US-12624099-B2 - Polypeptide improved in protein purity and affinity for antigen, conjugate thereof with antibody or antigen-binding fragment, and preparation method therefor

Abstract

The present invention relates to a polypeptide improved in protein purity and in affinity for a target antigen, a conjugate thereof with an antibody or antigen-binding fragment, and a preparation method of the polypeptide and the conjugate. The polypeptide or the conjugate thereof according to the present invention does not undergo glycosylation even when produced in a eukaryotic cell and thus has high protein purity and affinity for a target antigen, showing a very high value as a reagent for diagnosis or treatment of a disease.

Inventors

• Jong Ho Lee
• Hyun Jong LEE
• Bong Kook Ko
• Kyu Tae Kim
• Jong Seo Lee

Assignees

• ABCLON INC.

Dates

Publication Date

20260512

Application Date

20180418

Priority Date

20170418

Claims (10)

1. 1 . A polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 4.
2. 2 . A polypeptide complex comprising: i) the polypeptide of claim 1 ; and ii) an antibody or an antigen-binding fragment thereof that specifically binds to a target antigen, wherein i) and ii) are linked to each other.
3. 3 . The polypeptide complex of claim 2 , wherein the target antigen of ii) the antibody or the antigen-binding fragment thereof is selected from the group consisting of angiogenin 2 (Ang-2), vascular endothelial growth factor, tumor necrosis factor, TNF-α, TNFSF11, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, insulin-like growth factor, interleukin 1α, interleukin 1β, interleukin 10, interleukin 17A, interleukin 12, interleukin 23, interleukin 33, granulocyte macrophage colony-stimulating factor, granulocyte colony stimulating factor, high-mobility group protein B1, lipopolysaccharide, toll-like receptor 4, nerve growth factor, chemokine C-C motif ligand 19, chemokine C-C motif ligand 21, chemokine C-C motif ligand 4, and interferon alpha.
4. 4 . The polypeptide complex of claim 2 , wherein the target antigen of ii) the antibody or the antigen-binding fragment thereof is tumor necrosis factor-α (TNF-α).
5. 5 . The polypeptide complex of claim 2 , wherein in ii), the antibody or the antigen-binding fragment thereof is selected from the group consisting of adalimumab, infliximab, golimumab, and certolizumab pegol, and antigen-binding fragments thereof.
6. 6 . The polypeptide complex of claim 2 , wherein in ii), the antibody or the antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 5.
7. 7 . The polypeptide complex of claim 2 , wherein i) the polypeptide and ii) the antibody or the antigen-binding fragment thereof are linked to each other via at least one linker.
8. 8 . The polypeptide complex of claim 7 , wherein the linker consists of an amino acid sequence represented by the general formula (GnSm)p or (SmGn)p, wherein n, m, and p each are independent; n is an integer of 1 to 7; m is an integer of 0 to 7; the sum of n and m is an integer of 8 or less; and p is an integer of 1 to 7.
9. 9 . A nucleic acid consisting of a nucleotide sequence encoding the polypeptide of claim 1 .
10. 10 . A nucleic acid consisting of a nucleotide sequence encoding the polypeptide complex of claim 2 .

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a national phase application of PCT Application No. PCT/KR2018/004516, filed on Apr. 18, 2018, which claims benefit of Korean Patent Application 10-2017-0049732, filed on Apr. 18, 2017. The entire disclosure of the applications identified in this paragraph are incorporated herein by references. FIELD The present invention relates to a polypeptide improved in protein purity and affinity for an antigen, a complex of the polypeptide with an antibody or an antigen-binding fragment thereof, a method for producing the polypeptide, and a method for producing the complex. More specifically, the present invention relates to a polypeptide and the like, which are improved in protein purity and affinity for an antigen by substituting an amino acid residue at a specific position in the scaffold of a known polypeptide to thereby prevent glycosylation when the polypeptide and the like are expressed in eukaryotic cells. BACKGROUND Affibody® molecules are small proteins composed of 58 amino acid residues based on the Z domain, which is an affinity site for IgG in Protein A from Staphylococcus aureus. In protein sequencing of the Affibody molecules, 13 amino acids that form the binding surface with IgG can bind to various target antigens depending on the amino acid sequence thereof and can be randomly arranged to construct libraries. Similar to antibodies, Affibody molecules capable of binding to various target antigens can be screened from libraries through screening methods, such as phage display and yeast two hybrid (Y2H). Affibody molecules specifically binding to HER2 and amyloid-β have been recently developed using characteristics of Affibody molecules capable of binding to target antigens (Orlova et al. 2006, Cancer Res., and Gronwall et al., 2007, J. Biotechnol). Since the Affibody molecules have a very small molecular weight of 6 kDa, the Affibody molecules are systemically diffused and fast removed through kidney filtration when administered into the human body. Therefore, Affibody molecules are mainly applied to the research and development of diagnostic species (Goldstein R et al., 2013, Expert Rev Anticancer Ther). Affibody molecules have also been developed in the form of double antibodies binding to general IgG (Yu F et al., 2014, MAbs). Protein post-translation modification (PTM) occurs in cells of eukaryotic creatures including humans. Examples of post-translational modification are acetylation, phosphorylation, and the like, and such a post-translational modification affects protein diversity, plays an important role in intracellular signaling, and regulates cellular physiology. Such an abnormal post-translational modification occurring in intercellular proteins causes a variety of diseases including cancer. However, the sequence information of a particular protein alone makes it impossible to accurately predict whether the protein will undergo post-translational modification. Therefore, protein identification needs to be accompanied by a task of checking, through a variety of experiments, whether or not post-translational modification occurs. In cases where a protein to be expressed is expressed in eukaryotes, such as animal cells, but not prokaryotes, such as bacteria, there is a possibility of producing proteins having no desired characteristics by such post-translational modification. For example, it has been reported that the occurrence of glycosylation, which is a kind of post-translational modification, in an antibody variable region, may drop the homogeneity of antibodies and disturb target-specific binding thereof (Wright A et al., 1991, EMBO). PCT Publication WO95/19374 discloses first-generation Z variant-based polypeptide scaffolds and PCT Publication WO2009/080811 discloses second-generation Z variant-based polypeptide scaffolds. However, the cited documents do not disclose, teach, and suggest the occurrence or not of post-translational modification (i.e., glycosylation) as in the present invention and the effect of the post-translational modification on homogeneity or target-specific binding ability of polypeptides as a resultant product, and do not recognize the need of improvement thereof. Above all, there is a continuous need for improvement of protein purity (homogeneity) and target-specific binding ability in cases of medicinal products using polypeptides, especially, polypeptides using target-specific binding characteristics. Throughout the present specification, many papers and patent documents are used as references, and the citations thereof are represented. The disclosure of the cited papers and patent documents is incorporated in the present specification by reference in its entirety to describe the level of the technical field to which the present invention pertains and the content of the present invention more clearly. SUMMARY Technical Problem The present inventors found that in cases where Affibody® molecules, which are