IGF-1r and TSHR binding protein and pharmaceutical use thereof

Abstract

Provided are an IGF-1R and TSHR binding protein and a pharmaceutical use thereof. Specifically, provided are an IGF-1R / TSHR binding protein, an IGF-1R binding protein, a TSHR binding protein, a method for using same for treatment of an autoimmune disease (e.g. Graves' orbitopathy), and a related pharmaceutical use.

Description

IGF-1R and TSHR binding protein and its medical use

[0001] This application claims priority to Chinese patent application CN202311157087.9 filed on September 8, 2023. Technical Field

[0002] The present disclosure relates to IGF-1R / TSHR binding proteins, IGF-1R binding proteins, TSHR binding proteins, nucleic acids encoding the same, pharmaceutical compositions, and methods for use thereof in treating autoimmune diseases (eg, Graves' ophthalmopathy) and related pharmaceutical uses. Background Art

[0003] Graves' Orbitopathy (GO), also known as thyroid associated ophthalmopathy (TAO; Thyroid Eye Disease (TED),), is an organ-specific autoimmune disease that is both related to and independent of Graves' disease. In patients, the immune system overstimulates the thyroid gland, causing it to secrete excessive amounts of thyroid hormone. Orbital fibroblasts (OF) express the thyroid-stimulating hormone receptor (TSHR), insulin-like growth factor-1 receptor (IGF-1R), and immunomodulatory molecules. Upon appropriate stimulation, some OFs produce hyaluronic acid (HA), inflammatory mediators, and autoantibodies, while others differentiate into adipocytes, the primary target cells of autoimmunity in Graves' ophthalmopathy (Terry J. Smith, N Engl J Med 375; 16 (2016)). OF-derived adipocytes are the primary source of TSHR in orbital tissue in patients with GO. Studies have confirmed that TSHR mRNA and protein are expressed in orbital adipose tissue in patients with Graves' disease, both with and without ophthalmopathy. TSHR levels are indeed higher in patients with GO than in those without GO, suggesting that increased orbital TSHR expression may contribute to disease progression (Wang Y, Invest Ophthalmol Vis Sci, 55;3 (2014)). Studies have found that IGF-1R and TSHR are expressed in the same cellular location, synergizing to form a functional complex. Activated B cells produce thyroid-stimulating antibodies, which bind to TSHR. Together, TSHR and IGF-1R mediate the activation of downstream signaling pathways, leading to the production of numerous inflammatory factors in the orbital fat tissue, such as IL-8, prostaglandin E2, and monocyte chemoattractant protein. Furthermore, upon TSHR activation, the orbital fat tissue secretes large amounts of hyaluronic acid, leading to expansion, edema, and inflammation of the retroorbital adipose connective tissue. Furthermore, binding of IGF-1 to IGF-1R can further promote the proliferation of the orbital fat tissue.

[0004] Tepezza is currently the only marketed antibody drug targeting IGF-1R and has not yet been approved in China. K1-70, an inhibitory antibody against TSHR, is currently in Phase I clinical trials, with Phase I results showing effective relief of symptoms in patients with goiter (Jadwiga Furmaniak, Clinical Endocrinology, 96; 878-887 (2022)).

[0005] Single-domain antibodies, with a molecular weight of only 12-15 kDa and containing three CDRs, are the smallest known antibodies with complete antigen-binding activity. They offer numerous advantages, including high binding affinity, high specificity, ease of modification, high solubility, high stability, and high expression levels. This disclosure provides novel sequence-structured anti-TSHR single-domain antibodies and anti-IGF-1R single-domain antibodies, and constructs bispecific antibodies combining these two. Testing demonstrates synergistic effects between the two antibodies, potentially making them more effective and safer clinical treatments for Graves' ophthalmopathy.

[0006] Summary of the Invention

[0007] IGF-1R / TSHR binding protein, IGF-1R binding protein, TSHR binding protein, their encoding nucleic acids, pharmaceutical compositions, methods for treating diseases, and related pharmaceutical uses.

[0008] IGF-1R binding protein

[0009] The present disclosure provides an IGF-1R binding protein comprising an immunoglobulin single variable domain comprising:

[0010] 1) CDR1, CDR2, and CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 4, 20-24, and 34, or, 2) CDR1, CDR2, and CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 5, 25-30;

[0011] Wherein, the CDR1, CDR2 and CDR3 are numbered according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, for example, according to the Kabat numbering system.

[0012] In some embodiments, an IGF-1R binding protein is provided, comprising any one or any combination of the above-mentioned CDR1, CDR2, and CDR3.

[0013] In some embodiments, an IGF-1R binding protein is provided, comprising an immunoglobulin single variable domain, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are:

[0014] 1) as shown in SEQ ID NO: 7, 8, 35, respectively, as shown in SEQ ID NO: 7, 8, 9, respectively, or as shown in SEQ ID NO: 7, 8, 36, respectively;

[0015] 2) As shown in SEQ ID NO: 10, 11, and 12 respectively.

[0016] In some embodiments, the immunoglobulin single variable domain in the aforementioned IGF-1R binding protein is humanized, backmutated, affinity matured, T cell epitope (TCE) removed / reduced, antibody deamidation reduced, and / or antibody isomerization reduced.

[0017] In some embodiments, the immunoglobulin single variable domain is obtained by removing / reducing TCEs and has one or more changes in one or more CDRs, which result in reduced immunogenicity of the IGF-1R binding protein.

[0018] In some embodiments, the heavy chain framework region of the human germline template used for humanizing the immunoglobulin single variable domain in the aforementioned IGF-1R binding protein is derived from IGHV3-23, IGHV3-66, or IGJH4. For example, FR1, FR2, and FR3 are derived from IGHV3-66, and FR4 is derived from IGJH4.

[0019] In some embodiments, when the aforementioned immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 7, 8, 35, respectively, as shown in SEQ ID NO: 7, 8, 9, respectively, or as shown in SEQ ID NO: 7, 8, 36, respectively, one, more or any combination of mutations are present at amino acid residues 23, 27, 30, 37, 44, 45, 47, 74, 82, 94 (numbered according to the Kabat numbering system), for example, one, more or any combination of mutations are present selected from 23T, 27L, 30G, 37F, 44E, 45R, 47G, 74A, 82D or A, 94V.

[0020] In some embodiments, when the aforementioned immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 10, 11, 12, respectively, there are one, more or any combination of mutations at amino acid residues 26, 27, 28, 44, 45, 47, 48, 49, 73, 74, 77, 94, 103 (numbered according to the Kabat numbering system), for example, there are one, more or any combination of mutations selected from 26R, 27Y, 28I, 44E, 45R, 47E or G, 48I, 49A, 73G, 74A, 77F, 94A, 103L.

[0021] In some embodiments, the amino acid sequences of the immunoglobulin single variable domains in the aforementioned IGF-1R binding proteins are:

[0022] 1) as shown in any one of SEQ ID NOs: 4, 20-24, 34 or having a sequence identity of at least 80% (preferably at least 90%) to any one of SEQ ID NOs: 4, 20-24, 34;

[0023] 2) as shown in any one of SEQ ID NOs: 5, 25-30 or having a sequence identity of at least 80% (preferably at least 90%) to any one of SEQ ID NOs: 5, 25-30.

[0024] In the present disclosure, "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity.

[0025] "At least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity.

[0026] In some embodiments, the aforementioned IGF-1R binding protein comprises or is an antibody or antigen-binding fragment thereof that specifically binds to a human IGF-1R protein or a fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a camel antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is a recombinant antibody or a fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is a linear antibody, a single-chain antibody, a nanobody, a peptibody, a domain antibody and a diabody, a triabody and a tetrabody, a tandem di-scFv, a tandem tri-scFv). In some specific embodiments, the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody (e.g., a trispecific antibody).

[0027] In some embodiments, the immunoglobulin single variable domain in the aforementioned IGF-1R binding protein is a single domain antibody or VHH, or the IGF-1R binding protein is a single domain antibody or VHH.

[0028] In some embodiments, the present disclosure provides an IGF-1R binding protein comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains, wherein the immunoglobulin single variable domains may be the same or different and may form dimers or multimers.

[0029] In some embodiments, the aforementioned IGF-1R binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region is a human IgG1 Fc region, such as that set forth in SEQ ID NO:16, or having at least 80% (preferably at least 90%) sequence identity thereto. In some embodiments, the Fc region has enhanced stability or comprises mutations that increase the stability of the Fc region compared to a wild-type Fc region. In some embodiments, the Fc region enables the binding protein to form a dimeric molecule. In some specific embodiments, the Fc region comprises mutations that increase the in vivo half-life, which is determined by the FcRn binding affinity. This increased half-life can allow for a reduction in the amount of drug administered to a patient and / or less frequent dosing. For example, the Fc region comprises the M252Y, S254T, and / or T256E mutations. In some specific embodiments, the Fc region can be an Fc region with reduced effector function, for example, the Fc region can have a mutation, and exemplary IgG Fc regions with reduced effector function include those with the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); or S267E / L328F (IgG1).

[0030] In some embodiments, the immunoglobulin single variable domain in the aforementioned IGF-1R binding protein is connected to the Fc region directly or through a linker. The linker can be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure. For example, the linker is a flexible linker, such as G4S (SEQ ID NO: 53), GS, GAP, (G4S)2 (SEQ ID NO: 54), (G4S)3 (SEQ ID NO: 55), (G4S)4 (SEQ ID NO: 56), (G4S)5 (SEQ ID NO: 57), ASGS (SEQ ID NO: 58), etc., for example, (G4S)2; for example, (G m S n ) h or (G m Q n ) h or (GGNGT) h(SEQ ID NO: 59) or (YGNGT) h (SEQ ID NO: 60) or (EPKSS) h (SEQ ID NO: 61), wherein m and n are each independently selected from an integer of 1-8, and h is independently selected from an integer of 1-20.

[0031] In some embodiments, the IGF-1R binding protein of the present disclosure is an anti-IGF-1R antibody or an antigen-binding fragment thereof, or a conjugate or fusion protein comprising the antibody or antigen-binding fragment.

[0032] In some embodiments, an IGF-1R binding protein is provided that comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 17, 37, and 18, or an amino acid sequence that is at least 80% (preferably at least 90%) identical to any one of SEQ ID NOs: 17, 37, and 18.

[0033] In some embodiments, the aforementioned IGF-1R binding protein has at least one function or property selected from the group consisting of:

[0034] (a)≤10 -7 K D The K value (M) binds to human IGF-1R or its epitope. D Methods for detecting the value of is conventional in the art, such as the method in Example 3 of the present disclosure. In some embodiments, the binding K of the single domain antibody of the present disclosure to human IGF-1R or its epitope is D Value ≤ 10 -10 , the binding K of the single domain antibody of the present disclosure to human IGF-1R or its epitope D Value ≤ 10 -8 or ≤10 -9 .

[0035] (b) inhibiting IGF-1R downstream signaling pathways, such as pAKT. In some embodiments, in A549 cells, inhibition of pAKT IC 50 (nM) is less than 10, less than 5, less than 2, less than 1, less than 0.5, less than 0.2. In some embodiments, the IC 50 The value was detected by the method in Example 4 of the present disclosure.

[0036] In some embodiments, the aforementioned IGF-1R binding proteins of the present disclosure bind to the K D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0037] In some embodiments, the aforementioned IGF-1R binding proteins disclosed herein encompass variants, which have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to any one of SEQ ID NOs: 4, 20-24, 34, or 5, 25-30; the amino acid mutations may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function; the amino acid mutations may occur in the CDR region and / or the FR region.

[0038] In some embodiments, an anti-IGF-1R antibody or antigen-binding fragment thereof is provided that binds to or competes for binding to the same epitope as the immunoglobulin single variable domain in the aforementioned IGF-1R binding protein of the present disclosure.

[0039] In some embodiments, an anti-IGF-1R antibody or antigen-binding fragment thereof is provided that blocks the binding of the immunoglobulin single variable domain in the aforementioned IGF-1R binding protein of the present disclosure to IGF-1R (eg, human IGF-1R).

[0040] In some embodiments, an anti-IGF-1R antibody or antigen-binding fragment thereof is provided, wherein the binding of the antibody to IGF-1R (eg, human IGF-1R) is blocked by the immunoglobulin single variable domain in the IGF-1R binding protein of the present disclosure.

[0041] In some embodiments, a protein or molecule is provided that comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) immunoglobulin single variable domains of any of the IGF-1R binding proteins disclosed herein, wherein the immunoglobulin single variable domains are the same or different. For example, the protein or molecule is a conjugate, and the conjugate may comprise any detectable label.

[0042] TSHR binding protein

[0043] The present disclosure provides TSHR binding proteins comprising an immunoglobulin single variable domain.

[0044] In some embodiments, the immunoglobulin single variable domain in the TSHR binding protein comprises CDR1, CDR2, and CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 6, 31-33; wherein the CDR1, CDR2, and CDR3 are numbered according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems, for example, according to the Kabat numbering system.

[0045] In some embodiments, a TSHR binding protein is provided, comprising any one or any combination of the above-mentioned CDR1, CDR2 and CDR3.

[0046] In some embodiments, a TSHR binding protein is provided, which comprises an immunoglobulin single variable domain, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 13-15, respectively.

[0047] In some embodiments, the immunoglobulin single variable domain in the aforementioned TSHR binding protein is humanized, backmutated, affinity matured, T cell epitope (TCE) removed / reduced, antibody deamidation reduced, and / or antibody isomerization reduced.

[0048] In some embodiments, the immunoglobulin single variable domain is obtained by removing / reducing TCE, and has one or more changes in one or more CDRs, which result in reduced immunogenicity of the TSHR binding protein.

[0049] In some embodiments, the heavy chain framework region of the human germline template used for humanizing the immunoglobulin single variable domain in the aforementioned TSHR binding protein is derived from IGHV3-23, IGHV3-66, or IGJH4. For example, FR1, FR2, and FR3 are derived from IGHV3-23, and FR4 is derived from IGJH4.

[0050] In some embodiments, the immunoglobulin single variable domain has one, more or any combination of mutations at amino acid residues 24, 25, 27, 29, 37, 44, 45, 46, 47, 49, 71, 74, 93, 94 (numbered according to the Kabat numbering system), for example, one, more or any combination of mutations selected from 24G, 25P, 27Y, 29S, 37F, 44E, 45R, 46T, 47G, 49A, 71Q, 74A, 93W, 94A.

[0051] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the aforementioned TSHR binding protein is shown in any one of SEQ ID NOs: 6, 31-33, or has at least 80% (preferably at least 90%) sequence identity with any one of SEQ ID NOs: 6, 31-33.

[0052] In some embodiments, the aforementioned TSHR binding protein comprises or is an antibody or antigen-binding fragment thereof that specifically binds to a human TSHR protein or a fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a camel antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is a recombinant antibody or a fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is a linear antibody, a single-chain antibody, a nanobody, a peptide antibody, a peptibody, a domain antibody and a diabody, a triabody and a tetrabody, a tandem two-scFv, a tandem three-scFv). In some specific embodiments, the antibody is a monospecific antibody, a bispecific antibody, a multispecific antibody (e.g., a trispecific antibody).

[0053] In some embodiments, the immunoglobulin single variable domain in the aforementioned TSHR binding protein is a single domain antibody or VHH, or the TSHR binding protein is a single domain antibody or VHH.

[0054] In some embodiments, the present disclosure provides a TSHR binding protein comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains, wherein the immunoglobulin single variable domains may be the same or different and may form dimers or multimers.

[0055] In some embodiments, the aforementioned TSHR binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region is a human IgG1 Fc region, such as that set forth in SEQ ID NO: 16, or has at least 80% (preferably at least 90%) sequence identity thereto. In some embodiments, the Fc region has enhanced stability or comprises mutations that increase the stability of the Fc region compared to a wild-type Fc region. In some embodiments, the Fc region can enable the binding protein to form a dimeric molecule. In some specific embodiments, the Fc region comprises mutations that extend the in vivo half-life, which is determined by the FcRn binding affinity. This increased half-life can allow for a reduction in the amount of drug administered to a patient and / or a reduced dosing frequency. For example, the Fc region comprises M252Y, S254T, and / or T256E mutations. In some specific embodiments, the Fc region can be an Fc region with reduced effector function, for example, the Fc region can have a mutation, and exemplary IgG Fc regions with reduced effector function include those with the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); or S267E / L328F (IgG1).

[0056] In some embodiments, the immunoglobulin single variable domain in the aforementioned TSHR binding protein is connected to the Fc region directly or through a linker. The linker can be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure. For example, the linker is a flexible linker, such as G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, etc., and more preferably (G4S)2; for example (G m S n ) h or (G m Q n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h As shown, wherein m and n are each independently selected from an integer of 1-8, and h is independently selected from an integer of 1-20.

[0057] In some embodiments, the TSHR binding protein of the present disclosure is an anti-TSHR antibody or an antigen-binding fragment thereof, or a conjugate or fusion protein comprising the antibody or antigen-binding fragment.

[0058] In some embodiments, a TSHR binding protein is provided, comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 19 and 38, or having at least 80% (preferably at least 90%) identity to any one of SEQ ID NOs: 19 and 38.

[0059] In some embodiments, the aforementioned TSHR binding protein has at least one function or property selected from the group consisting of:

[0060] (a)≤10 -7 K D The K value (M) binds to human TSHR or its epitope. D The method for detecting the value is conventional in the art, such as the method in Example 3 of the present disclosure.

[0061] (b) Inhibition of cAMP signaling. In some embodiments, in HEK293 cells, the IC 50 (nM) is less than 20, less than 10, less than 5, less than 3. In some embodiments, the IC 50 The value was detected by the method in Example 5 of the present disclosure.

[0062] (c) Inhibition of serum total thyroxine T4 and / or free T4. In some embodiments, T4 is detected according to the method of Example 6 of the present disclosure.

[0063] In some embodiments, the aforementioned TSHR binding proteins of the present disclosure bind to the K D The value can be ≤1×10 - 8 M, for example, ≤1×10 -9 M, or ≤1×10 -10 M, or ≤1×10 -11 M, or ≤1×10 -12 M.

[0064] In some embodiments, the aforementioned TSHR binding proteins disclosed herein encompass variants, which have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to any one of SEQ ID NOs: 6, 31-33; the amino acid mutations may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function; the amino acid mutations may occur in the CDR region and / or the FR region.

[0065] In some embodiments, anti-TSHR antibodies or antigen-binding fragments thereof are provided that bind to or compete for binding to the same epitope as the immunoglobulin single variable domain in the aforementioned TSHR binding proteins of the present disclosure.

[0066] In some embodiments, anti-TSHR antibodies or antigen-binding fragments thereof are provided, which block the binding of the immunoglobulin single variable domain in the aforementioned TSHR binding protein of the present disclosure to TSHR (eg, human TSHR).

[0067] In some embodiments, an anti-TSHR antibody or antigen-binding fragment thereof is provided, wherein the binding of the anti-TSHR antibody to the TSHR (eg, human TSHR) is blocked by the immunoglobulin single variable domain in the TSHR binding protein of the present disclosure.

[0068] In some embodiments, a protein or molecule is provided that comprises an immunoglobulin single variable domain from any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) TSHR-binding proteins disclosed herein, wherein the immunoglobulin single variable domains are the same or different. For example, the protein or molecule is a conjugate, and the conjugate may, for example, comprise any detectable label.

[0069] Combination of IGF-1R binding protein and TSHR binding protein

[0070] The present disclosure provides for the combination or combined use of an IGF-1R binding protein and a TSHR binding protein.

[0071] In some embodiments, the IGF-1R binding protein is any of the aforementioned IGF-1R binding proteins disclosed herein, and the TSHR binding protein is any of the aforementioned TSHR binding proteins disclosed herein.

[0072] In some embodiments, in the combinations of the present disclosure, the IGF-1R binding protein and the TSHR binding protein are not covalently linked.

[0073] In some specific embodiments, the IGF-1R binding protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 20-24, 34, or at least 80% (preferably at least 90%) identical to any one of SEQ ID NOs: 4, 20-24, 34, or comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 25-30, or at least 80% (preferably at least 90%) identical to any one of SEQ ID NOs: 5, 25-30.

[0074] In some specific embodiments, the TSHR binding protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 6, 31-33, or an amino acid sequence that is at least 80% (preferably at least 90%) identical to any one of SEQ ID NOs: 6, 31-33.

[0075] In some specific embodiments, the IGF-1R binding protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 17 or 37, 18, or at least 80% (preferably at least 90%) identical to any one of SEQ ID NOs: 17 or 37, 18, and the TSHR binding protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 19, 38, or at least 80% (preferably at least 90%) identical to any one of SEQ ID NOs: 19, 38.

[0076] In some specific embodiments, the IGF-1R binding protein comprises the amino acid sequence of SEQ ID NO:34, and the TSHR binding protein comprises the amino acid sequence of SEQ ID NO:38.

[0077] In some specific embodiments, the IGF-1R binding protein comprises the amino acid sequence of SEQ ID NO: 37, and the TSHR binding protein comprises the amino acid sequence of SEQ ID NO: 33.

[0078] In some embodiments, the combination of the IGF-1R binding protein and the TSHR binding protein has the following functions or properties:

[0079] (a) Synergistic inhibition of pERK. In some embodiments, pERK is detected according to the method of Example 9 of the present disclosure; and / or

[0080] (b) Synergistically inhibiting the secretion of hyaluronic acid by cells (e.g., human orbital fibroblasts). In some embodiments, the inhibition of hyaluronic acid secretion by cells is detected according to the method in Example 10 of the present disclosure.

[0081] In some embodiments, the combination of the IGF-1R binding protein and the TSHR binding protein of the present disclosure is prepared in the form of a kit, wherein the IGF-1R binding protein and the TSHR binding protein are in different containers or in the same container.

[0082] Combined administration of IGF-1R binding protein and TSHR binding protein

[0083] In some embodiments, the present disclosure provides any of the aforementioned IGF-1R binding proteins for use in treating a disease (as an example, Graves' ophthalmopathy), wherein the IGF-1R binding protein is administered in combination with a TSHR binding protein of the present disclosure.

[0084] In other embodiments, the present disclosure provides any of the aforementioned TSHR binding proteins for use in treating a disease (as an example, Graves' ophthalmopathy), wherein the TSHR binding protein is administered in combination with an IGF-1R binding protein of the present disclosure.

[0085] In some embodiments, the present disclosure provides use of any of the aforementioned TSHR binding proteins in combination with any of the aforementioned IGF-1R binding proteins for the preparation of a medicament for treating or preventing a disease (as an example, Graves' ophthalmopathy).

[0086] In some embodiments, the present disclosure provides a method of treating or preventing a disease (as an example, Graves' ophthalmopathy) comprising administering to a patient any of the aforementioned TSHR binding proteins and any of the aforementioned IGF-1R binding proteins.

[0087] IGF-1R / TSHR binding protein

[0088] The present disclosure provides an IGF-1R / TSHR binding protein that can bind to IGF-1R (or an epitope thereof) and TSHR (or an epitope thereof) simultaneously or sequentially.

[0089] In some embodiments, the IGF-1R / TSHR binding protein comprises a first antigen binding domain that specifically binds to the TSHR and a second antigen binding domain that specifically binds to IGF-1R.

[0090] In some embodiments, the first antigen-binding domain that specifically binds to TSHR comprises an immunoglobulin single variable domain, and the immunoglobulin single variable domain in the first antigen-binding domain comprises CDR1, CDR2, and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 6 and 31-33.

[0091] In some embodiments, the second antigen-binding domain that specifically binds to IGF-1R comprises an immunoglobulin single variable domain, and the immunoglobulin single variable domain in the second antigen-binding domain comprises:

[0092] 1) CDR1, CDR2 and CDR3 in the amino acid sequence of any one of SEQ ID NOs: 4, 20-24, 34, or, 2) CDR1, CDR2 and CDR3 in the amino acid sequence of any one of SEQ ID NOs: 5, 25-30.

[0093] The aforementioned CDR1, CDR2 and CDR3 are numbered according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, for example, according to the Kabat numbering system.

[0094] In some embodiments, an IGF-1R / TSHR binding protein is provided, comprising any one or any combination of the above-mentioned CDR1, CDR2, and CDR3.

[0095] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the first antigen-binding domain that specifically binds to TSHR are shown in SEQ ID NOs: 13-15, respectively.

[0096] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the second antigen-binding domain that specifically binds to IGF-1R are:

[0097] 1) as shown in SEQ ID NO: 7, 8, 35, respectively, as shown in SEQ ID NO: 7, 8, 9, respectively, or as shown in SEQ ID NO: 7, 8, 36, respectively;

[0098] 2) As shown in SEQ ID NO: 10, 11, and 12 respectively.

[0099] In some embodiments, the immunoglobulin single variable domain in the first antigen-binding domain that specifically binds to TSHR and / or the immunoglobulin single variable domain in the second antigen-binding domain that specifically binds to IGF-1R is humanized, backmutated, affinity matured, removed / reduced T cell epitopes (TCEs), reduced antibody deamidation, and / or reduced antibody isomerization. Removal / reduction of TCEs can result in one or more changes in one or more CDRs of the immunoglobulin single variable domain, resulting in reduced immunogenicity.

[0100] In some embodiments, the heavy chain framework region of the human germline template used for humanization is derived from IGHV3-23, IGHV3-66, or IGJH4. For example, FR1, FR2, and FR3 are derived from IGHV3-23 or IGHV3-66, and FR4 is derived from IGJH4.

[0101] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the aforementioned first antigen-binding domain that specifically binds to TSHR is as shown in any one of SEQ ID NOs: 6, 31-33, or has at least 80% (preferably at least 90%) sequence identity with any one of SEQ ID NOs: 6, 31-33.

[0102] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the aforementioned second antigen-binding domain that specifically binds to IGF-1R is as shown in any one of SEQ ID NOs: 4, 20-24, 34, or has at least 80% (preferably at least 90%) sequence identity with any one of SEQ ID NOs: 4, 20-24, 34, or as shown in any one of SEQ ID NOs: 5, 25-30, or has at least 80% (preferably at least 90%) sequence identity with any one of SEQ ID NOs: 5, 25-30.

[0103] In some embodiments, the aforementioned IGF-1R / TSHR binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region is a human IgG1 Fc region, such as that set forth in SEQ ID NO: 16, or having at least 80% (preferably at least 90%) sequence identity thereto. In some embodiments, the Fc region has increased stability or comprises mutations that increase the stability of the Fc region compared to a wild-type Fc region. In some specific embodiments, the Fc region comprises mutations that extend the in vivo half-life, which is determined by FcRn binding affinity. This increased half-life can allow for a reduction in the amount of drug administered to a patient and / or a reduced frequency of dosing. For example, the Fc region comprises M252Y, S254T, and / or T256E mutations. In some specific embodiments, the Fc region can be an Fc region with reduced effector function, for example, the Fc region can have a mutation, and exemplary IgG Fc regions with reduced effector function include those with the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); or S267E / L328F (IgG1). In some embodiments, the Fc region can enable the binding protein to form a dimeric molecule.

[0104] In some embodiments, the Fc region comprises a first subunit (Fc1) and a second subunit (Fc2), and a mutation is introduced that causes the two subunits (Fc1, Fc2) of the Fc region to pair and form a dimer, or a mutation that reduces homodimerization. In some embodiments, the Fc region comprises a knob-into-hole mutation that promotes the association of the first and second subunits. For example, within the CH3 / CH3 interface, one, two, or more amino acid residues in the CH3 domain of Fc1 are mutated with one or more amino acid residues having a larger side chain volume, thereby generating a protrusion (or knob) on the surface of the CH3 domain of Fc1, and one, two, or more amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated with amino acid residues having a smaller side chain volume, thereby generating a depression (or hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1.

[0105] In some embodiments, the Fc1 has one or more amino acid substitutions at positions selected from 354, 356, 358, and 366, and the Fc2 has one or more amino acid substitutions at positions selected from 349, 356, 358, 366, 368, and 407. In some embodiments, the Fc1 contains a mutation at position 366, and the Fc2 contains a mutation at positions 366, 368, and 407, or any combination thereof. In some embodiments, the Fc1 contains a mutation at position 354 or 356, and the Fc2 contains a mutation at position 349. In some embodiments, the Fc1 contains a mutation at position 354 or 356, and the Fc2 contains a mutation at position 349, 366, 368, and 407.

[0106] In some embodiments, the Fc1 comprises one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the Fc2 comprises one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the Fc1 comprises a 366W mutation, and the Fc2 comprises a mutation selected from 366S, 368A, and 407V, or any combination thereof. In some embodiments, the Fc1 comprises a 354C or 356C mutation, and the Fc2 comprises a 349C mutation. Alternatively, in some embodiments, the Fc1 comprises 354C / 366W mutations, and the Fc2 comprises 349C / 366S / 368A / 407V mutations.

[0107] In some specific embodiments, the aforementioned IGF-1R / TSHR binding protein further comprises a linker. For example, the immunoglobulin single variable domain in the first antigen binding domain that specifically binds to TSHR and / or the immunoglobulin single variable domain in the second antigen binding domain that specifically binds to IGF-1R is connected to the Fc region via a linker. The linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary or higher structures. For example, the linker is a flexible linker, such as G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, and more for example (G4S)2; for example (G m S n ) h or (G m Q n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h As shown, wherein m and n are each independently selected from an integer of 1-8, and h is independently selected from an integer of 1-20.

[0108] In some embodiments, the aforementioned IGF-1R / TSHR binding protein comprises a sequence selected from the following:

[0109] [immunoglobulin single variable domain in the first antigen-binding domain]-linker 1-Fc region-linker 2-[immunoglobulin single variable domain in the second antigen-binding domain],

[0110] [immunoglobulin single variable domain in the second antigen-binding domain]-linker 1-Fc region-linker 2-[immunoglobulin single variable domain in the first antigen-binding domain],

[0111] The linker 1 and linker 2 may exist independently or not, and the linker 1 and linker 2 may be the same or different.

[0112] In some embodiments, the aforementioned IGF-1R / TSHR binding protein comprises the following combination of a first and a second polypeptide chain:

[0113] (1) First polypeptide chain: [immunoglobulin single variable domain in the first antigen-binding domain] 1-Linker 1-Fc1-Linker 2-[immunoglobulin single variable domain in the first antigen-binding domain] 2,

[0114] Second polypeptide chain: [immunoglobulin single variable domain in the second antigen-binding domain] 1-Linker 1-Fc2-Linker 2-[immunoglobulin single variable domain in the second antigen-binding domain] 2; or,

[0115] (2) First polypeptide chain: [immunoglobulin single variable domain in the first antigen-binding domain] 1-Linker 1-Fc2-Linker 2-[immunoglobulin single variable domain in the first antigen-binding domain] 2,

[0116] Second polypeptide chain: [immunoglobulin single variable domain in the second antigen-binding domain] 1-linker 1-Fc1-linker 2-[immunoglobulin single variable domain in the second antigen-binding domain] 2;

[0117] In which, the linker 1 and linker 2 may exist independently or not, and the linker 1 and linker 2 may be the same or different; [the immunoglobulin single variable domain in the first antigen-binding domain] 1 and [the immunoglobulin single variable domain in the first antigen-binding domain] 2 may be the same or different, and [the immunoglobulin single variable domain in the second antigen-binding domain] 1 and [the immunoglobulin single variable domain in the second antigen-binding domain] 2 may be the same or different.

[0118] In some specific embodiments, Fc1 has one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and Fc2 has one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. For example, Fc1 contains 354C / 366W mutations, and Fc2 contains 349C / 366S / 368A / 407V mutations.

[0119] In some embodiments, the amino acid sequences of linker 1 and linker 2 are as follows (G m S n ) h or (G m Q n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h As shown, wherein m and n are each independently selected from integers of 1-8, and h is independently selected from integers of 1-20; in some specific embodiments, linker 1 does not exist, and the amino acid sequence of linker 2 is as shown in (G4Q)3 and (G4S)3.

[0120] In some embodiments, the aforementioned IGF-1R / TSHR binding protein is an anti-IGF-1R / TSHR antibody or an antigen-binding fragment thereof. In some specific embodiments, the antibody is a bispecific or multispecific antibody. In some specific embodiments, the antibody is a camelid antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some specific embodiments, the antibody or antigen-binding fragment thereof is a recombinant antibody or fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is a linear antibody, a single-chain antibody, a nanobody, a peptibody, a domain antibody, a diabody, a triabody, a tetrabody, a tandem di-scFv, or a tandem tri-scFv.

[0121] In some embodiments, the immunoglobulin single variable domain in the aforementioned IGF-1R / TSHR binding protein is a single domain antibody or VHH.

[0122] In some embodiments, an IGF-1R / TSHR binding protein is provided, comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 49-52, or an amino acid sequence having at least 80% (preferably at least 90%) identity to any one of SEQ ID NOs: 49-52.

[0123] In some embodiments, the aforementioned IGF-1R / TSHR binding protein has at least one function or property selected from the group consisting of:

[0124] (a)≤10 -7 K D The value (M) binds to human TSHR or its epitope, and the -7 K D The K value (M) binds to human IGF-1R or its epitope. D The method for detecting the value is conventional in the art, such as the method in Example 3 of the present disclosure.

[0125] (b) inhibiting TSHR downstream signaling pathways, such as pAKT. In some embodiments, in A549 cells, inhibition of pAKT IC 50 (nM) is less than 10, less than 5, less than 2, less than 1, less than 0.5, less than 0.2. In some embodiments, the IC 50 The value was detected by the method in Example 4 of the present disclosure.

[0126] (c) Inhibition of cAMP signaling. In some embodiments, in HEK293 cells, the IC 50 (nM) is less than 20, less than 10, less than 5, less than 3. In some embodiments, the IC 50 The value was detected by the method in Example 5 of the present disclosure.

[0127] (d) Inhibition of serum total thyroxine T4 and / or free T4. In some embodiments, T4 is detected according to the method of Example 6 of the present disclosure.

[0128] (e) No or almost no ADCC effect is induced. In some embodiments, ADCC is detected according to the method in Example 8 of the present disclosure.

[0129] (f) The first binding domain that specifically binds to TSHR and the second binding domain that specifically binds to IGF-1R synergistically inhibit pERK. In some embodiments, pERK is detected according to the method in Example 9 of the present disclosure.

[0130] (g) The first binding domain that specifically binds to TSHR and the second binding domain that specifically binds to IGF-1R synergistically inhibit the secretion of hyaluronic acid by cells (e.g., human orbital fibroblasts). In some embodiments, the inhibition of cell hyaluronic acid secretion is detected according to the method in Example 10 of the present disclosure.

[0131] In some embodiments, the aforementioned IGF-1R / TSHR binding proteins of the present disclosure bind to the K D The value can be ≤1×10 -8 M, for example, ≤1×10 -9 M, or ≤1×10 -10 M, or ≤1×10 -11 M, or ≤1×10 -12 M. K of the aforementioned IGF-1R / TSHR binding protein binding to IGF-1R D Value ≤ 1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0132] In some embodiments, the aforementioned IGF-1R / TSHR binding proteins disclosed herein encompass variants having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to any one of SEQ ID NOs: 49-52; the amino acid mutations may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function; the amino acid mutations may occur in the CDR region and / or the FR region.

[0133] In some embodiments, an IGF-1R / TSHR binding protein (e.g., an anti-IGF-1R / TSHR antibody or antigen-binding fragment thereof) is provided that binds to or competes for binding to the same TSHR (e.g., human TSHR) and / or IGF-1R (e.g., human IGF-1R) epitope as the aforementioned IGF-1R / TSHR binding protein of the present disclosure.

[0134] In some embodiments, an IGF-1R / TSHR binding protein (e.g., an anti-IGF-1R / TSHR antibody or antigen-binding fragment thereof) is provided that blocks the binding of the aforementioned IGF-1R / TSHR of the present disclosure to TSHR (e.g., human TSHR) and / or IGF-1R (e.g., human IGF-1R).

[0135] In some embodiments, an IGF-1R / TSHR binding protein (e.g., an anti-IGF-1R / TSHR antibody or antigen-binding fragment thereof) is provided, wherein binding to TSHR (e.g., human TSHR) and / or IGF-1R (e.g., human IGF-1R) is blocked by the immunoglobulin single variable domain in the aforementioned IGF-1R / TSHR binding protein of the present disclosure.

[0136] In some embodiments, a protein or molecule is provided, which comprises an immunoglobulin single variable domain from any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) TSHR binding proteins disclosed herein (the immunoglobulin single variable domains are the same or different), and comprises an immunoglobulin single variable domain from any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) IGF-1R binding proteins disclosed herein (the immunoglobulin single variable domains are the same or different). For example, the protein or molecule is a conjugate, and the conjugate may, for example, comprise any detectable label.

[0137] Polynucleotides and vectors

[0138] The present disclosure provides polynucleotides encoding the IGF-1R / TSHR binding proteins, IGF-1R binding proteins, and TSHR binding proteins of the present disclosure. The nucleic acids of the present disclosure may be RNA, DNA, or cDNA. According to some embodiments of the present disclosure, the nucleic acids of the present disclosure are substantially isolated nucleic acids.

[0139] The nucleic acids disclosed herein may also be in the form of, present in, and / or part of, a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may particularly be an expression vector, i.e., a vector that provides for expression of the IGF-1R / TSHR binding protein, IGF-1R binding protein, or TSHR binding protein in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). The expression vector typically comprises at least one nucleic acid disclosed herein, operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the skill of the art. Regulatory and other elements useful or necessary for expression of the IGF-1R / TSHR binding protein, IGF-1R binding protein, or TSHR binding protein disclosed herein include, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.

[0140] The nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0141] host cells

[0142] The present disclosure provides recombinant host cells that express or are capable of expressing one or more IGF-1R / TSHR binding proteins, IGF-1R binding proteins, TSHR binding proteins, and / or polynucleotides or vectors containing the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0143] Examples of bacterial cells include cells of gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0144] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0145] Examples of mammalian cells include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0146] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0147] The host cells of the present disclosure are incapable of developing into complete plant or animal individuals.

[0148] Preparation method

[0149] The present disclosure provides a method for preparing IGF-1R / TSHR binding protein, IGF-1R binding protein, and TSHR binding protein, comprising: expressing the target protein in the host cell as described above, and isolating the target protein from the host cell. Optionally, a purification step may also be included, for example, purification using an A or G Sepharose FF column containing an adjusted buffer, washing away non-specifically bound components, and then eluting the bound antibodies using a pH gradient method, detecting with SDS-PAGE, and collecting. Optionally, conventional methods are used for filtration and concentration. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as at -70°C, or freeze-dried.

[0150] Methods for producing and purifying antibodies are well known in the art and can be found in, for example, the Cold Spring Harbor Manual of Antibody Laboratory Techniques (Chapters 5-8 and 15).

[0151] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded and cultured in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes the antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange.

[0152] Composition

[0153] The present disclosure provides compositions comprising the aforementioned IGF-1R / TSHR binding proteins, IGF-1R binding proteins, and / or TSHR binding proteins of the present disclosure. For example, pharmaceutical compositions are provided, comprising an effective amount of the aforementioned IGF-1R / TSHR binding proteins, IGF-1R binding proteins, and / or TSHR binding proteins for treating, alleviating, or preventing a disease, and at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0154] In some embodiments, the unit dosage of the pharmaceutical composition may contain 0.01 to 99% by weight of IGF-1R / TSHR binding protein, IGF-1R binding protein and / or TSHR binding protein, or the amount of IGF-1R / TSHR binding protein, IGF-1R binding protein and / or TSHR binding protein in a unit dose of the pharmaceutical composition is 0.1-2000 mg, and in some embodiments, 1-1000 mg.

[0155] In some embodiments, an article or product (as an example, a kit) is provided that includes the aforementioned IGF-1R / TSHR binding protein, IGF-1R binding protein, and / or TSHR binding protein. Optionally, the article comprises a container and a label. The container is, for example, a bottle, a syringe, and a test tube. The container holds a composition effective for treating a condition. The label on or associated with the container indicates that the composition is used to treat the selected condition.

[0156] In some embodiments, the aforementioned disease is an inflammatory disease or an autoimmune disease.

[0157] In some embodiments, the disease is Graves' ophthalmopathy.

[0158] Methods of treatment and pharmaceutical uses

[0159] The present disclosure provides the aforementioned IGF-1R / TSHR binding protein, IGF-1R binding protein, TSHR binding protein, their encoding polynucleotides, and compositions (including pharmaceutical compositions) for use in methods for treating, alleviating, preventing, and diagnosing diseases or disorders.

[0160] In some embodiments, a method for improving, alleviating, treating or preventing a disease is provided, comprising administering to a subject an effective amount of any one selected from the following:

[0161] 1) the aforementioned IGF-1R / TSHR binding protein or its encoding polynucleotide, or pharmaceutical composition;

[0162] 2) the aforementioned IGF-1R binding protein or its encoding polynucleotide, or pharmaceutical composition;

[0163] 3) the TSHR binding protein or its encoding polynucleotide, or pharmaceutical composition disclosed above;

[0164] 4) The combination of the aforementioned IGF-1R binding protein and TSHR binding protein disclosed herein, or the polynucleotides encoding the polynucleotides and pharmaceutical compositions thereof.

[0165] In some embodiments, there is provided the use of the aforementioned items 1) to 4) for preparing a drug for improving, alleviating, treating or preventing a disease.

[0166] In some embodiments, the aforementioned disease is a disease or condition caused by overexpression of IGF-1R and / or TSHR.

[0167] In some embodiments, the aforementioned disease is an autoimmune system disease.

[0168] In some embodiments, the aforementioned disease is an eye disease, such as Graves' Orbitopathy (GO).

[0169] Detection

[0170] The present disclosure provides uses of IGF-1R / TSHR binding proteins, IGF-1R binding proteins, and TSHR binding protein compositions for detection. The present disclosure also provides methods, systems, or devices for in vivo or in vitro detection of IGF-1R and / or TSHR, comprising treating a sample with the aforementioned binding proteins or compositions of the present disclosure.

[0171] In some embodiments, an in vitro detection method, system, or device may include, for example:

[0172] (1) contacting a sample with an IGF-1R / TSHR binding protein, an IGF-1R binding protein, a TSHR binding protein, or a composition of the present disclosure;

[0173] (2) detecting a complex formed between the binding protein and the sample; and / or

[0174] (3) contacting a reference sample (e.g., a control sample) with the binding protein; and

[0175] (4) Determining the extent of complex formation by comparison with a reference sample. A change (e.g., a statistically significant change) in complex formation in the sample compared to the control sample indicates the presence of IGF-1R and / or TSHR in the sample.

[0176] In some embodiments, kits are also provided, comprising the aforementioned IGF-1R / TSHR binding proteins, IGF-1R binding proteins, TSHR binding proteins, their encoding polynucleotides, compositions, and instructions for diagnostic use. The kits may also contain at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the IGF-1R / TSHR binding proteins, IGF-1R binding proteins, TSHR binding proteins, and their encoding polynucleotides can be formulated as pharmaceutical compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0177] FIG1A shows the inhibition of IGF1-induced pAKT signaling by the disclosed antibodies and positive antibodies in human non-small cell lung cancer cells (A549).

[0178] FIG1B shows the inhibition of IGF1-induced pAKT signaling by the antibodies of the present disclosure and positive antibodies in mouse myoblasts (C2C12).

[0179] FIG2A to FIG2C show the results of cellular cAMP inhibition assays performed after transient transfection of human / rat / mouse TSHR in HEK293 cells.

[0180] FIG3A and FIG3B show the efficacy test of TSHR antibody in Wistar rats, FIG3A shows the results of total T4 concentration in serum, and FIG3B shows the results of free T4 concentration in serum.

[0181] FIG4 shows the results of in vitro ADCC effect detection of bispecific antibodies and positive antibodies.

[0182] FIG5 shows the results of synergistic inhibition of ERK phosphorylation in HEK293-TSHR cells by IGF-1R / TSHR dual antibodies.

[0183] FIG6 shows the results of synergistic inhibition of hyaluronic acid production in primary orbital fibroblasts by IGF-1R / TSHR dual antibodies. DETAILED DESCRIPTION

[0184] the term

[0185] In order to make the present disclosure more easily understood, certain technologies and sciences are specifically defined below. Unless otherwise clearly defined elsewhere in the present disclosure, all other technologies and sciences used in the present disclosure have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0186] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0187] "IGF-1R" (insulin like growth factor-1 receptor) belongs to the tyrosine protein kinase receptor family and is a widely expressed heterotetrameric protein that is involved in regulating the proliferation and metabolic functions of many cell types and can be activated by IGF-1 and IGF-2 (both insulin growth factors). IGF-1R is a tyrosine kinase receptor composed of two subunits, IGF-IRα, which contains the ligand binding domain, and IGF-IRβ, which participates in signal transduction and contains tyrosine phosphorylation sites. For example, the sequence of human IGF-1R is found in Uniprot:P08069.

[0188] The TSHR (thyroid-stimulating hormone receptor) is a G protein-coupled receptor present on the membrane of thyroid follicular cells. Under normal physiological conditions, the TSHR regulates the growth, differentiation, and function of thyroid follicular cells by binding to thyroid-stimulating hormone (TSH) through the adenylate cyclase (AC-cAMP) pathway and the phospholipase C-diacylglycerol and phosphoinositide pathway. For example, the sequence of the human TSHR is found in Uniprot: P16473.

[0189] "IGF-1R binding protein" encompasses any molecule capable of specifically binding to an IGF-1R protein or an epitope thereof, including but not limited to antibodies, antigen-binding fragments thereof, or conjugates thereof directed against IGF-1R as defined herein.

[0190] "TSHR binding protein" encompasses any molecule that can specifically bind to a TSHR protein or an epitope thereof, including but not limited to antibodies, antigen-binding fragments thereof, or conjugates thereof directed against TSHR as defined in the present disclosure.

[0191] In some embodiments, an "IGF-1R binding protein" may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, or more) single-domain antibodies that specifically bind to IGF-1R according to the embodiments of the present disclosure. A "TSHR binding protein" may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, or more) single-domain antibodies that specifically bind to TSHR according to the embodiments of the present disclosure.

[0192] In some embodiments, the "IGF-1R binding protein" of the present disclosure may also comprise, in addition to the immunoglobulin single variable domain that binds to IGF-1R, a linker and / or a portion with effector function, such as a half-life extending portion (e.g., an immunoglobulin single variable domain that binds serum albumin) and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region.

[0193] In some embodiments, a "TSHR binding protein" may comprise, in addition to an immunoglobulin single variable domain that comprises the TSHR, a linker and / or a portion with effector function, such as a half-life extending portion (e.g., an immunoglobulin single variable domain that binds serum albumin) and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region.

[0194] "IGF-1R / TSHR binding protein" encompasses any protein that can specifically bind to IGF-1R and TSHR, or any molecule comprising the protein, including but not limited to antibodies, polypeptides, fusion proteins of antibodies and polypeptides, or conjugates thereof. In some embodiments, "IGF-1R / TSHR binding protein" encompasses the bispecific antibodies against IGF-1R and TSHR in the embodiments of the present disclosure.

[0195] "Antibodies" encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies may refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant regions of immunoglobulins' heavy chains differ, resulting in different antigenicity. Based on this, immunoglobulins can be divided into five classes, or so-called immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Igs of the same class can be further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of antibody heavy and light chains vary greatly in sequence and constitute the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0196] The antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic, or modified forms thereof, with monoclonal antibodies being particularly suitable for use in a number of embodiments. In general, the antibodies of the present disclosure are recombinant antibodies. As used herein, "recombinant" refers generally to products such as cells or nucleic acids, proteins, or vectors, indicating that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that the cells are derived from cells so modified. For example, recombinant cells express genes that are not present in the native (non-recombinant) cell form or express native genes that are abnormally expressed, underexpressed, or not expressed at all.

[0197] For the determination or definition of CDRs, the deterministic depiction of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions. The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the position of certain structural loop regions. (See, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd.). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from primary sequence (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In the conformational definition, the positions of the CDRs can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166).In addition, other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or residue group does not significantly affect antigen binding. As used in this disclosure, CDRs can refer to CDRs defined by any method known in the art (including combinations of methods). The correspondence between the various numbering systems is well known to those skilled in the art.

[0198] A "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for specific functional properties of a protein and in many cases can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain.

[0199] An "immunoglobulin domain" refers to a globular region of an antibody chain (eg, a chain of an antibody of conventional tetrapeptide chain structure or a heavy chain antibody), or to a polypeptide consisting essentially of such a globular region.

[0200] An "immunoglobulin variable domain" is an immunoglobulin domain that essentially consists of four "framework regions," referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3," respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity for an antigen by having an antigen-binding site.

[0201] "Antibody framework (FR)" refers to the portion of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain.

[0202] "Immunoglobulin single variable domain" is used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that can typically form a functional antigen binding site in the absence of interaction with other domains (variable or constant domains) (e.g., in the absence of the VH / VL interactions required between the VH and VL domains of conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, e.g., camelized human VH), IgNAR, domains, (single domain) antibodies that are VH domains or derived from VH domains (such as dAbs), and antibodies that are VH domains or derived from VH domains. TM ), and (single domain) antibodies (such as dAbs) as or derived from a VL domain. TM ). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

[0203] "VHH" is also called heavy chain single domain antibody, VHH, V H H domains, VHH antibody fragments, VHH antibodies, nanobodies, are variable domains of antigen-binding immunoglobulins known as "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). "VHH" is used to distinguish the variable domains from the heavy chain variable domains (referred to herein as "VH domains" or VH) and light chain variable domains (referred to herein as "VL domains" or VL) present in conventional tetrapeptide chain structure antibodies. The VHH domain specifically binds to an epitope without the need for additional antigen-binding domains (this is in contrast to the VH or VL domains in conventional tetrapeptide chain structure antibodies, in which case the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. H H domain", "VHH antibody fragment", "VHH antibody", as well as" "Nanobody" is a trademark of Ablynx NV, Ghent, Belgium. VHHs include, but are not limited to, natural antibodies produced by camelids, antibodies produced by camelids that have been humanized, or antibodies obtained by phage display technology. The total number of amino acid residues in a VHH will generally 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 in the present disclosure. 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, 17 June, 2009 and WO94 / 04678.

[0204] As is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions numbered according to Kabat may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Other numbering systems or coding conventions include Chothia, IMGT, and AbM.

[0205] "Humanized antibodies," also known as CDR-grafted antibodies, are antibodies produced by transplanting non-human CDR sequences into the human variable region framework. This can overcome the strong immune response induced by chimeric antibodies due to the presence of a large number of non-human protein components. To avoid a simultaneous decrease in immunogenicity and activity, minimal reverse mutations can be performed on the fully human variable region to maintain activity. Examples of "humanization" include "humanizing" a Camelidae-derived VHH domain by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in a VH domain of a conventional human tetrapeptide antibody (also referred to as "sequence optimization" in this disclosure; in addition to humanization, "sequence optimization" 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). A humanized VHH domain may contain one or more fully human framework region sequences, and in some embodiments, may contain human framework region sequences from IGHV3. Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework), that is, CDR "grafting" on other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and technologies suitable for the CDR transplantation are known in the art. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the VBase human germline sequence database, as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies after affinity maturation of CDRs by phage display. In addition, in order to avoid a decrease in immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0206] An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for the antigen, compared to a parent antibody that does not possess such alterations. For example, an "affinity matured" IGF-1R binding protein or anti-IGF-1R antibody has one or more alterations in one or more CDRs that result in increased affinity for the antigen compared to its parent antibody. Affinity matured antibodies can be prepared by methods known in the art, for example, as described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3):889-896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0207] Typically, the IGF-1R / TSHR binding proteins, IGF-1R binding proteins, TSHR binding proteins of the present disclosure will be expressed as measured in a Biacore or KinExA or Fortibio assay at a concentration of preferably 10 -7 to 10 -10 Mole / liter (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (K D ), and / or with at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen or target protein to be bound (i.e., IGF-1R, TSHR). Any -4 M's K DValues ​​are generally considered to indicate nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays) as described herein.

[0208] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules can be determined by determining the dissociation constant (K D K can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K values ​​were compared with those for non-target interactions (e.g., control antibodies known not to bind IGF-1R or TSHR). D The value is evaluated.

[0209] A "conservative substitution" refers to a substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0210] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0211] "Nucleic acid" or "polynucleotide" are used interchangeably in this disclosure to refer to any DNA or RNA molecule, whether single-stranded or double-stranded, and, in the case of single-stranded, its complementary sequence, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0212] "Host cell" includes individual cells or cell cultures that can be or have been recipients of vectors for incorporating polynucleotide inserts. Host cells include the progeny of a single host cell, and due to natural, accidental or intentional mutations, the progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of the present disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such designations include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included.

[0213] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. "Inhibit growth" (eg, involving cells) is intended to include any measurable decrease in cell growth.

[0214] "Administer," "apply," and "treat" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, for example, therapeutic, pharmacokinetics, diagnostic, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. When applied to humans, veterinary medicine, or research subjects, it refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0215] "Treatment" means administering an internal or external therapeutic agent, such as a binding protein or pharmaceutical composition thereof comprising any of the present disclosure, to a subject who has, is suspected of having, or is predisposed to having one or more autoimmune diseases or symptoms thereof, for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, either by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0216] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

[0217] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. "And / or" should be taken as specifically disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" used in phrases such as "A and / or B" in the present disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of "including but not limited to." In the context of mutations contained in the Fc region in the present disclosure, " / " means "and," for example, "354C / 366W" means "354C and 366W," that is, the Fc contains 354C and 366W mutations; the amino acid positions of the mutations in the Fc region of the present disclosure are all numbered according to the EU numbering system.

[0218] The "subject" and "patient" of the present disclosure refer to mammals, especially primates, and especially humans.

[0219] Example

[0220] The following examples further illustrate the present disclosure, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents where the specific source is not specified are commercially available.

[0221] Example 1. Screening and preparation of single-domain antibodies specifically binding to human IGF-1R and human TSHR

[0222] 1. Antigens and detection proteins

[0223] Using human IGF-1R (Uniprot: P08069) and human TSHR (Uniprot: P16473) as templates, the amino acid sequences of related proteins for screening and detection were designed.

[0224] >hIGF-1R-ECD-avi-his (human IGF-1R extracellular domain amino acid sequence)

[0225] (Note: The single underline is the human IGF-1R extracellular domain sequence; the italicized one is the GS linker; the double underline is the avi tag; the dotted underline is the his tag)

[0226] >hTSHR-ECD-avi-his (human TSHR extracellular domain amino acid sequence, 6 amino acid mutations)

[0227] (Note: The single underline indicates the human TSHR extracellular domain; the bold italicized text indicates six amino acid mutation sites used to optimize protein production; the italicized text indicates the GS linker; the double underlined text indicates the avi tag; the dotted line indicates the his tag) > hTSHR-ECD-camel Fc-avi-his (human TSHR extracellular domain amino acid sequence, six amino acid mutations)

[0228] (Note: The single underline indicates the human TSHR extracellular domain; bold italics indicate amino acid mutation sites used to optimize protein production; bold underline indicates the camel Fc; italics indicate the GS linker; double underline indicates the avi tag; and the dotted line indicates the his tag. SEQ ID NO: 3 is the human TSHR extracellular domain fused to the N-terminus of the camel Fc region for camel immunization.)

[0229] 2. Screening of single domain antibodies that specifically bind to human IGF-1R and human TSHR

[0230] Human IGF-1R (SEQ ID NO: 1) and human TSHR-camel Fc (SEQ ID NO: 3) were used as antigenic proteins to immunize a single Bactrian camel. 5 mL of pre-immune camel plasma was collected and serum was separated. Freund's complete adjuvant was mixed with the antigen at a 1:1 ratio, and multiple subcutaneous immunizations were performed every two weeks. The first immunization dose was 200 μg of protein, followed by four immunizations at a 100 μg protein dose each, for a total of five immunizations. Titers were then measured. Plates were coated with 2 μg / mL of human IGF-1R (SEQ ID NO: 1) and human TSHR (SEQ ID NO: 2) proteins (100 μL / well) at 4°C overnight. The next day, plates were washed and blocked with 4% skim milk powder and incubated at 37°C for 1 hour. After washing, camel serum of varying dilutions was added and incubated at 37°C for 1 hour. Negative controls included pre-immune serum (1:1000 dilution) and PBS solution. After incubation, the cells were washed three times with PBST and then incubated at 37°C for 1 hour with an HRP-conjugated rabbit anti-camelid polyclonal antibody (1:1000 dilution). After washing, alkaline phosphatase colorimetric solution was added, and the reaction was terminated with 2M sulfuric acid. The absorbance was read at 450 nm. The titer was determined after a 1:1,000,000 dilution. If the titer was acceptable, peripheral blood was collected from the camel for library preparation.

[0231] Lymphocytes were isolated from peripheral blood of camels, and the cell count was 1.2 × 10 8RNA was extracted and reverse transcribed to obtain cDNA for phage library construction. Single domain antibodies with high affinity to human IGF-1R and human TSHR antigen proteins were obtained by screening the phage library. 20 μg of biotinylated human IGF-1R (SEQ ID NO: 1) and human TSHR (SEQ ID NO: 2) were combined with 100 μL Dynabeads. TM M-280 Streptavidin was placed at 37°C for 1 hour and then blocked with 2% skim milk at room temperature for 1 hour. The camel heavy chain single domain antibody phage display library was added and allowed to act at room temperature for 1 hour. The plate was washed 9 times with PBST (PBS containing 0.05% Tween-20) to remove unbound phage. Phages that specifically bound to human IGF-1R and human TSHR were eluted with 1 mg / mL trypsin and infected with Escherichia coli TG1 in logarithmic phase growth to produce and purify phage for the next round of screening. Two rounds of screening were performed in total. 96 monoclonal colonies were picked from the positive clones enriched by screening and packaged into phage single chain antibodies for phage ELISA testing. 2 μg / mL of human IGF-1R and human TSHR antigens were coated on the ELISA plate, phage supernatant diluted with blocking solution was added, and detection was performed with anti-M13 HRP-labeled antibody. The clones with OD450 value / background value > 5 in the ELISA binding test results were sequenced to obtain enriched single domain antibody sequences.

[0232] Targeting TSHR, phage library screening at the cell level was also performed. HEK293 cells were transiently transfected with TSHR (37°C, 5% CO2), and 2×10 7 Cells, 210g, centrifugation for 6 minutes. Block the cells with PBS containing 5% BSA at room temperature for 1 hour (HEK293-TSHR) and the phage library for 1 hour. The HEK293-TSHR transiently transfected cells were then incubated with the phage library at room temperature for 1 hour. Wash with PBST (PBS containing 0.05% Tween-20) 5 times, wash with PBS 3 times, 210g, 6 minutes. Elute with 0.5mL citric acid (0.1M pH 2.2) for 10 minutes, then neutralize with 0.5mL 1M tris-HCl (pH 7.4), infect Escherichia coli TG1 in logarithmic phase growth, produce and purify phage for the next round of screening, and perform two rounds of screening. After NGS sequencing, the enriched sequences were selected for cellular binding (flow cytometry) and functional verification.

[0233] After affinity determination, we selected single-domain antibodies IG05 and IG06 targeting IGF-1R and single-domain antibody CB16 targeting TSHR.

[0234] >IG05

[0235] >IG06

[0236] >CB 16

[0237] In the above sequences SEQ ID NOs: 4-6, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the FR sequences are in italics, and the underlined sequences are CDR1, CDR2, and CDR3, respectively. The CDR sequences are summarized in Table 1.

[0238] Table 1. Single domain antibody CDRs (Kabat numbering convention)

[0239] 3. Preparation of complete antibodies from single domain antibodies

[0240] The aforementioned single-domain antibody (VHH) was used to construct a complete antibody. Specifically, the VHH sequence was fused to the human IgG1-Fc region (CH2-CH3) sequence and constructed into the pTT5 expression vector. The sequence of the connected human IgG1-Fc region can be as follows:

[0241] >Human IgG1 Fc region

[0242] The following are the full sequences of IG05, IG06, and CB16 fused to the human IgG1 Fc region: IG05-IgG1, IG06-IgG1, and CB16-IgG1. The sequences are as follows:

[0243] >IG05-IgG1

[0244] >IG06-IgG1

[0245] >CB16-IgG1

[0246] The aforementioned antibody sequence was cloned into the mammalian expression vector pTT5, transfected into HEK293E or ExpiCHO cells for expression, and the cell culture supernatant was harvested, filtered, purified, and tested to obtain the target protein.

[0247] Example 2. Modification of anti-IGF-1R and TSHR single domain antibodies

[0248] 1. Humanization

[0249] By performing three-dimensional homology modeling of the selected specific IGF-1R or TSHR single-domain antibodies and comparing the results with the V-base human germline sequence database and the IMGT human antibody heavy chain variable region germline gene database, the heavy chain variable region germline genes IGHV3-23 or IGHV3-66, which show high homology to the target sequence, were selected as templates for FR1, FR2, and FR3. IGJH4 was used as the template for FR4. The CDRs of the camelid single-domain antibody were transplanted into the corresponding human template, resulting in a variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. To retain the original activity of the humanized single-domain antibody, a series of back mutations were performed in the FR regions.

[0250] Table 2. Humanized back mutation sites and amino acid types (Kabat numbering rules)

[0251] The obtained humanized sequence is shown below, with the CDR regions underlined (Kabat numbering convention).

[0252] >IG05-hu1

[0253] >IG05-hu2

[0254] >IG05-hu3

[0255] >IG05-hu4

[0256] >IG05-hu5

[0257] >IG06-hu1

[0258] >IG06-hu2

[0259] >IG06-hu3

[0260] >IG06-hu4

[0261] >IG06-hu5

[0262] >IG06-hu6

[0263] >CB 16-hu1

[0264] >CB 16-hu2

[0265] >CB 16-hu3

[0266] Based on IG05-hu5, TCE and deamidation sites were modified to obtain IG05-hu5-1.

[0267] >IG05-hu5-1

[0268] That is, the CDR of IG05 disclosed in the present invention has the following general structure:

[0269] CDR1: TFGMA (SEQ ID NO: 7)

[0270] CDR2: AISASSGRTYYADSVKG (SEQ ID NO: 8)

[0271] CDR3: RSKX1GAGYGFWYEX2RLYGY, wherein X1 is N or Q, and X2 is A or G (SEQ ID NO: 35).

[0272] The CDR3 can be RSKNGAGYGFWYEARLYGY (SEQ ID NO:9) or RSKQGAGYGFWYEGRLYGY (SEQ ID NO:36).

[0273] The method described in Example 1 was used to construct a full antibody sequence in which the C-terminus of the engineered single-domain antibody VHH was directly fused to the N-terminus of the Fc region of hIgG1 (SEQ ID NO: 16). The antibody structures and corresponding names are as follows:

[0274] Table 3. Antibody structures and corresponding names

[0275] An exemplary complete antibody sequence is as follows: >IG05-hu5-1-IgG1

[0276] >CB16-hu3-IgG1

[0277] Example 3. Determination of affinity between anti-IGF-1R antibody, anti-TSHR antibody and antigen

[0278] 1. Determination of the affinity of anti-IGF-1R antibodies to human and mouse IGF-1R

[0279] The affinity of IG05 and IG06 related antibodies to IGF-1R was determined by Biacore T200 (GE Healthcare) instrument, and the positive controls were Tepezza, VRDN-001, and PHP1003.

[0280] Using a Series S sensor chip CM5 (GE Healthcare, Cat. 29149603), an anti-His antibody kit (GE Healthcare, Cat. 28-9950-56) was amino-coupled (amine coupling kit GE Healthcare, Cat. BR100050) to the chip surface, and then human IGF-1R (SEQ ID NO: 1) / mouse IGF-1R (Acro, Cat. IGR-M5223) antigens were captured. Different concentrations of IG05 and IG06-related antibodies and a positive control antibody were passed over the chip surface, and the reaction signals were detected in real time to obtain binding and dissociation curves. The binding constants were then obtained by fitting. The experimental solution used was HBS-EP solution. At the end of each experimental cycle, the chip was regenerated with pH 1.5 Glycine (GE Healthcare, Cat. BR-1003-54) solution. The antibody affinity results are shown in Tables 4 and 5.

[0281] Results showed that the intact antibodies IG05 and IG06 obtained through screening in this disclosure had superior affinity for human IGF-1R compared to the positive controls Tepezza, VRDN-001, and PHP1003. IG06 also exhibited strong species cross-reactivity with mouse IGF-1R, making it suitable as a surrogate antibody for in vivo efficacy testing in non-transgenic mice and rats.

[0282] Table 4. Affinity of IG05 and IG06-related single domain antibodies to human / mouse IGF-1R

[0283] Table 5. Affinity of IG05 and IG06 related intact antibodies to human / mouse IGF-1R

[0284] 2. Determination of the affinity of anti-TSHR antibodies to human TSHR

[0285] The affinity of CB16-related antibodies to human TSHR was determined using a Biacore T200 (GE Healthcare) instrument, with K1-70 and M22 as positive controls and HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) as negative control.

[0286] The antibody to be detected was captured onto the chip surface using a Series S sensor chip Protein A (GE Healthcare, Cat. 29127556). Human TSHR (SEQ ID NO: 2) at varying concentrations was then passed over the chip surface. The reaction signal was monitored in real time to generate binding and dissociation curves, which were then fitted to obtain the binding constant. The experimental solution used was HBS-EP solution. At the end of each experimental cycle, the chip was regenerated with pH 1.5 Glycine (GE Healthcare, Cat. BR-1003-54).

[0287] The affinity results of the antibodies are shown in Table 6. Later in vivo efficacy experiments demonstrated that CB16-hu3-IgG1 was superior to K1-70 (Figure 3A, Figure 3B).

[0288] Table 6. Affinity of anti-TSHR antibodies to human TSHR

[0289] In this disclosure, the positive controls for anti-IGF-1R antibodies are Tepezza (Horizon, US2022356257), VRDN-001 (Viridian, WO2023019171), and PHP1003 (Suzhou Pulekang, CN113512116); the positive controls for anti-TSHR antibodies are K1-70 (PDB: 7XW7, Av7), and the agonist antibody M22 (PDB: 3G04). The sequences are as follows:

[0290] >Tepezza antibody heavy chain

[0291] >Tepezza antibody light chain

[0292] >VRDN-001 antibody heavy chain

[0293] > VRDN-001 antibody light chain

[0294] >PHP1003 antibody heavy chain

[0295] >PHP1003 antibody light chain

[0296] >K1-70 antibody heavy chain

[0297] >K1-70 antibody light chain

[0298] >M22 antibody heavy chain

[0299] >M22 antibody light chain

[0300] Example 4. Phosphorylated AKT (pAKT) inhibition experiment with anti-IGF-1R antibodies

[0301] To examine the effects of anti-IGF-1R antibodies on human IGF-1R downstream signaling pathways, we tested the inhibitory effect of the antibodies on IGF1-induced pAKT signaling.

[0302] pAKT was detected using PHOSPHO-AKT (SER473) KITS (PerkinElmer, Cat. No. 64 AKS PET).

[0303] Human non-small cell lung cancer cells (A549, 15,000 cells / well) and mouse myoblasts (C2C12, 15,000 cells / well) were cultured overnight in a 96-well flat-bottom plate using DMEM (Gibco, Cat. No. 11995065) supplemented with 1% FBS. After the start of the experiment, the culture medium in the plate was discarded and replaced with 90 μL of HANKS balanced salt solution. The antibody to be tested was diluted, 10 μL was added to each well of cells, and mixed evenly. The plate was placed in a cell culture incubator and incubated at 37°C for 30 minutes. After the incubation was completed, 10 μL of human IGF1 (Beyotime, Cat. No. P5502) was added to each well to a final concentration of 100 ng / mL and incubated at 37°C for another 30 minutes.

[0304] After the incubation, discard all the liquid in the well plate and lyse the cells according to the instructions of PHOSPHO-AKT (SER473) KITS. Add 1x supplemented lysis buffer and shake at 400 rpm for 20 minutes at room temperature. After the end, transfer 16 μL of cell lysate to a white 96-well plate and add 4 μL of Phospho-AKT d2 and Phospho-AKT Eu Crytate antibody mixture. Cover with sealing film and let it stand at room temperature for 4 hours. Use Tecan Read the values ​​at 665 nm and 620 nm using a 200 PRO microplate reader. Calculate the values ​​using the following formula to compare the ability of the antibodies to inhibit pAKT signaling.

[0305] IG05-hu5-1-IgG1, IG06-IgG1, PHP1003, VRDN-001, and Tepezza were all compared using the above method in A549 cells. Because IG06-IgG1 is cross-species compatible with mouse, we also validated its ability to inhibit pAKT by binding to mouse IGF-1R in C2C12 mouse myoblasts.

[0306] As shown in Figure 1A and Table 7-1, in human non-small cell lung cancer cells (A549), Tepezza, VRDN-001, PHP1003, IG05-hu5-1-IgG1, and IG06-IgG1 all inhibited IGF1-induced pAKT signaling, with IG05-hu5-1-IgG1 performing best. As shown in Figure 1B and Table 7-2, in mouse myoblasts (C2C12), IG06-IgG1 and PHP1003 both inhibited IGF1-induced pAKT signaling, with IG06-IgG1 exhibiting superior inhibitory activity compared to PHP1003. Tepezza lacked cross-species activity with mice and exhibited no inhibitory effect.

[0307] Table 7-1. Experimental results of antibodies inhibiting IGF1-induced pAKT in A549 cells

[0308] Table 7-2. Experimental results of antibodies inhibiting IGF1-induced pAKT in C2C12 cells

[0309] Note: / indicates no pAKT inhibitory effect, NA indicates that the accurate IC was not obtained 50 Numeric value.

[0310] Example 5. Detection of the inhibitory effect of anti-TSHR antibodies on cAMP signaling

[0311] 1. cAMP inhibition experiment in HEK293-human TSHR overexpressing cells

[0312] To examine the effects of anti-TSHR antibodies on human TSHR downstream signaling pathways, we tested the inhibitory effect of the antibodies on TSH-induced cAMP signaling.

[0313] HEK293 suspension cells were transiently transfected with a plasmid expressing the full-length human TSHR using lipofectamine 2000 (ThermoFisher, Cat. No. 11668019). After 48 hours of culture, the cells were resuspended in HANKS balanced salt solution and plated at 50,000 cells / well in a 96-well U-bottom plate. Antibodies at various concentrations were added and preincubated for 1 hour. 5,000 cells per well were transferred to a 96-well HTRF plate (cisbio, Cat. No. 66PL96025) and stimulated for 30 minutes with bovine TSH (Sigma, Cat. No. T8931) or 100 ng / mL M22 at a final concentration of 0.1 μM. cAMP levels produced by lysed cells were measured using the CAMP-GSDYNAMIC KIT (cisbio, Cat. No. 62AM4PEB).

[0314] As shown in Figure 2A , both CB16-hu3-IgG1 and the positive control antibody K1-70 showed inhibitory function on cAMP in HEK293-human TSHR cells, IC 50 The values ​​were 2.202nM and 2.295nM, respectively, which were functionally equivalent, indicating that CB16-hu3-IgG1 and K1-70 could block the interaction between ligands and TSHR receptors and inhibit the activation of their downstream cAMP signaling pathways.

[0315] 2. cAMP inhibition experiment in HEK293-rat / mouse TSHR overexpression cells

[0316] This study aimed to examine the inhibitory effect of anti-TSHR antibodies on cAMP signaling downstream of rat / mouse TSHR receptors.

[0317] Plasmids expressing rat or mouse full-length TSHR were transiently transfected into HEK293 suspension cells. After 48 hours of culture, the cells were resuspended in HANKS balanced salt solution and plated at 100,000 cells / well in a 96-well U-bottom plate. Antibodies at various concentrations were added and preincubated for 1 hour. 10,000 cells per well were transferred to an HTRF 96-well plate and stimulated with bovine TSH at a final concentration of 0.1 μM for HEK293-rat TSHR cells or 0.1 μM for HEK293-mouse TSHR cells for 30 minutes. cAMP levels produced by lysed cells were measured using the CAMP-GSDYNAMIC Kit.

[0318] As shown in Figures 2B and 2C, both CB16-hu3-IgG1 and K1-70 significantly inhibited the cAMP production by bovine TSH-stimulated rat and mouse TSHR, and their activities were similar. 50The values ​​were 3.661nM (rat) and 5.941nM (mouse), respectively. The K1-70 inhibitory activity IC 50 The values ​​were 2.018 nM (rat) and 4.031 nM (mouse), respectively.

[0319] Example 6. Pharmacological efficacy of anti-TSHR antibodies in Wistar rats

[0320] To evaluate the inhibitory function of TSHR antibodies in vivo, the Wistar rat model was selected to explore the effects of antibodies on total thyroxine T4 and free T4 in rat serum under physiological conditions.

[0321] Five-week-old female Han Wistar rats (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were divided into three groups of five rats each based on body weight. On day 0, each rat was given an equal molar amount of K1-70 positive antibody (200 μg) and CB16-hu3-IgG1 (106.7 μg) by intramuscular injection. The control group was given 0.1 mL of PBS. Orbital blood was collected from all rats before administration and 4 h, 24 h, 48 h, 72 h, 96 h, and 120 h after administration, and serum was collected. After 120 h of blood collection, the rats were sacrificed. The T4 concentration in the serum of rats in each group was detected using total T4 (Cat. No.: EIAT4C, ThermoFisher) and free T4 ELISA (Cat. No.: CSB-E05079r, Cusabio) kits.

[0322] As shown in Figures 3A and 3B and Table 8, CB16-hu3-IgG1 demonstrated significant inhibition of serum total and free T4 levels starting 24 hours after administration. This inhibitory effect persisted until the end of the experiment, 5 days after administration, and the inhibitory effect was significantly superior to that of the positive control antibody K1-70. Serum free T4 is the active form of T4 that functions in vivo. At 120 hours after administration, serum free T4 levels in rats treated with K1-70 had returned to levels similar to those in the PBS control group, while serum free T4 levels in rats treated with CB16-hu3-IgG1 remained significantly lower. This suggests that CB16-hu3-IgG1 can block the TSHR receptor and inhibit downstream T4 secretion with a strong and sustained effect.

[0323] Table 8. Wistar rat model results

[0324] Example 7. Construction and preparation of anti-IGF-1R / TSHR bispecific antibodies

[0325] In this example, an anti-IGF-1R / TSHR bispecific antibody was constructed.

[0326] Exemplarily, IG05-hu5-1 and CB16-hu3 were linked to the N-terminus and C-terminus of the IgG1 Fc region (SEQ ID NO: 16) via a (G4S)3 linker, respectively, to construct bispecific antibody 0031-0024. IG06 and CB16-hu3 were linked to the N-terminus and C-terminus of the IgG1 Fc region (SEQ ID NO: 16) via a (G4Q)3 linker, respectively, to construct bispecific antibody 0031-0053. Similarly, 0031-0060 and 0031-0070 were constructed. The sequences are as follows:

[0327] >0031-0024

[0328] >0031-0053

[0329] >0031-0060

[0330] >0031-0070

[0331] The bispecific antibody sequence was cloned into the mammalian expression vector pTT5, transfected into ExpiCHO cells, expressed, purified, and tested to obtain the desired bispecific antibody.

[0332] Example 8. Detection of Antibody ADCC Effect

[0333] This example aims to detect the ability of antibodies to mediate cytotoxicity in effector cells to kill target cells.

[0334] HEK293 suspension cells were transiently transfected with a plasmid expressing the full-length human TSHR using lipofectamine 2000. After 48 hours of culture, the cells were resuspended in phenol red-free RPMI 1640 medium (Gibco, Cat. No. 11835030) containing 2% fetal bovine serum and 10 ng / mL recombinant human IL-2 (Biolegend, Cat. No. 589102). HEK293-human TSHR target cells were plated at 20,000 cells / well in a 96-well U-bottom plate and preincubated with various concentrations of antibodies for 1 hour. 50,000 NK92-FcγRIIIA-176V3 effector cells were then added to each well and incubated in a 37°C cell culture incubator for 5 hours. After centrifugation of the cells, the supernatant was collected and assayed using a Cytotoxicity Assay Kit (Promega, Cat. No. G1780) with substrate. The readings were read at 490 nm using a SpectraMax M5 microplate reader. The values ​​were calculated using the following formula to compare the ADCC activity of the antibodies.

[0335] As shown in Figure 4, the positive control antibody (0031-0024, with S228D / I332E mutations in the Fc region) with Fc-modified enhanced ADCC activity exhibited significant cytotoxicity. In comparison, the cytotoxicity induced by monoclonal antibody CB16-IgG1 was weaker, and no ADCC activity was detected for bispecific antibody 0031-0024, monoclonal antibody IG05-hu5-1-IgG1, and positive antibodies such as K1-70, Tepezza, VRDN-001, and PHP1003. This indicates that bispecific antibody 0031-0024 does not induce ADCC, suggesting that it has a low risk of inducing cytotoxicity in vivo and a good safety profile.

[0336] Example 9. Synergistic Experiment of Antibody Inhibition of ERK Phosphorylation (pERK)

[0337] In this example, the ability of the anti-IGF-1R / TSHR bispecific antibody to inhibit pERK signaling in HEK293-human TSHR cells was tested.

[0338] First, HEK293 cells in suspension culture were transiently transfected with a human TSHR plasmid and lipofectamine 2000, resulting in cell surface expression of both endogenous human IGF-1R and overexpression of human TSHR. Twenty-four hours after transfection, a pERK costimulation assay was performed using the following protocol:

[0339] Count the cells and plate HEK293-human TSHR cells at 1.25x10 6 Resuspend the cells at a density of 100 μL / mL in HANKS balanced salt solution and place 40 μL per well in a 96-well U-bottom plate. Dilute the anti-IGF-1R / TSHR bispecific antibody to be tested in HANKS balanced salt solution to the desired concentration and add 10 μL per well. After mixing the antibody and cells, place the 96-well plate in a cell culture incubator and incubate at 37°C for 30 minutes.

[0340] Dilute human IGF1 (Beyotime, Cat. No. P5502) and bovine TSH (Sigma, Cat. No. T8931) to the desired concentrations and add 10 μL of IGF1, TSH, or both to each well for costimulation. After mixing, return the 96-well plate to the cell culture incubator and incubate at 37°C for 6 minutes. The final concentrations of IGF1 and TSH in each well are 100 ng / mL and 1000 ng / mL, respectively.

[0341] Immediately after the stimulation, the cells were resuspended again, and 6 μL (about 5000 cells) of cells were transferred to a white 96-well microplate, and 2 μL 4x lysis buffer was added for lysis. The cells were shaken at 400 rpm at room temperature for 30 minutes. Then, referring to the instructions in PHOSPHO-ERK1 / 2 (THR202 / TYR204) DETECTION KITS (PerkinElmer, Cat. No. 64ERKPEG), 2 μL of Phospho-ERK1 / 2 d2 antibody and Phospho-ERK1 / 2 Eu Cryptate antibody mixture were added to each well, covered with a sealing film, and incubated at room temperature for 4 hours. Using Tecan Read the values ​​at 665 nm and 620 nm using a 200 PRO microplate reader. Calculate the values ​​using the following formula to compare the ability of the antibodies to inhibit pERK signaling under IGF1 and TSH co-stimulation.

[0342] The results in Figure 5 and Table 9 show that only the dual antibody 0031-0024 produced a significant dose-dependent inhibitory effect on the pERK signal synergistically generated under IGF1 / TSH co-stimulation, which was significantly better than other control monoclonal antibodies.

[0343] Table 9. Results of antibody inhibition of pERK signal synergistically generated by IGF1 / TSH co-stimulation

[0344] Note: / indicates no ERK phosphorylation inhibitory effect, no dose-dependent effect

[0345] Example 10. Synergistic Experiment on Inhibition of Hyaluronic Acid Secretion

[0346] Orbital fibroblasts from TAO patients can secrete hyaluronic acid and cause tissue edema under the stimulation of IGF-1R / TSHR autoimmune antibodies. This example tests the ability of IGF-1R / TSHR antibodies to inhibit the secretion of hyaluronic acid (HA) from orbital fibroblasts (OF) from TAO patients.

[0347] Retrobulbar adipose connective tissue was obtained from patients undergoing orbital decompression surgery for TAO. The surgical specimens were then separated and purified to obtain OF cells for experiments. The specific experimental methods are as follows:

[0348] Count the cells and divide the OF cells into 5x10 4Resuspend at a density of 100 μL / mL in DMEM supplemented with 1% fetal bovine serum and place 100 μL per well in a 96-well plate. Starve for 24 hours. Dilute the IGF-1R / TSHR antibody to be tested to the desired concentration in reduced serum medium, add 50 μL per well, and incubate in a 37°C cell culture incubator for 30 minutes.

[0349] Bovine TSH or M22 was diluted to the desired concentration, and 50 μL of bovine TSH was added to each well for stimulation. The final concentration of bovine TSH in each well was 2 μg / mL. The cells were incubated in a 37°C cell culture incubator for 72 hours. Hyaluronan levels in the culture supernatant were measured using the Hyaluronan Quantikine ELISA Kit (Cat. No. DHYAL0, R&D Systems).

[0350] The results in Figure 6 and Table 10 show that both TSHR and IGF1R monoclonal antibodies can inhibit the secretion of HA stimulated by bovine TSH alone. In TAO patient donors, at an antibody concentration of 50 nM, the inhibitory activity of the monoclonal antibody combination and the bispecific antibody 0031-0024 was stronger than that of the control monoclonal antibody, showing a synergistic inhibitory trend, and the bispecific antibody 0031-0024 had the strongest inhibitory activity.

[0351] Table 10. Antibody inhibition of hyaluronic acid production under TSH stimulation

Claims

1. A TSHR binding protein comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 6 and 31-33, wherein The CDR1, CDR2 and CDR3 are numbered according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 13, 14, and 15, respectively.

2. The TSHR binding protein according to claim 1, wherein the immunoglobulin single variable domain is modified by any one of the following or a combination thereof: humanization, back mutation, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, and reduction of antibody isomerization; Preferably, the heavy chain framework region of the human germline template used in the humanization process is derived from IGHV3-23 or IGJH4.

3. The TSHR binding protein according to claim 1 or 2, wherein the amino acid sequence of the immunoglobulin single variable domain is as shown in any one of SEQ ID NOs: 6, 31-33 or has at least 80% sequence identity thereto, Preferably, the TSHR binding protein is an anti-TSHR antibody or an antigen-binding fragment thereof; More preferably, the TSHR binding protein is an anti-TSHR single domain antibody or VHH.

4. The TSHR binding protein according to any one of claims 1 to 3, further comprising an immunoglobulin Fc region; Preferably, the Fc region is the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4; More preferably, the Fc region is the Fc region of human IgG1.

5. An IGF-1R / TSHR binding protein comprising: A first antigen binding domain that specifically binds to TSHR; and A second antigen binding domain that specifically binds to IGF-1R; Preferably, the first antigen binding domain comprises an immunoglobulin single variable domain, and the immunoglobulin single variable domain in the first antigen binding domain comprises: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 6 and 31-33, in, The CDR1, CDR2 and CDR3 are numbered according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems. 6 . The IGF-1R / TSHR binding protein according to claim 5 , wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain in the first antigen binding domain are shown in SEQ ID NOs: 13, 14 and 15, respectively.

7. The IGF-1R / TSHR binding protein according to claim 5 or 6, wherein The second antigen binding domain comprises an immunoglobulin single variable domain, and the immunoglobulin single variable domain in the second antigen binding domain comprises: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 4, 20-24, 34, or CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 5, 25-30, wherein the CDR1, CDR2 and CDR3 are numbered according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the immunoglobulin single variable domain in the second antigen binding domain comprises: CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 7, 8, 35, respectively, or, CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 10, 11 and 12, respectively; More preferably, the immunoglobulin single variable domain in the second antigen binding domain comprises: CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 7, 8, 9, respectively, or, CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 7, 8 and 36 respectively.

8. The IGF-1R / TSHR binding protein according to any one of claims 5 to 7, wherein the immunoglobulin single variable domain is modified by any one of the following or a combination thereof: humanization, back mutation, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, and reduction of antibody isomerization; Preferably, the heavy chain framework region of the human germline template used in the humanization process is derived from IGHV3-23, IGHV3-66 and IGJH4.

9. The IGF-1R / TSHR binding protein according to any one of claims 5 to 8, wherein the amino acid sequence of the immunoglobulin single variable domain in the first antigen binding domain is as shown in any one of SEQ ID NOs: 6, 31-33 or has at least 80% sequence identity thereto.

10. The IGF-1R / TSHR binding protein according to any one of claims 5 to 9, wherein The amino acid sequence of the immunoglobulin single variable domain in the second antigen binding domain is as shown in any one of SEQ ID NOs: 4, 20-24, 34, or has at least 80% sequence identity thereto, or, as shown in any one of SEQ ID NOs: 5, 25-30, or has at least 80% sequence identity thereto; Preferably, the immunoglobulin single variable domain in the second antigen binding domain is an anti-IGF-1R single domain antibody or VHH.

11. The IGF-1R / TSHR binding protein according to any one of claims 5 to 10, wherein The immunoglobulin single variable domain is a single domain antibody or VHH.

12. The IGF-1R / TSHR binding protein according to any one of claims 5 to 11, further comprising an Fc region of an immunoglobulin; Preferably, the Fc region is the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4; More preferably, the Fc region is the Fc region of human IgG1; Most preferably, the Fc region comprises an amino acid sequence as shown in SEQ ID NO: 16, or at least 90% identical thereto.

13. The IGF-1R / TSHR binding protein according to any one of claims 5 to 12, further comprising a linker; Preferably, the amino acid sequence of the linker is as follows (G m S n ) h or (G m Q n ) h or (GGNGT) h or (YGNGT) h As shown, m and n are each independently selected from an integer of 1-8, and h is independently selected from an integer of 1-20; More preferably, the amino acid sequence of the linker is as shown in (G4Q)3, (G4S)3.

14. The IGF-1R / TSHR binding protein according to any one of claims 5 to 13, which is selected from the following structures from amino terminus to carboxyl terminus: [immunoglobulin single variable domain in the first antigen-binding domain]-Linker 1-Fc region-Linker 2-[immunoglobulin single variable domain in the second antigen-binding domain], [The immunoglobulin single variable domain in the second antigen-binding domain]-Linker 1-Fc region-Linker 2-[The immunoglobulin single variable domain in the first antigen-binding domain], in, - is a peptide bond, the linker 1 and linker 2 may exist independently or not, and the linker 1 and linker 2 may be the same or different; Preferably, the amino acid sequences of the linker 1 and the linker 2 are each independently selected from (G m S n ) h or (G m Q n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h , wherein m and n are each independently selected from an integer of 1-8, and h is independently selected from an integer of 1-20; More preferably, the linker 1 does not exist, and the amino acid sequence of the linker 2 is as shown in (G4Q)3 or (G4S)3.

15. The IGF-1R / TSHR binding protein of any one of claims 5 to 14, comprising an amino acid sequence as shown in any one of SEQ ID NOs: 49-52, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 49-52. 16 . The IGF-1R / TSHR binding protein according to claim 5 , which is an anti-IGF-1R / TSHR antibody or an antigen-binding fragment thereof.

17. An IGF-1R binding protein comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 4, 20-24, 34, or CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 5, 25-30, wherein: The CDR1, CDR2 and CDR3 are numbered according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the immunoglobulin single variable domain comprises: CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 7, 8, 35, respectively, or, CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 10, 11 and 12, respectively; More preferably, the immunoglobulin single variable domain comprises: CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 7, 8, 9, respectively, or, CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 7, 8 and 36 respectively.

18. The IGF-1R binding protein according to claim 17, wherein the immunoglobulin single variable domain is modified by any one of the following or a combination thereof: humanization, back mutation, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, and reduction of antibody isomerization; Preferably, the heavy chain framework region of the human germline template used in the humanization process is derived from IGHV3-66 or IGJH4.

19. The IGF-1R binding protein of claim 17 or 18, wherein the amino acid sequence of the immunoglobulin single variable domain is as shown in any one of SEQ ID NOs: 5, 20-24, 34 or has at least 80% sequence identity thereto, or, as shown in any one of SEQ ID NOs: 4, 25-30 or has at least 80% sequence identity thereto; Preferably, the IGF-1R binding protein is an anti-IGF-1R antibody or an antigen-binding fragment thereof; More preferably, the IGF-1R binding protein is an anti-IGF-1R single domain antibody or VHH.

20. The IGF-1R binding protein of any one of claims 17 to 19, further comprising an immunoglobulin Fc region; Preferably, the Fc region is the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4; More preferably, the Fc region is the Fc region of human IgG1.

21. Combination, including: The TSHR binding protein according to any one of claims 1 to 4; and The IGF-1R binding protein according to any one of claims 17 to 20.

22. A polynucleotide encoding the TSHR binding protein of any one of claims 1 to 4, the IGF-1R / TSHR binding protein of any one of claims 5 to 16, the IGF-1R binding protein of any one of claims 17 to 20, or any combination thereof; Preferably, the polynucleotide is DNA or RNA.

23. A vector comprising the polynucleotide according to claim 22, Preferably, the vector expresses the polynucleotide of claim 22.

24. A host cell containing or expressing the polynucleotide of claim 22 or the vector of claim 23.

25. A method for preparing a TSHR binding protein, an IGF-1R / TSHR binding protein, or an IGF-1R binding protein, comprising: Expressing the polynucleotide of claim 22 or the vector of claim 23 in the host cell of claim 24, and isolating the expressed TSHR binding protein, IGF-1R / TSHR binding protein, or IGF-1R binding protein from the host cell; Optionally, the method further comprises the step of purifying the TSHR binding protein, IGF-1R / TSHR binding protein, or IGF-1R binding protein.

26. A pharmaceutical composition comprising: The TSHR binding protein of any one of claims 1 to 4, the IGF-1R / TSHR binding protein of any one of claims 5 to 16, the IGF-1R binding protein of any one of claims 17 to 20, or any combination thereof; and, At least one pharmaceutically acceptable excipient, diluent or carrier.

27. Methods for treating or preventing disease, including: Administering a therapeutically or prophylactically effective amount of the TSHR binding protein of any one of claims 1 to 4, the IGF-1R / TSHR binding protein of any one of claims 5 to 16, the IGF-1R binding protein of any one of claims 17 to 20, or any combination thereof, or the polynucleotide of claim 22, the vector of claim 23, or the pharmaceutical composition of claim 26 to a subject in need thereof; Preferably, the disease is an autoimmune disease; More preferably, the disease is Graves' ophthalmopathy.

28. Use of the TSHR binding protein according to any one of claims 1 to 4, the IGF-1R / TSHR binding protein according to any one of claims 5 to 16, the IGF-1R binding protein according to any one of claims 17 to 20, or any combination thereof, or the polynucleotide according to claim 22, the vector according to claim 23, or the pharmaceutical composition according to claim 26 for preparing a drug for treating or preventing a disease; Preferably, the disease is an autoimmune disease; More preferably, the disease is Graves' ophthalmopathy.

29. Use of the TSHR binding protein according to any one of claims 1 to 4 in combination with the IGF-1R binding protein according to any one of claims 17 to 20 for preparing a drug for treating or preventing a disease; Preferably, the disease is an autoimmune disease; More preferably, the disease is Graves' ophthalmopathy.

30. A method for treating or preventing a disease, comprising administering to a patient the TSHR binding protein according to any one of claims 1 to 4 and the IGF-1R binding protein according to any one of claims 17 to 20; Preferably, the disease is an autoimmune disease; More preferably, the disease is Graves' ophthalmopathy.

31. The TSHR binding protein of any one of claims 1 to 4 for use in treating or preventing a disease, wherein the TSHR binding protein is administered in combination with the IGF-1R binding protein of any one of claims 17 to 20; preferably, the disease is an autoimmune disease; More preferably, the disease is Graves' ophthalmopathy.

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