The bispecific antibody according to any one of claims 8-14, wherein, The binding valence ratio of the first antigen-binding region and the second antigen-binding region is 1:1, 2:1 or 1:2, preferably 1:

1.

16. The bispecific antibody according to any one of claims 8-15, wherein the bispecific antibody comprises: (i) a heavy chain, which from the N-end to the C-end includes the VH, heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3); and (ii) A light chain that includes the VL and the light chain constant region (CL) from the N end to the C end; The VHH is connected to the N or C end of the heavy chain or the light chain via an optional connector.

17. The bispecific antibody according to claim 16, wherein: (i) The heavy chain from N-end to C-end comprises: VHH-connector-VH-CH1-CH2-CH3; and the light chain from N-end to C-end comprises: VL-CL; or (ii) The heavy chain from N-end to C-end comprises: VH-CH1-CH2-CH3-connector-VHH; and the light chain from N-end to C-end comprises: VL-CL; or (iii) The heavy chain from the N-end to the C-end comprises: VH-CH1-CH2-CH3; and the light chain from the N-end to the C-end comprises: VL-CL-connector-VHH; or (iv) The heavy chain from the N end to the C end comprises: VH-CH1-CH2-CH3; and the light chain from the N end to the C end comprises: VHH-connector-VL-CL.

18. The bispecific antibody of claim 17, wherein the linker comprises an amino acid sequence selected from (G4S)n, wherein n is an integer selected from 1 to 5.

19. The bispecific antibody of claim 18, wherein the adapter comprises an amino acid sequence such as any one of SEQ ID NO:47-49.

20. The bispecific antibody according to any one of claims 8-19, wherein (i) the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:37; or (ii) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:39; or (iii) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:40; or (iv) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:41; or (v) The light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:43, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

42.

21. A nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein according to any one of claims 1-7 or a bispecific antibody according to any one of claims 8-20.

22. A vector comprising the nucleic acid according to claim 21.

23. A host cell comprising the nucleic acid according to claim 21 or the vector according to claim 22.

24. A pharmaceutical composition comprising (i) an antigen-binding protein according to any one of claims 1-7, or a bispecific antibody according to any one of claims 8-20; and (ii) a pharmaceutically acceptable carrier or excipient.

25. The pharmaceutical composition of claim 24, further comprising a second therapeutic agent.

26. The pharmaceutical composition of claim 25, wherein the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.

27. A conjugate or immunofusion compound comprising an antigen-binding protein according to any one of claims 1-7, or a bispecific antibody according to any one of claims 8-20, and a chemical moiety thereto.

28. The conjugate or immunofusion compound of claim 27, wherein the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.

29. A chimeric antigen receptor (CAR) comprising an antigen-binding protein according to any one of claims 1-7, or a bispecific antibody according to any one of claims 8-20.

30. A method for treating a disease in a subject, comprising administering to the subject an effective amount of an antigen-binding protein according to any one of claims 1-7, a bispecific antibody according to any one of claims 8-20, a pharmaceutical composition according to any one of claims 24-26, a conjugate or immunofusion according to claim 27 or 28, or a chimeric antigen receptor according to claim 29, wherein the disease is an IGF-1R and / or TSHR-mediated disease.

31. The method of claim 30, wherein the disease is an autoimmune disease, such as Graves' disease or thyroid eye disease (TED).

32. The method of claim 31, wherein the method further comprises administering a second therapeutic agent.

33. The method of claim 32, wherein the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.

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