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KR-20260068079-A - IGF-1R and TSHR binding proteins and their medicinal uses

KR20260068079AKR 20260068079 AKR20260068079 AKR 20260068079AKR-20260068079-A

Abstract

The present invention provides IGF-1R and TSHR binding proteins and their medicinal uses. Specifically, the invention provides IGF-1R/TSHR binding proteins, IGF-1R binding proteins, TSHR binding proteins, and methods for treating autoimmune diseases (e.g., Graves' disease of the eye) and related pharmaceutical uses.

Inventors

  • 쉬, 진징
  • 왕, 레이
  • 잔, 이캉
  • 류, 샤오

Assignees

  • 베이징 투오 지에 바이오파마수티컬 컴퍼니 리미티드

Dates

Publication Date
20260513
Application Date
20240906
Priority Date
20230908

Claims (20)

  1. As a TSHR binding protein, it comprises an immunoglobulin monovariable domain, and said immunoglobulin monovariable domain is: It includes CDR1, CDR2, and CDR3 among the amino acid sequences presented in any one of SEQ ID NO: 6, 31~33, said CDR1, CDR2, and CDR3 are numbered according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; Preferably, the immunoglobulin monovariable domain comprises a TSHR binding protein comprising CDR1, CDR2, and CDR3 as presented in SEQ ID NO: 13, 14, and 15, respectively.
  2. In claim 1, the immunoglobulin single variable domain is modified by any one selected from humanization, reverse mutation, affinity maturation, T cell epitope removal, reduction of antibody deamidation, reduction of antibody isomerization, or a combination thereof; Preferably, the heavy chain framework region of the human germline template used in the humanization modification process is a TSHR binding protein derived from IGHV3-23 or IGHJ4.
  3. In claim 1 or 2, the amino acid sequence of the immunoglobulin single variable domain is the same as that presented in any one of SEQ ID NO: 6, 31 to 33, or has at least 80% sequence identity with it, and 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 a VHH, a TSHR binding protein.
  4. In any one of paragraphs 1 to 3, it also includes 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, a TSHR binding protein.
  5. As an IGF-1R/TSHR binding protein, this is: A first antigen-binding domain that specifically binds to TSHR; and It includes a second antigen-binding domain that specifically binds to IGF-1R; Preferably, the first antigen-binding domain comprises an immunoglobulin monovariable domain, and the immunoglobulin monovariable domain among the first antigen-binding domains is: Includes CDR1, CDR2, and CDR3 among the amino acid sequences presented in any one of SEQ ID NO: 6, 31~33, and The above CDR1, CDR2, and CDR3 are IGF-1R/TSHR binding proteins numbered according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.
  6. In claim 5, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain among the first antigen-binding domains are IGF-1R/TSHR binding proteins as presented in SEQ ID NO: 13, 14, and 15, respectively.
  7. In claim 5 or 6, the second antigen-binding domain comprises an immunoglobulin single variable domain, and the immunoglobulin single variable domain among the second antigen-binding domains is: CDR1, CDR2, and CDR3 among the amino acid sequences presented in any one of SEQ ID NO: 4, 20–24, 34, or CDR1, CDR2, and CDR3 among the amino acid sequences presented in any one of SEQ ID NO: 5, 25–30, and The above CDR1, CDR2, and CDR3 are numbered according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; Preferably, the immunoglobulin monovariable domain among the second antigen-binding domains is: CDR1, CDR2, and CDR3 as presented in SEQ ID NO: 7, 8, 35, respectively, or, Includes CDR1, CDR2, and CDR3 as presented in SEQ ID NO: 10, 11, and 12, respectively; More preferably, the immunoglobulin monovariable domain among the second antigen-binding domains is: CDR1, CDR2, and CDR3 as presented in SEQ ID NO: 7, 8, and 9, respectively, or, IGF-1R/TSHR binding proteins, including CDR1, CDR2, and CDR3 as presented in SEQ ID NO: 7, 8, and 36, respectively.
  8. In any one of claims 5 to 7, the immunoglobulin single variable domain is modified by any one selected from humanization, reverse mutation, affinity maturation, T cell epitope removal, reduction of antibody deamidation, reduction of antibody isomerization, or a combination thereof; Preferably, the heavy chain framework region of the human germline template used in the humanization modification process is an IGF-1R/TSHR binding protein derived from IGHV3-23, IGHV3-66, and IGHJ4.
  9. An 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 presented in any one of SEQ ID NO: 6, 31 to 33, or has at least 80% sequence identity with respect to it.
  10. In any one of paragraphs 5 through 9, where, The amino acid sequence of the immunoglobulin single variable domain among the second antigen-binding domains is the same as that presented in any one of SEQ ID NO: 4, 20~24, 34, or has at least 80% sequence identity therewith, or is the same as that presented in any one of SEQ ID NO: 5, 25~30, or has at least 80% sequence identity therewith; Preferably, the immunoglobulin single variable domain among the second antigen-binding domains is an anti-IGF-1R single domain antibody or an IGF-1R/TSHR binding protein, VHH.
  11. In any one of claims 5 to 10, the immunoglobulin single variable domain is an IGF-1R/TSHR binding protein that is a single domain antibody or VHH.
  12. In any one of paragraphs 5 to 11, it also includes 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; Most preferably, the Fc region comprises an amino acid sequence that is the same as or at least 90% identical to that presented in SEQ ID NO: 16, an IGF-1R/TSHR binding protein.
  13. In any one of claims 5 to 12, the binding protein also has a linker; Preferably, the amino acid sequence of the linker is as presented in (G m S n ) h or (G m Q n ) h or (GGNGT) h or (YGNGT) h , wherein m and n are each independently selected from integers 1 to 8, and h is independently selected from integers 1 to 20; More preferably, the amino acid sequence of the linker is an IGF-1R/TSHR binding protein as presented in (G 4 Q) 3 , (G 4 S) 3 .
  14. In any one of claims 5 to 13, from the amino group to the carboxyl group, the structure is selected from the structures shown below: [Immunoglobulin monovariable domain in the first antigen-binding domain]-linker 1-Fc region-linker 2-[Immunoglobulin monovariable domain in the second antigen-binding domain], [Immunoglobulin monovariable domain in the second antigen-binding domain]-linker 1-Fc region-linker 2-[Immunoglobulin monovariable domain in the first antigen-binding domain], Here, - is a peptide bond, and the linker 1 and linker 2 may exist independently or not exist, and linker 1 and linker 2 may be the same or different; Preferably, the amino acid sequences of linker 1 and 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 integers 1 to 8, and h is independently selected from integers 1 to 20; More preferably, the linker 1 is absent, and the amino acid sequence of the linker 2 is an IGF-1R/TSHR binding protein as presented in (G 4 Q) 3 or (G 4 S) 3 .
  15. An IGF-1R/TSHR binding protein according to any one of claims 5 to 14, comprising an amino acid sequence as presented in any one of SEQ ID NO: 49 to 52 or an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 49 to 52.
  16. In any one of claims 5 to 15, the IGF-1R/TSHR binding protein is an anti-IGF-1R/TSHR antibody or an antigen-binding fragment thereof.
  17. As an IGF-1R binding protein, it comprises an immunoglobulin monovariable domain, and said immunoglobulin monovariable domain is: It comprises CDR1, CDR2, and CDR3 among the amino acid sequences presented in any one of SEQ ID NO: 4, 20–24, 34, or CDR1, CDR2, and CDR3 among the amino acid sequences presented in any one of SEQ ID NO: 5, 25–30, wherein said CDR1, CDR2, and CDR3 are numbered according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; Preferably, the immunoglobulin monovariable domain is: CDR1, CDR2, and CDR3 as presented in SEQ ID NO: 7, 8, 35, respectively, or, Includes CDR1, CDR2, and CDR3 as presented in SEQ ID NO: 10, 11, and 12, respectively; More preferably, the immunoglobulin monovariable domain is: CDR1, CDR2, and CDR3 as presented in SEQ ID NO: 7, 8, and 9, respectively, or, IGF-1R binding proteins, including CDR1, CDR2, and CDR3 as presented in SEQ ID NO: 7, 8, and 36, respectively.
  18. In claim 17, the immunoglobulin monovariable domain is modified by any one or a combination thereof selected from humanization, reverse mutation, affinity maturation, T cell epitope removal, reduction of antibody deamidation, and reduction of antibody isomerization; Preferably, the heavy chain framework region of the human germline template used in the humanization modification process is an IGF-1R binding protein derived from IGHV3-66 or IGHJ4.
  19. In claim 17 or 18, the amino acid sequence of the immunoglobulin single variable domain is the same as that presented in any one of SEQ ID NO: 5, 20–24, 34, or has at least 80% sequence identity therewith, or is the same as that presented in any one of SEQ ID NO: 4, 25–30, or has at least 80% sequence identity therewith; 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, an IGF-1R binding protein.
  20. In any one of claims 17 to 19, it also comprises the Fc region of the 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, an IGF-1R binding protein.

Description

IGF-1R and TSHR binding proteins and their medicinal uses This application claims priority to Chinese patent application CN202311157087.9 filed on September 8, 2023. The present disclosure relates to an IGF-1R/TSHR binding protein, an IGF-1R binding protein, a TSHR binding protein, a coding nucleic acid thereof, a pharmaceutical composition, and a method for treating an autoimmune disease (e.g., Graves' disease of the eye) and related pharmaceutical uses. Graves' Orbitopathy (GO), also known as Thyroid-associated Ophthalmopathy (TAO; Thyroid Eye Disease, TED), is an organ-specific autoimmune disease that is associated with but relatively independent of Graves' disease, in which the patient's immune system overstimulates the thyroid gland, causing it to overproduce thyroid hormones. Orbital fibroblasts (OF) express thyroid-stimulating hormone receptors (TSHR), insulin-like growth factor-1 receptors (IGF-1R), and immunomodulatory molecules; upon appropriate stimulation, some can produce hyaluronic acid (HA), inflammatory mediators, and autoantibodies, while others can differentiate into adipocytes, which are the primary target cells of autoimmunity in Graves' Orbitopathy (Terry J. Smith, n engl j med 375;16(2016)). Adipocytes differentiated by OF are the primary source of TSHR in the orbital tissue of GO patients. According to research, TSHR mRNA and protein are expressed in the orbital adipose tissue of Graves' disease patients with or without ocular disease, and it has been demonstrated that TSHR levels in GO patients are significantly higher than in patients without GO, indicating that increased TSHR expression in the orbit may be involved in disease progression (Wang Y, Invest Ophthalmol Vis Sci, 55;3(2014)). Research indicates that IGF-1R and TSHR are expressed at the same intracellular location, form a synergistic functional complex, and activate B cells to produce thyroid-stimulating antibodies capable of binding to TSHR. Additionally, TSHR and IGF-1R co-mediate downstream signaling pathways, causing OF to produce large amounts of inflammatory factors such as IL-8, prostaglandin E2, and mononuclear chemotactic proteins. Simultaneously, after TSHR is activated, OF secretes large amounts of hyaluronic acid, inducing swelling, edema, and inflammation of the posterior orbital adipose connective tissue. In addition, after the binding of IGF-1 and IGF-1R, the massive proliferation of OF can be further promoted. Tepezza is currently the only IGF-1R targeted antibody drug on the market worldwide, although it has not yet been approved in China. K1-70 is an inhibitory antibody against TSHR and is currently undergoing Phase I clinical trials; Phase I results indicate that it can effectively alleviate symptoms associated with GO patients (Jadwiga Furmaniak, Clinical Endocrinology, 96; 878-887(2022)). Single-domain antibodies have a molecular weight of only 12–15 kDa and contain three CDRs; they are the smallest known antibodies with full antigen-binding activity and possess many advantages such as high binding affinity, high specificity, easy modification, high solubility, high stability, and high expression levels. The present disclosure provides an anti-TSHR single-domain antibody with a novel sequence structure and an anti-IGF-1R single-domain antibody with a novel sequence structure, and constructs a bispecific antibody of both; according to detection results, a synergistic effect of the bispecific antibody is achieved, and there is a prospect that it will become a more effective and safer drug for the clinical treatment of Graves' disease of the eye. IGF-1R/TSHR binding protein, IGF-1R binding protein, TSHR binding protein, the coding nucleic acid thereof, pharmaceutical composition, and the method for treating diseases thereof and related pharmaceutical uses. IGF-1R binding protein The present disclosure provides an IGF-1R binding protein comprising an immunoglobulin monovariable domain, wherein the immunoglobulin monovariable domain is: 1) CDR1, CDR2, and CDR3 among the amino acid sequences presented in any one of SEQ ID NO: 4, 20–24, 34, or 2) CDR1, CDR2, and CDR3 among the amino acid sequences presented in any one of SEQ ID NO: 5, 25–30; Here, 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. In some embodiments, an IGF-1R binding protein comprising any one of the above-described CDR1, CDR2, and CDR3 or any combination thereof is provided. In some embodiments, an IGF-1R binding protein comprising an immunoglobulin monovariable domain is provided, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin monovariable domain are each as follows: 1) As presented in SEQ ID NO: 7, 8, 35, respectively, as presented in SEQ ID NO: 7, 8, 9, respectively, or as presented in SEQ ID NO: 7, 8, 36, respectively; 2) As presented in SEQ ID NO: 10, 11, and 12, respectively. In some embodiment