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US-12624077-B2 - CXCR3 ligand

US12624077B2US 12624077 B2US12624077 B2US 12624077B2US-12624077-B2

Abstract

The present disclosure relates to CXCR3 ligands having resistance to DPPIV and having CXCR3-expressing cell migration-inducing activity. The present disclosure specifically relates to N-terminal amino acid modifications and N-terminal amino acid sequences important for resistance to DPPIV and CXCR3-expressing cell migration-inducing activity.

Inventors

  • Yohei Yamamoto
  • Mika Sakurai
  • Kenta Haraya

Assignees

  • CHUGAI SEIYAKU KABUSHIKI KAISHA

Dates

Publication Date
20260512
Application Date
20191204
Priority Date
20181204

Claims (10)

  1. 1 . A C-X-C Motif Chemokine Receptor 3 (CXCR3) ligand, wherein the CXCR3 ligand comprises: a human C-X-C motif chemokine 10 (CXCL10) variant derived from human CXCL10 (SEQ ID NO: 60), wherein the N-terminal sequence of the CXCL10 variant is selected from the group consisting of: (a1) V-X1-L, wherein X1 is F, G, I, K, L, M, T, V, W, or Y and wherein V-X1-L is substituted for the N-terminal V-P-L amino acid residues of human CXCL10; (a2) X2-V-P, wherein X2 is A, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y and wherein X2 is an amino acid residue added at the N-terminus of the N-terminal V-P amino acid residues of human CXCL10; (a3) V-X3-P, wherein X3 is A, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y and wherein X3 is an amino acid residue inserted between the N-terminal V-P amino acid residues of human CXCL10; and, (a4) P-L-S wherein the valine residue at the N-terminus of human CXCL10 is deleted; or, a human C-X-C motif chemokine 11 (CXCL11) variant derived from human CXCL11 (SEQ ID NO: 61) or a human CXCL10-human CXCL11 chimeric protein (ITIP) variant derived from ITIP (SEQ ID NO: 63), wherein the N-terminal sequence of the CXCL11 variant or the chimeric protein ITIP variant is selected from the group consisting of: (a5) X4-F-P, wherein X4 is A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y and wherein X4 is an amino acid residue added at the N-terminus of the N-terminal F-P amino acid residues of human CXCL11 or the chimeric protein ITIP; (a6) F-X5-M, wherein X5 is A, D, E, G, H, I, M, N, Q, R, S, T, V, W, or Y and wherein X5 is an amino acid residue substitution of proline (P) at the N-terminal F-P-M amino acid residues of human CXCL11 or the chimeric protein ITIP variant; and, (a7) F-X6-P, wherein X6 is A, D, E, F, G, H, L, M, N, P, Q, S, T, W, or Y and wherein X6 is an amino acid residue inserted between the N-terminal F-P amino acid residues of human CXCL11 or the chimeric protein ITIP variant.
  2. 2 . A CXCR3 ligand comprising an N-terminal sequence according to claim 1 , wherein the CXCR3 ligand has comprises a sequence at the C-terminus of the C-X-C motif selected from the group consisting of: (c1) the sequence from the 12th amino acid to the 77th amino acid of SEQ ID NO: 60; (c2) the sequence from the 12th amino acid to the 73rd amino acid of SEQ ID NO: 61; (c3) the sequence from the 12th amino acid to the 103rd amino acid of SEQ ID NO: 62; (c4) the sequence from the 12th amino acid to the 77th amino acid of SEQ ID NO: 1; and (c5) the sequence from the 12 th amino acid to the 77 th amino acid of SEQ ID NO: 63.
  3. 3 . A CXCR3 ligand comprising an N-terminal sequence according to claim 1 , wherein a C-terminal sequence of the CXCR3 ligand comprises a sequence selected from the group consisting of: (d1) any one of SEQ ID NOs: 2 to 49, 51 to 57, 92 to 147, and 149 to 204; (d2) a sequence comprising 95% or more sequence identity to SEQ ID NO: 60; (d3) a sequence comprising 95% or more sequence identity to SEQ ID NO: 61; (d4) a sequence comprising 95% or more sequence identity to SEQ ID NO: 62; (d5) a sequence comprising 95% or more sequence identity to SEQ ID NO: 63; (d6) a sequence comprising 95% or more sequence identity to SEQ ID NO: 1; and (d7) a sequence comprising 3 or less amino acid substitutions, insertions, or deletions in a sequence selected from any one of SEQ ID NOs: 1 to 49, 51 to 57, 60 to 63, 92 to 147, and 149 to 204, wherein differences in percent sequence identity or amino acid substitutions are conservative amino acid substitutions, wherein said ligand has an activity to cause migration of cells expressing CXCR3.
  4. 4 . The CXCR3 ligand according to claim 1 , wherein the CXCL10 variant further comprises a substitution of arginine to alanine at amino acid position 75.
  5. 5 . A fusion protein comprising the CXCR3 ligand according to claim 1 .
  6. 6 . A pharmaceutical composition comprising the CXCR3 ligand according to claim 1 .
  7. 7 . A C-X-C Motif Chemokine Receptor 3 (CXCR3) ligand, wherein the CXCR3 ligand comprises a human C-X-C motif chemokine 10 (CXCL10) variant derived from human CXCL10 (SEQ ID NO: 60) a comprising substitution of the V-P-L-S-R-T-V-R sequence at the N-terminus of human CXCL10 with a sequence selected from the group consisting of: (b1) V-X1-L-S-R-T-V-R, wherein X1 is F, G, I, K, L, M, T, V, W, or Y (SEQ ID NO: 205); (b2) X2-V-P-L-S-R-T-V-R, wherein X2 is A, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y (SEQ ID NO: 206); (b3) V-X3-P-L-S-R-T-V-R, wherein X3 is A, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y (SEQ ID NO: 207); and, (b4) P-L-S-R-T-V-R (SEQ ID NO: 208), or a human C-X-C motif chemokine 11 (CXCL1l) variant derived from human CXCL11 (SEQ ID NO: 61) or a human CXCL10-human CXCL11 chimeric protein (ITIP) variant a derived from ITIP (SEQ ID NO: 63), wherein the F-P-M-F-K-R-G-R sequence at the N-terminus of the human CXCL11 or human CXCL10-human CXCL11 chimeric protein (ITIP) is substituted with a sequence selected from the group consisting of: (b5) X4-F-P-M-F-K-R-G-R, wherein X4 is A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y (SEQ ID NO: 209); (b6) F-X5-M-F-K-R-G-R, wherein X5 is A, D, E, G, H, I, M, N, Q, R, S, T, V, W, or Y (SEQ ID NO: 210); and (b7) F-X6-P-M-F-K-R-G-R, wherein X6 is A, D, E, F, G, H, L, M, N, P, Q, S, T, W, or Y (SEQ ID NO: 211).
  8. 8 . The CXCR3 ligand according to claim 7 , wherein the CXCL10 variant further comprises a substitution of arginine to alanine at amino acid position 75.
  9. 9 . A fusion protein comprising the CXCR3 ligand according to claim 7 .
  10. 10 . A pharmaceutical composition comprising the CXCR3 ligand according to claim 7 .

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a U.S. National Phase of PCT Application No. PCT/JP2019/047382, filed Dec. 4, 2019, which claims the benefit of Japanese Patent Application No. 2018-227353, filed Dec. 4, 2018, each of which is incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY The content of the electronically submitted sequence listing (Name: 6663_0175 Substitute_Sequence_Listing.txt; Size: 178 kilobytes; and Date of Creation: May 24, 2024) filed with the application is incorporated herein by reference in its entirety. TECHNICAL FIELD The present disclosure relates to CXCR3 ligands, methods of producing CXCR3 ligands, use of CXCR3 ligands, and methods of conferring resistance to DPPIV on CXCR3 ligands. BACKGROUND ART Chemokine receptor CXCR3 (also called G Protein-coupled Receptor 9 (GPR9) and CD183) belongs to the CXC chemokine receptor family and is a G protein-coupled receptor that binds to chemokines CXCL9, CXCL10, and CXCL11. CXCR3 is expressed primarily in activated T-helper type 1 (Th1) lymphocytes and cytotoxic T cells, but is also present in natural killer cells, macrophages, dendritic cells, and B lymphocyte subsets. The chemokines CXCL9, CXCL10, and CXCL11 are three naturally-occurring CXCR3 ligands. The interaction of CXCR3 and its ligands is involved in guiding receptor-bearing cells to specific parts of the body, especially sites of inflammation, immune impairment, and immune dysfunction. CXCL10 (C-X-C motif chemokine 10) is also called “IP10 (interferon gamma-induced protein 10)” or “small inducible cytokine B10” and is a chemokine belonging to the CXC subfamily. C-X-C motif chemokine 10 (CXCL10) is known to promote T cell migration activity via CXCR3 (NPL 1). Furthermore, it has been reported that direct administration of CXCL10 into a mouse tumor increases T cells in the tumor (NPL 2). On the other hand, it is known that the two amino acids from N-terminal of CXCL10 are cleaved by Dipeptidyl Peptidase-4 (DPPIV, DPP4) (NPL 1). Cleaved CXCL10 is present even in human blood and is also reported to be present in human tumors, indicating that CXCL10 is cleaved by DPPIV in human blood and tumors (NPLs 3 and 4). Furthermore, since it has been reported that the migration-inducing activity of DPPIV-cleaved CXCL10 is significantly reduced, it is thought that the cleavage could be one of the CXCL10-inactivating mechanisms (NPL 1). The three-dimensional structure of CXCL10 has been elucidated (NPLs 5 and 6), and a predicted structure of a model of a complex with CXCR3 has also been reported (NPL 7). In this complex model, the N-terminus of CXCL10 is arranged in such a way that it penetrates into the inside of CXCR3, and this interaction is thought to be important for CXCR3 activation. C-X-C motif chemokine 11 (C-X-C motif chemokine ligand 11, CXCL11) is a C-X-C chemokine which is also called I-TAC (Interferon-inducible T-cell alpha chemoattractant) or IP-9 (Interferon-gamma-inducible protein 9), and naturally-occurring CXCL11 is said to bind to CXCR3 more strongly than naturally-occurring CXCL10 and naturally-occurring CXCL9 (NPLs 8 and 9). It is known that the N-terminal sequence of naturally-occurring human CXCL11 is also cleaved by DPPIV (NPL 1). C-X-C motif chemokine 9 (C-X-C motif chemokine ligand 9, CXCL9) is a C-X-C chemokine which is also called Monokine induced by gamma-interferon (MIG), and the N-terminal sequence of naturally-occurring human CXCL9 is also known to be cleaved by DPPIV (NPL 1). CITATION LIST Non-Patent Literature [NPL 1] Proost P, Blood. 2001 Dec. 15; 98(13):3554-61.[NPL 2] Wang P, Cancer Immunol Immunother. 2010 November; 59(11):1715-26.[NPL 3] Decalf J, EMBO Mol Med. 2016 Jun. 1; 8(6):679-83.[NPL 4] Rainczuk A, Int J Cancer. 2014 Feb. 1; 134(3):530-41.[NPL 5] Booth V, Biochemistry. 2002 Aug. 20; 41(33):10418-25.[NPL 6] Swaminathan G J, Structure. 2003 May; 11(5):521-32.[NPL 7] Trotta T, Mol Immunol. 2009 December;47(2-3):332-9.[NPL 8] Cole K E, The Journal of Experimental Medicine. 187 (12): 2009-21.[NPL 9] Tensen CP, The Journal of Investigative Dermatology. 112 (5): 716-22. SUMMARY OF INVENTION Technical Problem The present disclosure provides CXCR3 ligands having resistance to DPPIV and having CXCR3-expressing cell migration-inducing activity, methods of using these CXCR3 ligands, and methods of producing these CXCR3 ligands. The present disclosure also provides methods of conferring DDPIV resistance on CXCR3 ligands. Solution to Problem The present inventors discovered CXCR3 ligands having resistance to DPPIV and CXCR3-expressing-cell migration-inducing activity, and completed the present disclosure. Specifically, the inventors discovered N-terminal amino acid modifications and N-terminal amino acid sequences that are important for resistance to DPPIV and CXCR3-expressing-cell migration-inducing activity. The present disclosure specifically includes the embodiments exemplified below: [A-1]