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KR-20260065533-A - Short peptides with pH selective binding property against FcRn

KR20260065533AKR 20260065533 AKR20260065533 AKR 20260065533AKR-20260065533-A

Abstract

The present invention relates to a peptide of a very short sequence having pH-selective binding ability to FcRn. The peptide of the present invention has the advantage of being able to easily penetrate target cells and/or tissues (e.g., solid tumors) because it is not large in size like the Fc site, and can be produced in inexpensive E. coli rather than animal cells because N-glycosylation is not essential, and can be produced without off-target toxicity because it does not bind to FcγR, while maximizing production efficiency by eliminating the possibility of unwanted combination formation.

Inventors

  • 주만석
  • 한성구
  • 백승일

Assignees

  • (주)앱파인더 테라퓨틱스

Dates

Publication Date
20260508
Application Date
20251027
Priority Date
20241031

Claims (20)

  1. A polypeptide comprising the amino acid sequence of SEQ ID NO. 1, wherein the polypeptide binds to FcRn (neonatal Fc receptor) at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.8, a pH-selective binding polypeptide.
  2. In claim 1, the pH-selective binding polypeptide comprises a sequence of 4 to 400, 4 to 300, 4 to 200, 4 to 100, 4 to 90, 4 to 80, 4 to 70, 4 to 60, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 19, 4 to 18, 4 to 16, or 4 to 11 amino acids.
  3. In claim 1, the pH-selective binding polypeptide comprises the amino acid sequence of the following general formula 1 (from the N-terminus to the C-terminus): <General Formula 1> DWQW-Xa In the above general formula 1, Xa is a sequence comprising one or more amino acids selected from the group consisting of arginine (R), lysine (K), histidine (H), glutamic acid (E), aspartic acid (D), glutamine (Q), asparagine (N), leucine (L), isoleucine (I), valine (V), methionine (M), phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), alanine (A), serine (S), threonine (T), proline (P), and cysteine (C).
  4. A pH-selective binding polypeptide according to claim 3, wherein Xa in general formula 1 is a sequence of 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5 amino acids.
  5. The pH-selective binding polypeptide of claim 1, characterized in that the pH-selective binding polypeptide comprises the amino acid sequence of the following general formula 2 (from the N-terminus to the C-terminus): <General Formula 2> Xb-DWQW-Xa In the above general formula 2, Xa and Xb are sequences comprising one or more amino acids selected from the group consisting of arginine (R), lysine (K), histidine (H), glutamic acid (E), aspartic acid (D), glutamine (Q), asparagine (N), leucine (L), isoleucine (I), valine (V), methionine (M), phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), alanine (A), serine (S), threonine (T), proline (P), and cysteine (C).
  6. A pH-selective binding polypeptide according to claim 5, wherein in the above general formula 2, Xa and Xb are each an amino acid sequence of 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5.
  7. In claim 1, the pH selectively binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 41 and 65 to 111.
  8. A pH-selective binding polypeptide according to claim 1, wherein one or more of the amino acids are D-type amino acids.
  9. In claim 1, the pH-selective binding polypeptide is a pH-selective binding polypeptide that does not substantially bind to FcγR (Fc gamma receptor) at pH 7.0 to 7.8.
  10. A fusion protein in which an additional polypeptide is fused to the pH-selective binding polypeptide of claim 1.
  11. In paragraph 10, the additional polypeptide is a fusion protein fused with the pH-selective binding polypeptide through a linker.
  12. In paragraph 10, the additional polypeptide is a fusion protein fused to the N-terminus, C-terminus, or both ends of the pH-selective binding polypeptide.
  13. In claim 10, the additional polypeptide fused to the N-terminus and the additional polypeptide fused to the C-terminus of the pH-selective binding polypeptide are different sequences, forming a fusion protein.
  14. In claim 10, the additional polypeptide is a fusion protein that does not include Fc or a functional fragment of Fc having the ability to selectively bind to hFcRn at pH 5.6 to 6.2 and to dissociate from FcRn at pH 7.0 to 7.8.
  15. In paragraph 10, the additional polypeptide is a fusion protein that is a polypeptide comprising the amino acid sequence of an antibody or its antigen-binding fragment.
  16. In paragraph 15, the antibody is a fusion protein that is a polyclonal antibody, a monoclonal antibody, a minibody, a domain antibody, a bispecific antibody, an antibody mimic, a chimeric antibody, an antibody conjugate, a human antibody or a humanized antibody, or an antigen-binding fragment thereof.
  17. In paragraph 15, the fusion protein wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, short-chain Fv(scFv), scAb, or sdAb.
  18. In paragraph 10, the additional polypeptide is a fusion protein that is a polypeptide comprising the amino acid sequence of a therapeutic protein.
  19. A pH-selective binding polypeptide of claim 1 or a nucleic acid encoding a fusion protein comprising said pH-selective binding polypeptide.
  20. A vector containing the nucleic acid of claim 19.

Description

Short peptides with pH selective binding property against FcRn The present invention relates to a pH-selective binding polypeptide that exhibits pH-dependent FcRn binding ability despite its very short length, which can be utilized to extend the blood half-life of a protein or antibody therapeutic agent. The Fc region of the antibody mediates interactions with the neonatal receptor FcRn, and its binding recirculates antibodies intracellularly introduced from the endosome into the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12: 181-220; Ghetie et al., 2000, Annu Rev Immunol 18: 739-766). Through this process, antibodies have a favorable antibody serum half-life in the range of 1 to 3 weeks, unlike other proteins that have short half-lives. The recycling of the above antibody is caused by the coordination of binding and dissociation of the Fc site to FcRn, and is due to the property of exhibiting increased binding affinity to FcRn at low pH (5.6 to 6.2) of the cell and dissociating from FcRn (neonatal Fc receptor) at high pH (7.0 to 7.8) outside the cell. Various technologies are being developed to increase the half-life of existing protein therapeutics with short half-lives by utilizing the beneficial property of the Fc site, which is the aforementioned increase in half-life. However, despite the aforementioned advantages, the production of antibodies or protein therapeutics containing the Fc site requires N-glycosylation and must be expressed in animal cells, resulting in relatively high production costs. Furthermore, due to the high molecular weight of the Fc protein, it is difficult to infiltrate solid tumors, and there is a problem of off-target toxicity as it possesses binding ability not only to FcRn but also to FcγR. Additionally, the production of bispecific antibodies is accompanied by the problem of unwanted combination formation, leading to increased production costs and difficulties in purification. Therefore, there is an urgent need to develop short peptides with Fc functions that are not large like the Fc region, making them easy to penetrate into solid tumors, can be produced in inexpensive E. coli rather than animal cells, do not possess off-target toxicity, and have no possibility of forming unwanted combinations. The matters described as background technology above are intended only to enhance understanding of the background of the present invention and should not be construed as an acknowledgment that they constitute prior art already known to those skilled in the art. Figure 1 shows a peptide library with a random loop capable of phage display constructed to discover specific peptide sequences that selectively bind to human FcRn at pH 6.0 and dissociate at pH 7.4. Figure 2 shows the results of selecting only clones from a selected library pool that have no binding affinity to B2M and GST under pH 6.0 conditions and exhibit absorbance only in human FcRn-GST-B2M. Figure 3 shows the results confirming that the selected clones do not bind to human FcRn recombinant protein, B2M, and GST under pH 7.4 conditions. Figure 4 shows the results of gene sequencing analysis for the two finally selected clones. Figure 5 shows the three-dimensional structure of Trastuzumab scAb-ECSE predicted using an artificial intelligence-based protein structure prediction algorithm. Figure 6 shows the SDS-PAGE analysis results of purified Trastuzumab scAb-ECSE. Figure 7 shows the results of measuring the binding affinity between Tastuzumab IgG1, Trastuzumab scAb, Trastuzumab scAb-ECSE, and FcRn using ELISA under pH 6.0 and pH 7.4 conditions. Figure 8 shows the results of analyzing the association constant (K on ), dissociation constant (K dis ), and equilibrium dissection constant (K D ) for IgG antibodies and Trastuzumab scAb-ECSE antibodies using OCTET Data Analysis software. Figure 9 shows the results of verifying the binding affinity between Trastuzumab IgG1, Trastuzumab scAb, and Trastuzumab scAb-ECSE (PepFc) antibodies and FcγR1A using ELISA under pH 6.0 and pH 7.4 conditions. Figure 10 shows the results of verifying the binding affinity between Trastuzumab scAb and Trastuzumab scAb-ECSE (PepFc) antibodies and FcγR2A, FcγR2B, FcγR3A, and FcγR3B using ELISA under pH 6.0 and pH 7.4 conditions. Figure 11 shows the results of verifying the binding affinity between Trastuzumab IgG1, Trastuzumab scAb, and Trastuzumab scAb-ECSE (PepFc) antibodies and the human Her2 receptor using ELISA. Figure 12 shows the results of using a flow cytometer to determine whether Trastuzumab IgG and Trastuzumab scAb-ECSE (PepFc) bind to human breast cancer cell line SK-BR-3 that overexpresses human Her2. Figure 13 shows fluorescence images obtained using a live cell real-time analyzer for the analysis of the internalization activity of Trastuzumab IgG and Trastuzumab scAb-ECSE (PepFc) on human Her2-overexpressing human breast cancer cell line SK-BR-3. Figure 14 shows the results of real-time fluorescence analysis performed