CXCR3 ligand having enhanced CXCR3-expressing cell migration activity

Abstract

The present disclosure relates to a CXCR3 ligand having CXCR3-expressing cell migration activity, and specifically relates to amino acid modification and an amino acid sequence that are important for CXCR3-expressing cell migration activity.

Description

A CXCR3 ligand that enhances the migration activity of CXCR3-expressing cells.

[0001] The present disclosure relates to CXCR3 ligands, methods for producing CXCR3 ligands, uses of CXCR3 ligands, methods for enhancing the chemotactic activity of CXCR3 ligands for CXCR3-expressing cells, and methods for improving the stability of CXCR3 ligands in blood.

[0002] The chemokine receptor CXCR3 (also known as G protein-coupled receptor 9 (GPR9) or CD183) is a G protein-coupled receptor (GPCR) that belongs to the CXC chemokine receptor family and binds to the chemokines CXCL9, CXCL10, and CXCL11. CXCR3 is primarily expressed on activated T helper type 1 (Th1) lymphocytes and cytotoxic T cells, but is also present on 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 with its ligands is involved in guiding receptor-bearing cells to specific sites in the body, particularly areas of inflammation, immune disorders, and immune dysfunction (Non-Patent Document 1: Tokunaga, Zhang et al. 2017).

[0003] CXC motif chemokine 10 (CXCL10), also known as "IP10 (interferon gamma-induced protein 10)" or "small inducible cytokine B10," is a chemokine belonging to the CXC subfamily. CXC motif chemokine 10 (CXCL10) is known to promote T cell migration via CXCR3. Furthermore, direct administration of CXCL10 into mouse tumors has been reported to increase T cell numbers in the tumor (Rainczuk, Rao et al. 2014). C-X-C motif chemokine 11 (C-X-C motif chemokine ligand 11, CXCL11) is a type of C-X-C chemokine, also known as I-TAC (Interferon-inducible T-cell alpha chemoattractant) or IP-9 (Interferon-gamma-inducible protein 9). Native CXCL11 is believed to bind more strongly to CXCR3 than native CXCL10 or CXCL9 (Non-Patent Document 3: Aguilera-Duran and Romo-Mancillas 2020). C-X-C motif chemokine 9 (C-X-C motif chemokine ligand 9, CXCL9) is also known as monokine-induced by gamma interferon (MIG).

[0004] Chemokines are known to bind to GPCRs as well as glycosaminoglycans (GAGs). GAGs are abundant on epithelial cell surfaces, and chemokines are thought to localize there by binding to GAGs. Chemokines that localize by binding to GAGs are in equilibrium between binding and dissociation from GAGs, and dissociated chemokines are thought to exert their function by binding to GPCRs (Non-Patent Document 4: Graham, Handel et al. 2019). Binding to GAGs is also known to be important for the function of CXCL10, and it has been reported that substitution of amino acids at positions 12, 14, 20, and 25 with K or R improves the GAG-binding activity of CXCL10, thereby enhancing T cell migration (Patent Document 1: ANTAGONIS BIOTHERAPEUTICS GMBH, WO2021 / 038033) (Non-Patent Document 5: Gerlza, Nagele et al. 2019).

[0005] WO2021 / 038033

[0006] Tokunaga, R et al., (2017). Cancer Treat Rev 63: 40-47. Rainczuk, A. et al, (2014). Int J Cancer 134(3): 530-541. Aguilera-Duran, G et al. (2020). Molecules 25(19). Graham, GJ et al, (2019). Trends Immunol 40(6): 472-481.Gerlza, T., M et al. (2019). Protein Eng Des Sel. 31;32(8):367-373

[0007] The present disclosure provides a CXCR3 ligand, a method for producing the CXCR3 ligand, and a method for using the CXCR3 ligand. The present disclosure also provides a method for enhancing the chemotactic activity of a CXCR3 ligand on CXCR3-expressing cells, and a method for improving the stability of a CXCR3 ligand in blood.

[0008] The present inventors have discovered amino acid modifications and amino acid sequences of CXCR3 ligands that are important for enhancing the migratory activity of CXCR3-expressing cells, and have also discovered amino acid modifications that improve the stability of CXCR3 ligands in blood.

[0009] The present disclosure specifically encompasses the following exemplary embodiments: [A] (A1) A CXCR3 ligand in which the amino acid sequence of a parent CXCR3 ligand has been modified by at least one of (i) introducing a disulfide bond and (ii) modifying an amino acid in the N-terminal region, or both, and the CXCR3-expressing cell migration activity is enhanced compared to the parent CXCR3 ligand. (A2) The CXCR3 ligand according to (A1), wherein the parent CXCR3 ligand has a CXC motif. (A3) The CXCR3 ligand according to (A2), wherein the CXC motif is CTC (Cys-Thr-Cys), CLC (Cys-Leu-Cys), or CSC (Cys-Ser-Cys). (A4) The CXCR3 ligand according to any one of (A1) to (A3), wherein the parent CXCR3 ligand comprises at least a portion of the amino acid sequence of a native human CXCR3 ligand. (A5) The CXCR3 ligand according to (A4), wherein the native human CXCR3 ligand is native human CXCL10. (A6) The CXCR3 ligand according to (A4) or (A5), wherein the disulfide bond is introduced at a site other than the β-sheet region of the parent CXCR3 ligand. (A7) The CXCR3 ligand according to any one of (A2) to (A6), wherein the parent CXCR3 ligand has three β-strands, and the disulfide bond is formed by substituting, with Cys, at least one amino acid contained in any two regions selected from the following in the amino acid sequence of the parent CXCR3 ligand: (i) a first loop between the CXC motif and the first β-strand located most N-terminally; (ii) a second loop between the first β-strand and the second β-strand located second most N-terminally; and (iii) a third loop between the second β-strand and the third β-strand located third most N-terminally.(A8) The CXCR3 ligand according to any one of (A5) to (A7), wherein the disulfide bond is introduced by substituting, with Cys, amino acids corresponding to the following amino acid positions (i) and (ii) in the amino acid sequence of native human CXCL10: (i) at least one selected from the group consisting of positions 18, 14, 21, 25, and 41; (ii) at least one selected from the group consisting of positions 60, 55, 67, 46, and 56. (A9) The CXCR3 ligand according to any one of (A5) to (A8), wherein the disulfide bond is introduced at an amino acid position corresponding to at least one selected from the group consisting of combinations of the following amino acid positions in the amino acid sequence of native human CXCL10: (i) positions 18 and 60; (ii) positions 14 and 55; (iii) positions 21 and 67; (iv) positions 25 and 46; (v) positions 41 and 56. (A10) The CXCR3 ligand according to any one of (A5) to (A9), wherein the disulfide bonds are introduced at amino acid positions corresponding to positions 18 and 60 in the amino acid sequence of native human CXCL10. (A11) The CXCR3 ligand according to any one of (A1) to (A10), wherein the amino acids at positions 18 and 60 in the amino acid sequence of the parent CXCR3 ligand are substituted with Cys. (A12) The CXCR3 ligand according to any one of (A1) to (A11), wherein the amino acid at position 18 from the N-terminus in the amino acid sequence of the parent CXCR3 ligand is substituted from P to C and the amino acid at position 60 from A to C. (A13) The CXCR3 ligand according to any one of (A2) to (A12), wherein the amino acid modification in the N-terminal region is an amino acid substitution on the N-terminal side of the CXC motif of the parent CXCR3 ligand. (A14) A CXCR3 ligand described in any of (A5) to (A13), wherein the amino acid modification in the N-terminal region is an amino acid substitution at an amino acid position corresponding to positions 1 to 8 in the amino acid sequence of the native human CXCL10.(A15) The CXCR3 ligand according to any one of (A1) to (A14), wherein the amino acid modification in the N-terminal region is a substitution of an amino acid in the N-terminal region with Y, F, H, T, or M. (A16) The CXCR3 ligand according to any one of (A1) to (A15), wherein the amino acid modification in the N-terminal region is a substitution of Tyr for the amino acid at position 1 in the amino acid sequence of the parent CXCR3 ligand. (A17) The CXCR3 ligand according to any one of (A1) to (A16), wherein the first amino acid from the N-terminus in the amino acid sequence of the parent CXCR3 ligand is V, and the amino acid modification in the N-terminal region comprises a V1Y amino acid substitution. (A18) The CXCR3 ligand according to any one of (A1) to (A17), wherein the second amino acid from the N-terminus in the amino acid sequence of the parent CXCR3 ligand is P, and the amino acid modification in the N-terminal region comprises a P2V amino acid substitution. (A19) The CXCR3 ligand according to (A1) to (A18), further comprising at least one modification selected from amino acid substitution, deletion, and insertion. (A20) A CXCR3 ligand having enhanced CXCR3-expressing cell migration activity compared to a parent CXCR3 ligand comprising a portion of the amino acid sequence of native human CXCL10, wherein the amino acids at positions corresponding to amino acids 18 and 60 in the amino acid sequence of native human CXCL10 are substituted with Cys. (A21) The CXCR3 ligand according to (A20), further comprising the amino acid at position corresponding to amino acid 1 in the amino acid sequence of native human CXCL10 is substituted with Tyr. (A22) A CXCR3 ligand having enhanced CXCR3-expressing cell migration activity compared to a parent CXCR3 ligand comprising a portion of the amino acid sequence of native human CXCL10, wherein the amino acid at position corresponding to amino acid 1 in the amino acid sequence of native human CXCL10 is substituted with Tyr. (A23) The CXCR3 ligand according to any one of (A20) to (A22), wherein the amino acid at the position corresponding to the second amino acid in the amino acid sequence of native human CXCL10 is substituted with Val. (A24) The CXCR3 ligand according to any one of (A1) to (A23), wherein the parent CXCR3 ligand is resistant to Furin protease.(A25) The CXCR3 ligand according to any one of (A1) to (A24), wherein the parent CXCR3 ligand is a CXCL10 variant obtained by adding an R75A modification to the amino acid sequence of native human CXCL10. (A26) The CXCR3 ligand according to any one of (A1) to (A17), wherein the seventh amino acid from the N-terminus in the amino acid sequence of the parent CXCR3 ligand is V, and the amino acid modification in the N-terminal region includes an amino acid substitution of V7P. (A27) The CXCR3 ligand according to any one of (A26), wherein the amino acid substitution of V7P increases blood stability compared to native human CXCL10 or a parent CXCR3 ligand comprising a part of the amino acid sequence of native human CXCL10.

[0010] [B] (B1) A polypeptide in which the amino acids at positions 18 and 60 of the amino acid sequence of native human CXCL10 or a CXCL10 variant, where the N-terminal amino acid of native human CXCL10 is defined as position 1, are substituted with Cys. (B2) The polypeptide of (B1), further comprising Tyr as the amino acid at position 1 of the native human CXCL10. (B3) The polypeptide of (B1) or (B2), further comprising Val as the amino acid at position 2 of the native human CXCL10. (B4) A polypeptide in which the N-terminal amino acid (position 1) of the amino acid sequence of native human CXCL10 or a CXCL10 variant is substituted with Tyr. (B5) The polypeptide of (B4), further comprising Val as the amino acid at position 2 of the native human CXCL10. (B6) The polypeptide of (B1) to (B5), further comprising Pro as the amino acid at position 7 of the native human CXCL10. (B7) A polypeptide which is a CXCR3 ligand and has the sequence of YPL or YVL at its N-terminus. (B8) A polypeptide which is a CXCR3 ligand and has the sequence of YPLSRT (SEQ ID NO: 19) or YVLSRT (SEQ ID NO: 20) at its N-terminus. (B9) A polypeptide having any of the amino acid sequences (a1) to (a3) ​​below: (a1) a sequence set forth in any one of SEQ ID NOs: 4 to 12; (a2) a sequence which shows 90% or more sequence identity with the sequence set forth in any one of SEQ ID NOs: 4 to 12 and is a CXCR3 ligand; (a3) ​​a sequence which contains 10 or less amino acid substitutions selected from amino acid substitutions, insertions, deletions, and additions relative to the sequence set forth in any one of SEQ ID NOs: 4 to 12 and is a CXCR3 ligand. (B10) The polypeptide according to (B9), wherein amino acid position 1 is Tyr. (B11) The polypeptide according to (B9) or (B10), wherein amino acid positions 18 and 60 are Cys. (B12) The polypeptide according to any one of (B9) to (B11), wherein the amino acid at position 2 is Val. (B13) The polypeptide according to any one of (B9) to (B12), wherein the amino acid at position 7 is Pro.(B14) The polypeptide according to any one of (B1) to (B13), further comprising a substitution of Ala for the amino acid corresponding to position 75 of the native human CXCL10. (B15) The polypeptide according to any one of (B1) to (B14), comprising a CXC motif and further comprising any one of the following (b1) to (b6) at the C-terminus of the CXC motif: (b1) the sequence from amino acid 12 to amino acid 77 of any one of SEQ ID NOs: 4 to 12; (b2) the sequence from amino acid 12 to amino acid 77 of SEQ ID NO: 25; (b3) the sequence from amino acid 12 to amino acid 73 of SEQ ID NO: 26; (b4) the sequence from amino acid 12 to amino acid 103 of SEQ ID NO: 27; (b5) the sequence from amino acid 12 to amino acid 77 of SEQ ID NO: 1; (b6) the sequence from amino acid 12 to amino acid 77 of SEQ ID NO: 28. (B16) A polypeptide having the following amino acid sequence: (X1)(X2)LSRTVRCTCISISNQ(X3)VNPRSLEQLEIIPASQFCPRVEIIATMKKKGEKRCLNPESK(X4)IKNLLKAVSKERSK(X5)SP (SEQ ID NO: 53), wherein (X1) is V or Y, (X2) is P or V, (X3) and (X4) are C, and (X5) is R or A. (B17) A polypeptide having the following amino acid sequence: (X1)(X2)LSRTVRCTCISISNQ(X3)VNPRSLEQLEIIPASQFCPRVEIIATMKKKGEKRCLNPESK(X4) IKNLLKAVSKERSK(X5)SP (SEQ ID NO: 54), wherein (X1) is Y, (X2) is P or V, (X3) is P or C, (X4) is A or C, and (X5) is R or A. (B18) The polypeptide according to any one of (B1) to (B17), further comprising at least one modification selected from amino acid substitution, deletion, and insertion. (B19) The polypeptide according to any one of (B1) to (B18), having CXCR3-expressing cell migration activity. (B20) The polypeptide according to any one of (B1) to (B19), wherein the CXCR3-expressing cell migration activity of the polypeptide is equal to or higher than the CXCR3-expressing cell migration activity of native human CXCL10.(B21) The polypeptide according to any one of (B1) to (B20), wherein the CXCR3-expressing cell migration activity of the polypeptide is higher than that of the variant obtained by adding the R75A modification to native human CXCL10. (B22) The polypeptide according to any one of (B1) to (B21), wherein the polypeptide concentration at which the CXCR3-expressing cell migration activity of the polypeptide is maximized is equal to or lower than the native human CXCL10 concentration at which the CXCR3-expressing cell migration activity of native human CXCL10 is maximized. (B23) The polypeptide according to any one of (B1) to (B22), wherein the polypeptide concentration at which the CXCR3-expressing cell migration activity of the polypeptide is maximized is lower than the native human CXCL10 concentration at which the CXCR3-expressing cell migration activity of the variant obtained by adding the R75A modification to native human CXCL10 is maximized. (B24) The polypeptide according to any one of (B20) to (B23), wherein the CXCR3-expressing cell migration activity of the polypeptide is measured by cell migration rate. (B25) The polypeptide according to any one of (B19) to (B24), wherein the CXCR3-expressing cell is a transfectant expressing CXCR3 or a cell isolated from a living body. (B26) The polypeptide according to any one of (B19) to (B25), wherein the CXCR3-expressing cell is a cell expressing human CXCR3.

[0011] [C] (C1) An isolated nucleic acid encoding the CXCR3 ligand described in [A] or the polypeptide described in [B]. (C2) A vector having the nucleic acid described in (C1). (C3) A host cell comprising the nucleic acid described in (C1) or the vector described in (C2). (C4) A method for producing the CXCR3 ligand described in [A] or the polypeptide described in [B], comprising culturing the host cell described in (C3) so as to produce the CXCR3 ligand or polypeptide. (C5) The method described in (C4), further comprising the step of recovering the polypeptide or the CXCR3 ligand from the host cell. (C6) A fusion protein comprising the CXCR3 ligand described in [A] or the polypeptide described in [B]. (C7) The fusion protein described in (C6), in which the CXCR3 ligand described in [A] or the polypeptide described in [B] is fused with an antibody Fc region. (C8) The fusion protein described in (C7), in which the CXCR3 ligand described in [A] or the polypeptide described in [B] is fused with a complete antibody or an antibody fragment. (C9) The fusion protein according to (C7) or (C8), wherein the CXCR3 ligand or the polypeptide and the antibody Fc region, or the CXCR3 ligand or the polypeptide and the complete antibody or antibody fragment, are fused via a linker. (C10) A pharmaceutical composition comprising the CXCR3 ligand according to [A], the polypeptide according to [B], or the fusion protein according to any one of (C6) to (C9). (C11) A method for treating a disease, comprising the step of administering the CXCR3 ligand according to [A], the polypeptide according to [B], or the fusion protein according to any one of (C6) to (C9).

[0012] [D] (D1) A method for enhancing the chemotactic activity of a CXCR3 ligand for CXCR3-expressing cells, comprising the step of modifying the amino acid sequence of a parent CXCR3 ligand by at least one of (i) introducing a disulfide bond and (ii) modifying an amino acid in the N-terminal region, or both. (D2) The method according to (D1), wherein the parent CXCR3 ligand has a CXC motif. (D3) The method according to (D2), wherein the CXC motif is CTC (Cys-Thr-Cys), CLC (Cys-Leu-Cys), or CSC (Cys-Ser-Cys). (D4) The method according to (D3), wherein the parent CXCR3 ligand comprises a portion of the amino acid sequence of a native human CXCR3 ligand. (D5) The method according to (D4), wherein the native human CXCR3 ligand is native human CXCL10. (D6) The method of any one of (D4) and (D5), wherein the modification comprises the introduction of a disulfide bond, and the disulfide bond is introduced into a site other than the beta-sheet region of the parent CXCR3 ligand. (D7) The method of any one of (D2) to (D6), wherein the parent CXCR3 ligand has three beta strands, and the disulfide bond is formed by substituting, with Cys, at least one amino acid in any two of the following regions in the amino acid sequence of the parent CXCR3 ligand selected from the following: (i) a first loop between the CXC motif and the first beta strand located most N-terminally; (ii) a second loop between the first beta strand and the second beta strand located second most N-terminally; and (iii) a third loop between the second beta strand and the third beta strand located third most N-terminally. (D8) The method according to any one of (D5) to (D7), wherein the disulfide bond is introduced by substituting Cys for amino acids corresponding to the following amino acid positions (i) and (ii) in the amino acid sequence of native human CXCL10: (i) at least one selected from the group consisting of positions 18, 14, 21, 25, and 41; (ii) at least one selected from the group consisting of positions 60, 55, 67, 46, and 56.(D9) The method of any of (D5) to (D8), wherein the disulfide bond is introduced at an amino acid position corresponding to at least one selected from the group consisting of combinations of the following amino acid positions in the amino acid sequence of native human CXCL10: (i) positions 18 and 60; (ii) positions 14 and 55; (iii) positions 21 and 67; (iv) positions 25 and 46; and (v) positions 41 and 56. (D10) The method of any of (D2) to (D9), wherein the modification comprises an amino acid modification in the N-terminal region, and the amino acid modification in the N-terminal region is an amino acid substitution N-terminal to the CXC motif of the parent CXCR3 ligand. (D11) The method of any of (D5) to (D10), wherein the amino acid modification in the N-terminal region is an amino acid substitution at an amino acid position corresponding to positions 1 to 8 in the amino acid sequence of native human CXCL10. (D12) The method of any of (D1) to (D10), wherein the amino acid modification in the N-terminal region is a substitution of an amino acid in the N-terminal region with Y, F, H, T, or M. (D13) The method of any of (D1) to (D12), wherein the first amino acid from the N-terminus in the amino acid sequence of the parent CXCR3 ligand is V, and the method comprises the step of adding an amino acid substitution of V1Y. (D14) The method of any of (D1) to (D13), wherein the 18th amino acid from the N-terminus in the amino acid sequence of the parent CXCR3 ligand is P and the 60th amino acid from the N-terminus is A, and the method comprises the step of adding an amino acid substitution of P18C or A60C. (D15) The method of any of (D1) to (D14), further wherein the second amino acid from the N-terminus in the amino acid sequence of the parent CXCR3 ligand is P, and the method comprises the step of adding an amino acid substitution of P2V. (D16) The method according to any one of (D1) to (D15), further comprising the step of adding at least one modification selected from amino acid substitution, deletion, and insertion.(D17) A method for enhancing the CXCR3-expressing cell migration activity of a parent CXCR3 ligand comprising a portion of the amino acid sequence of native human CXCL10, the method comprising substituting Cys for the amino acids at positions corresponding to the 18th and 60th amino acids in the amino acid sequence of native human CXCL10. (D18) The method according to (D17), further comprising substituting Tyr for the amino acid at position corresponding to the 1st amino acid in the amino acid sequence of native human CXCL10. (D19) A method for enhancing the CXCR3-expressing cell migration activity of a parent CXCR3 ligand comprising a portion of the amino acid sequence of native human CXCL10, the method comprising substituting Tyr for the amino acid at position corresponding to the 1st amino acid in the amino acid sequence of native human CXCL10. (D20) The method according to any one of (D17) to (D19), further comprising substituting Val for the amino acid at position corresponding to the 2nd amino acid in the amino acid sequence of native human CXCL10. (D21) The method according to any one of (D1) to (D20), wherein the parent CXCR3 ligand is resistant to Furin protease. (D22) The method according to any one of (D1) to (D21), wherein the parent CXCR3 ligand is a CXCL10 variant obtained by adding an R75A modification to the amino acid sequence of native human CXCL10. (D23) The method according to any one of (D1) to (D22), further comprising adding a V7P amino acid substitution at the seventh amino acid position from the N-terminus in the amino acid sequence of the parent CXCR3 ligand.

[0013] [E] (E1) A method for producing a CXCR3 ligand having enhanced CXCR3-expressing cell migration activity compared to the parent ligand, the method comprising at least one or both of the following steps: (i) introducing a disulfide bond and (ii) modifying an amino acid in the N-terminal region of the amino acid sequence of the parent CXCR3 ligand. (E2) The method according to (E1), wherein the parent CXCR3 ligand has a CXC motif. (E3) The method according to (E2), wherein the CXC motif is CTC (Cys-Thr-Cys), CLC (Cys-Leu-Cys), or CSC (Cys-Ser-Cys). (E4) The method according to (E2), wherein the parent CXCR3 ligand comprises a portion of the amino acid sequence of a native human CXCR3 ligand. (E5) The method according to (E3), wherein the native human CXCR3 ligand is native human CXCL10. (E6) The method according to (E4) or (E5), wherein the disulfide bond is introduced into a region other than a beta-sheet region of the parent CXCR3 ligand. (E7) The method according to any one of (E2) to (E6), wherein the parent CXCR3 ligand has three beta strands, and the disulfide bond is formed by substituting, with Cys, at least one amino acid in any two regions selected from the following in the amino acid sequence of the parent CXCR3 ligand: (i) a first loop between the CXC motif and the first beta strand located most N-terminally; (ii) a second loop between the first beta strand and the second beta strand located second most N-terminally; and (iii) a third loop between the second beta strand and the third beta strand located third most N-terminally. (E8) The method according to any one of (E5) to (E7), wherein the disulfide bond is introduced by substituting Cys for amino acids corresponding to the following amino acid positions (i) and (ii) in the amino acid sequence of native human CXCL10: (i) at least one selected from the group consisting of positions 18, 14, 21, 25, and 41; (ii) at least one selected from the group consisting of positions 60, 55, 67, 46, and 56.(E9) The method of any of (E5) to (E8), wherein the disulfide bond is introduced at an amino acid position corresponding to at least one selected from the group consisting of combinations of the following amino acid positions in the amino acid sequence of native human CXCL10: (i) positions 18 and 60; (ii) positions 14 and 55; (iii) positions 21 and 67; (iv) positions 25 and 46; and (v) positions 41 and 56. (E10) The method of any of (E1) to (E9), wherein the amino acid at position 18 and the amino acid at position 60 in the amino acid sequence of the parent CXCR3 ligand are P and A, respectively, and the method comprises amino acid substitutions of P18C and A60C. (E11) The method of any of (E3) to (E10), wherein the amino acid modification in the N-terminal region is an amino acid substitution on the N-terminal side of the CXC motif of the parent CXCR3 ligand. (E12) The method of any of (E5) to (E11), wherein the amino acid modification in the N-terminal region is performed between positions 1 and 8 in the amino acid sequence of native human CXCL10. (E13) The method of any of (E1) to (E12), wherein the amino acid modification in the N-terminal region is performed by substituting an amino acid in the N-terminal region with Y, F, H, T, or M. (E14) The method of any of (E1) to (E13), wherein the amino acid at position 1 in the amino acid sequence of the parent CXCR3 ligand is V, and the method comprises a V1Y amino acid substitution. (E15) The method of any of (E1) to (E14), wherein the amino acid at position 2 in the amino acid sequence of the parent CXCR3 ligand is P, and the method further comprises a P2V amino acid substitution. (E16) The method of any of (E1) to (E15), wherein the CXCR3 ligand has a chemotactic activity for CXCR3-expressing cells that is equivalent to or higher than that of a native human CXCR3 ligand. (E17) A method for producing a CXCR3 ligand having enhanced CXCR3-expressing cell migration activity from a parent CXCR3 ligand containing a portion of the amino acid sequence of native human CXCL10, the method comprising substituting the amino acids at positions corresponding to the 18th and 60th amino acids in the amino acid sequence of native human CXCL10 with Cys.(E18) The method of (E15), further comprising substituting Tyr for the amino acid at position 1 in the amino acid sequence of native human CXCL10. (E19) A method for producing a CXCR3 ligand having enhanced CXCR3-expressing cell migration activity from a parent CXCR3 ligand comprising a portion of the amino acid sequence of native human CXCL10, the method comprising substituting Tyr for the amino acid at position 1 in the amino acid sequence of native human CXCL10. (E20) The method of any of (E15) to (E17), further comprising substituting Val for the amino acid at position 2 in the amino acid sequence of native human CXCL10. (E21) The method of any of (E1) to (E20), wherein the parent CXCR3 ligand is resistant to Furin protease. (E22) The method of any of (E1) to (E21), wherein the parent CXCR3 ligand is a CXCL10 variant obtained by adding an R75A modification to the amino acid sequence of native human CXCL10. (E23) The method according to any one of (E1) to (E22), further comprising the step of substituting V7P for the seventh amino acid from the N-terminus in the amino acid sequence of the parent CXCR3 ligand. (E24) A CXCR3 ligand produced by the method according to any one of (E1) to (E23).

[0014] [F] (F1) A CXCR3 ligand having improved stability in blood compared to the parent CXCR3 ligand, wherein the amino acid at position 7, where the N-terminal amino acid in the amino acid sequence of the parent CXCR3 ligand is position 1, is substituted with Pro. (F2) The CXCR3 ligand according to (F1), wherein the parent CXCR3 ligand has a CXC motif. (F3) The CXCR3 ligand according to (F2), wherein the CXC motif is CTC (Cys-Thr-Cys), CLC (Cys-Leu-Cys), or CSC (Cys-Ser-Cys). (F4) The CXCR3 ligand according to any one of (F1) to (F3), wherein the parent CXCR3 ligand comprises at least a portion of the amino acid sequence of a native human CXCR3 ligand. (F5) The CXCR3 ligand according to (F4), wherein the native human CXCR3 ligand is native human CXCL10. (F6) The CXCR3 ligand according to any one of (F1) to (F5), wherein the 7th amino acid from the N-terminus in the amino acid sequence of the parent CXCR3 ligand is substituted from V to P. (F7) The CXCR3 ligand according to any one of (F1) to (F6), wherein the amino acid sequence of the parent CXCR3 ligand is further modified by either or both of (i) introducing a disulfide bond and (ii) modifying an amino acid in the N-terminus region, thereby enhancing the CXCR3-expressing cell migration activity compared to the parent CXCR3 ligand. (F8) The CXCR3 ligand according to (F7), wherein the disulfide bond is introduced at amino acid positions corresponding to positions 18 and 60 in the amino acid sequence of native human CXCL10. (F9) The CXCR3 ligand according to (F7) or (F8), wherein the amino acids at positions 18 and 60 in the amino acid sequence of the parent CXCR3 ligand are substituted with Cys. (F10) The CXCR3 ligand according to any one of (F7) to (F9), wherein the amino acid at position 18 from the N-terminus in the amino acid sequence of the parent CXCR3 ligand is substituted from P to C, and the amino acid at position 60 is substituted from A to C. (F11) The CXCR3 ligand according to any one of (F7) to (F10), wherein the amino acid modification in the N-terminal region is a substitution of the amino acid at position 1 in the amino acid sequence of the parent CXCR3 ligand with Tyr.(F12) The CXCR3 ligand according to any one of (F7) to (F11), wherein the first amino acid from the N-terminus in the amino acid sequence of the parent CXCR3 ligand is V, and the amino acid modification in the N-terminal region comprises a V1Y amino acid substitution. (F13) The CXCR3 ligand according to any one of (F1) to (F13), wherein the second amino acid from the N-terminus in the amino acid sequence of the parent CXCR3 ligand is P, and the amino acid modification in the N-terminal region comprises a P2V amino acid substitution. (F14) The CXCR3 ligand according to (F1) to (F13), further comprising at least one modification selected from amino acid substitution, deletion, and insertion. (F15) The CXCR3 ligand according to any one of (F1) to (F14), wherein the parent CXCR3 ligand is resistant to Furin protease. (F16) The CXCR3 ligand according to any one of (F1) to (F15), wherein the parent CXCR3 ligand is a CXCL10 variant obtained by modifying the amino acid sequence of native human CXCL10 by R75A. (F17) A method for improving the blood stability of a CXCR3 ligand, comprising substituting Pro for the amino acid at position 7 of the amino acid sequence of the parent CXCR3 ligand, where the N-terminal amino acid is defined as position 1. (F18) The method according to (F17), wherein the parent CXCR3 ligand comprises at least a portion of the amino acid sequence of native human CXCL10. (F19) The method according to (F17) or (F18), further comprising the step of adding at least one modification selected from amino acid substitution, deletion, and insertion. (F20) A method for producing a CXCR3 ligand having enhanced blood stability compared to the parent ligand, comprising the step of substituting Pro for the amino acid at position 7 of the amino acid sequence of the parent CXCR3 ligand, where the N-terminal amino acid is defined as position 1. (F21) The method according to (F20), wherein the parent CXCR3 ligand comprises at least a portion of the amino acid sequence of native human CXCL10. (F22) The method according to (F20) or (F21), further comprising a step of adding at least one modification selected from amino acid substitution, deletion, and insertion. (F23) A CXCR3 ligand produced by the method according to any one of (F20) to (F22).

[0015] [G] (G1) A polypeptide comprising the amino acid at position 7 of the amino acid sequence of native human CXCL10 or a CXCL10 variant, where the N-terminal amino acid of native human CXCL10 is defined as position 1, substituted with Pro. (G2) The polypeptide of (G1), further comprising the amino acids at positions 18 and 60 of native human CXCL10 substituted with Cys. (G3) The polypeptide of (G1) or (G2), further comprising Tyr for the amino acid at position 1 of native human CXCL10. (G4) The polypeptide of any of (G1) to (G3), further comprising Val for the amino acid at position 2 of native human CXCL10. (G5) A polypeptide that is a CXCR3 ligand and has the sequence of YPLSRTP (SEQ ID NO: 67), YVLSRTP (SEQ ID NO: 68), VPLSRTP (SEQ ID NO: 70), or VVLSRTP (SEQ ID NO: 71) at its N-terminus. (G6) A polypeptide having the following amino acid sequence: (X1)(X2)LSRT(X6)RCT CISISNQ(X3)VN PRSLEKLEII PASQFCPRVE IIATMKKKG EKRCLNPESK(X4) IKNLLKAVSK ERSK(X5)SP (SEQ ID NO: 69), wherein (X1) is V or Y, (X2) is P or V, (X3) is P or C, (X4) is A or C, (X5) is R or A, and (X6) is P.

[0016] [H] (H1) An isolated nucleic acid encoding the CXCR3 ligand according to [F] or the polypeptide according to [G]. (H2) A vector having the nucleic acid according to (H1). (H3) A host cell comprising the nucleic acid according to (H1) or the vector according to (H2). (H4) A method for producing the CXCR3 ligand according to [F] or the polypeptide according to [G], comprising culturing the host cell according to (H3) so as to produce the CXCR3 ligand or polypeptide. (H5) The method according to (H4), further comprising the step of recovering the polypeptide or the CXCR3 ligand from the host cell. (H6) A fusion protein comprising the CXCR3 ligand according to [F] or the polypeptide according to [G]. (H7) The fusion protein according to (H6), in which the CXCR3 ligand according to [F] or the polypeptide according to [G] is fused with an antibody Fc region. (H8) The fusion protein according to (H6), in which the CXCR3 ligand according to [F] or the polypeptide according to [G] is fused with a complete antibody or an antibody fragment. (H9) The fusion protein according to (H8), wherein the complete antibody or antibody fragment has at least one cleavage site, and the binding of the complete antibody or antibody fragment to the CXCR3 ligand or the polypeptide is attenuated when the complete antibody or antibody fragment is cleaved at at least one cleavage site. (H10) The fusion protein according to (H9), wherein the ligand or the polypeptide is released from the complete antibody or antibody fragment when the cleavage site is cleaved. (H11) The fusion protein according to (H9) or (H10), wherein the cleavage site comprises a protease cleavage sequence. (H12) The fusion protein according to (H11), wherein the protease is a target tissue-specific protease. (H13) The fusion protein according to (H12), wherein the target tissue is cancer tissue or inflamed tissue, and the target tissue-specific protease is a cancer tissue-specific protease or an inflamed tissue-specific protease.(H14) The fusion protein according to any one of (H11) to (H13), wherein the protease is at least one protease selected from metalloproteases, serine proteases, aspartic acid proteases, cysteine ​​proteases, threonine proteases, matriptase, and urokinase (uPA). (H15) The fusion protein according to any one of (H9) to (H14), wherein a flexible linker is added to one or both ends of the cleavage site or the protease cleavage sequence. (H16) The fusion protein according to any one of (H9) to (H15), wherein the complete antibody or antibody fragment has an antibody constant region, an antibody VH, and an antibody VL, and the cleavage site or the protease cleavage sequence is located near the boundary between the antibody constant region and the antibody VH, or near the boundary between the antibody constant region and the antibody VL. (H17) The fusion protein according to (H16), wherein the cleavage site or the protease cleavage sequence is inserted into at least one of (i) any position in the sequence from amino acid 109 (Kabat numbering) of the antibody VH to amino acid 122 (EU numbering) of the antibody heavy chain constant region, and (ii) any position in the sequence from amino acid 104 (Kabat numbering) of the antibody VL to amino acid 113 (EU numbering) of the antibody light chain constant region. (H18) The fusion protein according to (H16) or (H17), wherein the antibody VL and the antibody VH of the complete antibody or antibody fragment are associated, and the association is terminated by cleavage of the cleavage site or the protease cleavage sequence with a protease. (H19) The fusion protein according to any one of (H7) to (H18), wherein the CXCR3 ligand or the polypeptide and the antibody Fc region, or the CXCR3 ligand or the polypeptide and the complete antibody or antibody fragment, are fused via a linker. (H20) A pharmaceutical composition comprising the CXCR3 ligand according to [A], the polypeptide according to [B], or the fusion protein according to any one of (H6) to (H19).(H21) A method for treating a disease, comprising the step of administering a CXCR3 ligand described in [A], a polypeptide described in [B], or a fusion protein described in any one of (H6) to (H19).

[0017] Figure 1 shows the molecular structure of CXCL10 variants. Figure 1A is a ribbon model showing a model structure in which disulfide bonds are formed by two naturally occurring disulfide bonds (conventional SS bond, Cys9-Cys36 and Cys11-Cys53) and two cysteine ​​substitutions (introduced SS bond by P18C / A60C substitutions). Figure 1B is a schematic diagram showing the structure of the hCXCL10 mutant Fc fusion prepared in Example 1. Figure 2 is a graph showing the results of comparing the BaF3 / mCXCR3 cell induction ability of CXCL10 variants (#1, #5, #6, and #7). The amino acid substitutions introduced into each variant are listed in Table 3. In the figure, the vertical axis represents the migration rate, and the horizontal axis represents the chemokine (CXCL10 variant) concentration (nM). The migration rate was calculated as the relative value of the luminescence in the lower chamber to the luminescence obtained from the entire amount of cells loaded in the upper chamber (luminescence value / total cells). Figure 3 shows the results of a comparison of the BaF3 / mCXCR3 cell induction ability of CXCL10 variants (#1, #7, #8, #10). The amino acid substitutions introduced into each variant are listed in Table 4. In the figure, the vertical axis represents the migration rate, and the horizontal axis represents the chemokine (CXCL10 variant) concentration (nM). The migration rate was calculated as the relative value of the luminescence in the lower chamber to the luminescence obtained from the entire amount of cells loaded in the upper chamber (luminescence value / total cells). Figure 4 shows the results of a comparison of the BaF3 / mCXCR3 cell induction ability of CXCL10 variants (#1, #4, #8, #9). The amino acid substitutions introduced into each variant are listed in Table 5. In the figure, the vertical axis represents the migration rate, and the horizontal axis represents the concentration (nM) of the chemokine (CXCL10 variant). The migration rate was calculated as the relative value of the luminescence in the lower chamber to the luminescence obtained from the total amount of cells loaded in the upper chamber (luminescence value / luminescence value of all cells). Figure 5 shows the results of comparing the induction ability of CXCL10 variants (#1, #4, #7, #12) to BaF3 / mCXCR3 cells. The amino acid substitutions introduced into each mutant are shown in Table 6.In the figure, the vertical axis represents the migration rate, and the horizontal axis represents the concentration (nM) of the chemokine (CXCL10 variant). The migration rate was calculated as the relative value of the luminescence in the lower chamber to the luminescence obtained from the total amount of cells loaded in the upper chamber (luminescence value / luminescence value for all cells). Figure 6 is a schematic diagram showing the molecular structure of the hCXCL10-fused anti-hCXCL10 antibody prepared in Example 3. Figure 7 shows the quantification of each monitor peptide using antibodies (A) hCXCL10R75A.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0, (B) hCXCL10R75A.0016.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0, or (C) 1 is a graph showing the time course of plasma concentration after administration of hCXCL10R75A.0303.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0. Figure 8 is a graph showing the cell migration activity of Fc fusions of hCXCL10 variants: hCXCL10R75A (having R75A), hCXCL10R75A.0670 (having V1Y / P2V / P18C / A60A), and hCXCL10R75A.0613 (having V1Y / P2V / V7P / P18C / A60A). Figure 9 is a graph showing the time course of plasma concentration after administration of the hCXCL10-fused anti-hCXCL10 antibody hCXCL10R75A.0659.G4SGGGG.G7HFR0039H.0004.N0222-G1T4h / G7L.R38E-LT0 / / IC17HdK-G1T4k.H435R / G7L.R38E-LT0, quantified using each monitor peptide.

[0018] The following definitions and detailed descriptions are provided to facilitate understanding of the present disclosure as set forth herein: Amino Acids Amino acids are referred to herein by one-letter or three-letter codes, or both, e.g., Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, Val / V.

[0019] Amino Acid Modification To modify an amino acid in the amino acid sequence of a CXCR3 ligand, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed. Furthermore, several known methods can also be used to modify amino acids by substituting amino acids other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which a non-natural amino acid is bound to an amber suppressor tRNA complementary to the UAG codon (amber codon), a type of stop codon, can also be used.

[0020] As used herein, the term "and / or" used to describe the site of amino acid modification includes any combination of "and" and "or." Specifically, for example, "amino acids at positions 33, 55, and / or 96 have been substituted" includes the following amino acid modification variations: (a) 33, (b) 55, (c) 96, (d) 33 and 55, (e) 33 and 96, (f) 55 and 96, and (g) 33, 55, and 96. Alternatively, "amino acids at positions 33, 55, and / or 96 have been substituted" is synonymous with "amino acids at at least one (one, two, or three) positions selected from the group consisting of 33, 55, and 96 have been substituted." Furthermore, as used herein when describing the site of an amino acid modification, the term "amino acid corresponding to position X" refers to an amino acid residue that is homologous when the amino acid position is shifted by a modification, such as an insertion or deletion, and the amino acid sequences are aligned. Specifically, for example, in a variant of a wild-type ligand, the amino acid corresponding to amino acid position X in the amino acid sequence of the wild-type ligand is an amino acid residue that is homologous when the amino acid sequences of the wild-type ligand and the variant are aligned. This may be the same as position X in the amino acid sequence of the variant, or it may be different if the amino acid position has been changed by insertion or deletion.

[0021] As used herein, amino acid modification refers not only to changing the structure of an amino acid but also to mutating a portion or all of the amino acid sequence to produce a different amino acid sequence. Amino acid sequence modification can be achieved by introducing appropriate codon substitutions into a nucleotide sequence encoding the amino acid sequence, or by peptide synthesis. Amino acid modification includes, for example, deletion from the amino acid sequence, insertion into the amino acid sequence, and / or substitution of a residue within the amino acid sequence. When appropriate codon substitutions are introduced into a nucleotide sequence encoding an amino acid sequence, a peptide consisting of the substituted amino acid sequence can be obtained by translating the substituted nucleotide sequence into the peptide encoded thereby. Alternatively, when substitutions are achieved by peptide synthesis, a peptide consisting of the substituted amino acid sequence can be obtained directly. Amino acid modification can change the function and properties of the final construct. Any combination of deletion, insertion, and substitution can be performed to arrive at the final construct, provided that the final construct has the desired characteristics. In other words, in the present invention, amino acid modification can include any of the following: amino acid deletion, insertion into the amino acid sequence, and substitution of a residue within the amino acid sequence.

[0022] Herein, expressions for amino acid substitutions may be appropriately used, in which a number representing a specific position is followed by the one-letter or three-letter code for the amino acid before and after the modification. For example, the modification P2A or Pro2Ala used to substitute an amino acid contained in a CXCR3 ligand represents a substitution of Pro with Ala at position 2 from the N-terminus of the CXCR3 ligand (also referred to herein as the second amino acid from the N-terminus, position 2 in the amino acid sequence, or the second amino acid in the amino acid sequence). That is, the number represents the position of the amino acid as counted from the N-terminus of the CXCR3 ligand, the one-letter or three-letter code for the amino acid written before the number represents the amino acid before substitution, and the one-letter or three-letter code for the amino acid written after the number represents the amino acid after substitution.

[0023] Polypeptide: In the present invention, a polypeptide generally refers to a peptide or protein having a length of about four amino acids or more. That is, a polypeptide is a series of peptides linked by amide bonds, or a protein containing a plurality of series of polypeptides linked by amide bonds. Furthermore, the polypeptide of the present invention is generally a polypeptide consisting of an artificially designed sequence, but is not particularly limited thereto and may be, for example, a polypeptide derived from a living organism. It may also be a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, or the like. Furthermore, fragments of the above polypeptides are also included in the polypeptide of the present invention.

[0024] The CXCR3 chemokine receptor (CXCR3, also known as G protein-coupled receptor 9 (GPR9) or CD183) is a G protein-coupled receptor that belongs to the CXC chemokine family and binds to the chemokines CXCL9, CXCL10, and CXCL11. CXCR3 is primarily expressed on activated T helper type 1 (Th1) lymphocytes, but is also present on natural killer cells, macrophages, dendritic cells, and subsets of B lymphocytes. The chemokines CXCL9, CXCL10, and CXCL11 are three naturally occurring CXCR3 ligands. The interaction of CXCR3 with its ligands (hereafter referred to as the CXCR3 axis) is involved in guiding receptor-bearing cells to specific sites in the body, particularly sites of inflammation, immune disorder, and immune dysfunction. The term "CXCR3," as used herein, unless otherwise indicated, refers to any native CXCR3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The full amino acid sequence of human CXCR3 is set forth in Refseq:NP_001495. In certain embodiments, the CXCR3 is native human CXCR3 (SEQ ID NO: 51), and the cells expressing CXCR3 are cells expressing native human CXCR3.

[0025] Chemokines, CXC Chemokines Chemokines are a family of homogeneous serum proteins between 7 and 16 kDa, originally characterized by their ability to induce leukocyte migration. Most chemokines contain four characteristic cysteines (Cys), and are classified into the CXC (or alpha, CXC), CC (or beta), C (or gamma), and CX3C (or delta) chemokine classes, depending on the motif represented by the first two cysteines. Two disulfide bonds are formed between the first and third cysteines and between the second and fourth cysteines. Disulfide bridges are generally considered necessary, and Clark-Lewis and coworkers reported that disulfide bonds are crucial for chemokine activity, at least for CXCL10 (Clark-Lewis et al., J. Biol. Chem. 269:16075-16081, 1994). Furthermore, the CXC (or alpha, CXC) subfamily has been divided into two groups: ELR-CXC chemokines and non-ELR-CXC chemokines, depending on the presence of an ELR motif (Glu-Leu-Arg) preceding the first cysteine ​​(see, e.g., Clark-Lewis, supra, and Belperio et al., J. Leukoc. Biol. 68:1-8, 2000). CXCL10, CXCL11, and CXCL9 are all non-ELR-CXC chemokines.

[0026] CXC motif chemokine 10 (CXC motif chemokine ligand 10, CXCL10) is a CXC chemokine, also known as interferon-inducible protein-10 (IP-10). It is induced by interferon-γ and tumor necrosis factor-α and is produced by keratinocytes, endothelial cells, fibroblasts, and monocytes. CXCL10 is thought to play a role in the recruitment of activated T cells to sites of tissue inflammation (Dufour, et al., J Immunol., 168:3195-204, 2002). CXCL10 may also play a role in hypersensitivity responses and the development of inflammatory demyelinating neuropathy (Kieseier, et al., Brain, 125:823-34, 2002).

[0027] Studies have shown that CXCL10 may be useful in stem cell engraftment following transplantation (Nagasawa, T., Int. J. Hematol. 72:408-11, 2000), stem cell mobilization (Gazitt, Y., J. Hematother Stem Cell Res 10:229-36, 2001; Hattori et al., Blood 97:3354-59, 2001), and enhancing antitumor immunity (Nomura et al., Int. J. Cancer 91:597-606, 2001; Mach and Dranoff, Curr. Opin. Immunol. 12:571-75, 2000). For example, the biological activities of chemokines have been discussed in reports known to those skilled in the art (Bruce, L. et al., Methods in Molecular Biology (2000) vol. 138, pp. 129-134; Raphaele, B. et al., Methods in Molecular Biology (2000) vol. 138, pp. 143-148; Paul D. Ponath et al., Methods in Molecular Biology (2000) vol. 138, pp. 113-120). The biological activity of CXCL10 is exerted by binding to the chemokine receptor CXCR3 expressed on the cell surface (Booth V. et al., Biochemistry. 41 (33): 10418-25).

[0028] As used herein, the term "CXCL10," unless otherwise indicated, refers to any native CXCL10 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term refers to mature CXCL10 that is secreted extracellularly as a result of intracellular processing, rather than to the "full-length" unprocessed form. As used herein, the unprocessed form is referred to as the CXCL10 precursor. This term also encompasses naturally occurring variants of CXCL10, such as splice variants and allelic variants. Native human CXCL10 is expressed as the CXCL10 precursor (Refseq Accession number: NP_001556) and then secreted extracellularly as a protein of the sequence set forth in SEQ ID NO: 25. The complete amino acid sequence of the rhesus monkey CXCL10 precursor is shown under Refseq Accession number AKK95955, and the complete amino acid sequence of the mouse CXCL10 precursor is shown under Refseq Accession number NP_067249. The term "animal source" refers not only to a molecule of interest isolated from biological material obtained from an animal, but also to an artificially synthesized protein molecule having the same amino acid sequence as native CXCL10. Native CXCL10 or naturally occurring CXCL10 mutants with artificial amino acid modifications are referred to as "CXCL10 variants." CXCL10 variants may also be referred to as recombinant CXCL10 polypeptides.

[0029] CXCL11 CXC motif chemokine 11 (CXC motif chemokine ligand 11, CXCL11) is a type of CXC chemokine, also known as I-TAC (Interferon-inducible T-cell alpha chemoattractant) or IP-9 (Interferon-gamma-inducible protein 9). CXCL11 gene expression is strongly induced by IFN-γ and IFN-β, as well as by IFN-α (Rani MR, The Journal of Biological Chemistry. 271 (37): 22878-84).

[0030] CXCL11 has the biological activity of activating T cells. CXCL11 exerts its biological activity by binding to the cell surface-expressed chemokine receptor CXCR3, and natural CXCL11 is known to bind to CXCR3 more strongly than natural CXCL10 or CXCL9 (Cole KE, The Journal of Experimental Medicine. 187 (12): 2009-21; Tensen CP, The Journal of Investigative Dermatology. 112 (5): 716-22).

[0031] As used herein, the term "CXCL11," unless otherwise specified, refers to any native CXCL11 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term refers to mature CXCL11 that is secreted extracellularly as a result of intracellular processing, rather than to the "full-length" unprocessed form. As used herein, the unprocessed form is referred to as the CXCL11 precursor. This term also encompasses naturally occurring variants of CXCL11, such as splice variants and allelic variants. Native human CXCL11 is expressed as the CXCL11 precursor (Refseq Accession number: NP_005400) and then secreted extracellularly as a protein of the sequence set forth in SEQ ID NO: 26. Native CXCL11 or naturally occurring CXCL11 variants with artificial amino acid modifications are referred to as "CXCL11 variants." The CXCL11 variant may also be referred to as a recombinant CXCL11 polypeptide.

[0032] CXC motif chemokine 9 (CXC motif chemokine ligand 9, CXCL9) is a CXC chemokine, also known as monokine-induced by gamma interferon (MIG). CXCL9 is a T-cell chemoattractant induced by IFN-γ and exerts its biological activity by binding to the cell surface chemokine receptor CXCR3.

[0033] As used herein, the term "CXCL9," unless otherwise specified, refers to any native CXCL9 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term refers to mature CXCL9 secreted extracellularly as a result of intracellular processing, rather than to "full-length" unprocessed CXCL9. Herein, the unprocessed form is referred to as the CXCL9 precursor. This term also encompasses naturally occurring CXCL9 variants, such as splice variants and allelic variants. Native human CXCL9 is expressed as the CXCL9 precursor (Refseq Accession number: NP_002407) and then secreted extracellularly as a protein of the sequence set forth in SEQ ID NO: 27. Native CXCL9 or naturally occurring CXCL9 variants with artificial amino acid modifications are referred to as "CXCL9 variants." CXCL9 variants may also be referred to as recombinant CXCL9 polypeptides.

[0034] Chimeric proteins can be prepared by fusing portions of CXCL10, CXCL11, CXCL9, etc. to each other. For example, a human CXCL10-human CXCL11 chimeric protein (hITIP) (SEQ ID NO: 28) can be prepared by combining amino acid residues 1 to 24 of human CXCL11 (SEQ ID NO: 26) with amino acid residues 25 to 77 of a human CXCL10 variant (SEQ ID NO: 1). Such chimeric proteins can also be referred to as CXCR3 ligands as long as they can bind to CXCR3. A human CXCL10-human CXCL11 chimeric protein (hITIP) (SEQ ID NO: 28) combining amino acid residues 1 to 24 of human CXCL11 (SEQ ID NO: 26) with amino acid residues 25 to 77 of a human CXCL10 variant (SEQ ID NO: 1) with further amino acid modifications is called a "hITIP variant."

[0035] Interaction In the context of a compound (e.g., a peptide compound) that interacts with a specific amino acid residue on a target protein, "interaction" refers to a non-covalent interaction, such as a hydrogen bond, an ionic bond, a hydrophobic interaction, or a van der Waals interaction, or a bond via other molecules such as water molecules. Herein, an interaction between an amino acid residue and a compound can be determined if the interatomic distance between the non-hydrogen atom in the specific amino acid residue on the protein and the non-hydrogen atom in the compound (in the case of a bond via other molecules such as water molecules, the interatomic distance with the other molecule) is 4 angstroms (Å) or less. The interatomic distance can be measured, for example, using an X-ray analyzer and structural analysis software. A "change in interaction" refers to a change in the mode of non-covalent interaction or bond via other molecules such as water molecules. Examples include a change in the distance between interacting residues, a change to another residue, or a change in the type of bond involved (e.g., from a hydrogen bond to an ionic bond).

[0036] Chemokine-mediated receptor activation can be explained by a two-step model. The first step is receptor binding, in which the ligand binds to the N-terminal region of the receptor. The second step is activation, in which the N-terminal region of the ligand interacts with the receptor, inducing the receptor to its active form. CXC chemokine receptor activation also follows a two-step model. The region of the ligand important for the first step of binding is called the docking domain, which consists of the N-loop (first loop) from the conserved cysteine ​​pair (CXC motif) to β-strand 1 (first β-strand), the 30s loop (second loop) between β-strand 1 and β-strand 2 (second β-strand), and the 40s loop (third loop) between β-strand 2 and β-strand 3 (third β-strand). As mentioned above, CXC chemokines, such as CXCL10, have three β-strands: β-strand 1 is the most N-terminal, β-strand 2 is the second most N-terminal, and β-strand 3 is the third most N-terminal. In the second step, the N-terminal region of the ligand, called the triggering domain, interacts with the transmembrane domain of CXCR3, inducing the receptor into an activated conformation. Because the N-terminal region of the ligand directly contributes to the receptor's conformational change, substitution or deletion of amino acid residues in this region is known to reduce the efficiency of receptor activation (Booth, Keizer et al. 2002, Clark-Lewis, Mattioli et al. 2003, Booth, Clark-Lewis et al. 2004, Allen, Crown et al. 2007, Aguilera-Duran and Romo-Mancillas 2020). As used herein, the N-terminal region of a CXCR chemokine refers to the region N-terminal to the CXC motif, i.e., the amino acids in the N-terminal region are those located N-terminal to the N-terminal C of the cysteine ​​pair conserved in CXC chemokines.For example, when the CXCR chemokine is native human CXCL10 or a variant thereof, the amino acids in the N-terminal region are amino acids between amino acid positions 1 and 8.

[0037] The three-dimensional structure of the ligand is known to be important for the first step of receptor binding. XC motif chemokine ligand 1 (XCL1) is a chemokine that binds to the receptor XCR1 and contributes to the activation of dendritic cells and T cells. XCL1 lacks the N-terminal cysteine ​​residue conserved in other chemokine families and forms only one disulfide bond within the molecule. Chemokines generally adopt a chemokine fold (α+β fold) consisting of three β-strands and one α-helix. However, due to the lack of disulfide bonds, XCL1 is more unstable than other chemokine families, and is known to exist in an equilibrium between a chemokine form that can bind and activate receptors and an alternate form that cannot (Dishman, Tyler et al. 2021). Therefore, a technique has been reported in which XCL1 is modified by introducing additional disulfide bonds so that it preferentially forms a chemokine fold, similar to other chemokines. Compared to wild-type XCL1, modified XCL1 with additional disulfide bonds can activate XCR1-expressing cells at lower concentrations, and subcutaneous administration in mice has been shown to increase the accumulation of XCR1-expressing dendritic cells at the injection site (Tuinstra, Peterson et al. 2007, Matsuo, Kitahata et al. 2018). Although not directly mentioned, the enhanced activity of XCL1 due to structural confinement can be explained by the conformational selection model of protein-protein interactions. In this model, proteins have a certain degree of flexibility in solution, and only when the respective protein structures are optimally aligned can they bind to their partners. Therefore, confining the protein structure to a state close to the bound conformation promotes protein binding (Boehr, Nussinov et al. 2009).However, CXCR3 ligands such as CXCL10 contain a cysteine ​​residue in the N-terminal region and have a chemokine fold (α+β fold) formed by three β-strands and one α-helix, and an alternate fold like XCL1 has not been confirmed. Therefore, the introduction of a disulfide bond, which was useful for enhancing the activity of XCL1, does not appear to be very useful for enhancing the activity of CXCL10.

[0038] The inventors of the present invention considered the possibility that the N-terminal region, the C-terminal α-helical region, and the docking domain centered on the N-loop may be locally in equilibrium between the CXCR3-binding structure and the non-binding structure, and investigated modifications that would stabilize these regions of CXCL10 in the CXCR3-binding state based on conformational selection.

[0039] In the second activation step, the N-terminal region of the ligand interacts with the transmembrane domain of the receptor, and this interaction induces a conformational change and activation of the receptor. Therefore, the N-terminal region of the ligand has a major impact on receptor activation. It is known that ligands cleaved in this region often lose their activity. On the other hand, it has been reported that some chemokines, such as CXCL8 and CCL3L1, whose original sequences are inactive, become active upon protease cleavage. Based on this background, protease cleavage of the N-terminal region of chemokines is thought to regulate chemokine activity (Allen, Crown et al. 2007, Mortier, Van Damme et al. 2008). Cleavage of the N-terminal region of chemokines has little effect on receptor binding. Therefore, this region can be considered to regulate chemokine activity independently of receptor binding. Numerous techniques for converting inactive chemokines to active ones have been reported, including those mentioned above. It is also known that CXCL10 can be inactivated by N-terminal truncation (Proost, Schutyser et al. 2001, Repnik, Starr et al. 2015). However, to our knowledge, there have been no reports of enhancing activity through modification. It is clear from the activation mode that modifying residues important for the interaction between the N-terminal region of the ligand and the receptor during the activation step does not induce sufficient structural changes, resulting in reduced activity. On the other hand, it is difficult to achieve modifications that further induce an activated structure by substituting residues already involved in the interaction. The inventors of the present invention explored substitutions of amino acid residues in the N-terminal region, which contributes to the activation step, by adjusting the hydrophobicity and side chain size of the N-terminal region, possibly stabilizing the N-terminal region's insertion into the transmembrane domain and subsequent interaction.

[0040] (Cited References) Allen, S. J., S. E. Crown and T. M. Handel (2007). "Chemokine: receptor structure, interactions, and antagonism." Annu Rev Immunol 25: 787-820. Boehr, D. D., R. Nussinov and P. E. Wright (2009). "The role of dynamic conformational ensembles in biomolecular recognition." Nat Chem Biol 5(11): 789-796. Booth, V., I. Clark-Lewis and B. D. Sykes (2004). "NMR structure of CXCR3 binding chemokine CXCL11 (ITAC)." Protein Sci 13(8): 2022-2028. Booth, V., D. W. Keizer, M. B. Kamphuis, I. Clark-Lewis and B. D. Sykes (2002). "The CXCR3 Binding Chemokine IP-10 / CXCL10: Structure and Receptor Interactions." Biochemistry 41(33): 10418-10425. Clark-Lewis, I., I. Mattioli, J. H. Gong and P. Loetscher (2003). "Structure-function relationship between the human chemokine receptor CXCR3 and its ligands." J Biol Chem 278(1): 289-295. Dishman, A. F., R. C. Tyler, J. C. Fox, A. B. Kleist, K. E. Prehoda, M. M. Babu, F. C. Peterson and B. F. Volkman (2021)."Evolution of fold switching in a metamorphic protein." Science 371(6524): 86-90. Matsuo, K., K. Kitahata, F. Kawabata, M. Kamei, Y. Hara, S. Takamura, N. Oiso, A. Kawada, O. Yoshie and T. Nakayama (2018). "A Highly Active Form of XCL1 / Lymphotactin Functions as an Effective Adjuvant to Recruit Cross-Presenting Dendritic Cells for Induction of Effector and Memory CD8(+) T Cells." Front Immunol 9: 2775. Mortier, A., J. Van Damme and P. Proost (2008). "Regulation of chemokine activity by posttranslational modification." Pharmacol Ther 120(2): 197-217. Proost, P., E. Schutyser, P. Menten, S. Struyf, A. Wuyts, G. Opdenakker, M. Detheux, M. Parmentier, C. Durinx and A.-M. Lambeir (2001). "Amino-terminal truncation of CXCR3 agonists impairs receptor signaling and lymphocyte chemotaxis, while preserving antiangiogenic properties." Blood, The Journal of the American Society of Hematology 98(13): 3554-3561. Repnik, U., A. E. Starr, C. M. Overall and B. Turk (2015)."Cysteine Cathepsins Activate ELR Chemokines and Inactivate Non-ELR Chemokines." J Biol Chem 290(22): 13800-13811. Tuinstra, R. L., F. C. Peterson, E. S. Elgin, A. J. Pelzek and B. F. Volkman (2007). "An Engineered Second Disulfide Bond Restricts Lymphotactin / XCL1 to a Chemokine-like Conformation with XCR1 Agonist Activity." Biochemistry 46(10): 2564-2573.

[0041] Measurement of CXCR3 Ligand-CXCR3 Binding The binding of CXCR3 ligands to CXCR3 can be assessed using well-known methods, such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE (using surface plasmon resonance (SPR)), and BLI (Bio-Layer Interferometry) (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). ALPHA screens are performed using ALPHA technology, which uses two beads, donor and acceptor, based on the following principle: A luminescent signal is detected only when a molecule bound to the donor bead interacts with a molecule bound to the acceptor bead and the two beads are in close proximity. A photosensitizer within the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. Singlet oxygen diffuses around the donor beads and, upon reaching nearby acceptor beads, triggers a chemiluminescent reaction within the beads, ultimately emitting light. If the molecules bound to the donor and acceptor beads do not interact, the singlet oxygen produced by the donor beads does not reach the acceptor beads, and no chemiluminescent reaction occurs. For example, a biotin-labeled CXCR3 ligand is bound to the donor beads, and a glutathione S-transferase (GST)-tagged CXCR3 peptide is bound to the acceptor beads. The N-terminal extracellular domain region (residues 1-53) or a fragment thereof (22-42, Biochemistry (2002) 41, 10418-10425) can be used as the CXCR3 peptide. Furthermore, it is more preferable that the Y residue at position 27 and / or the Y residue at position 29 of CXCR3 be modified by sulfate (MOLECULAR AND CELLULAR BIOLOGY, Aug. 2006, pp. 5838-5849). In the absence of a competing untagged CXCR3 ligand, the CXCR3 ligand and the CXCR3 peptide interact to generate a signal at 520-620 nm.Untagged CXCR3 ligands compete with the interaction between tagged CXCR3 ligands and CXCR3 peptides. Relative binding affinity can be determined by quantifying the resulting decrease in fluorescence. Biotinylation of CXCR3 ligands, such as CXCL10, using Sulfo-NHS-biotin or similar is well known. GST-tagging of CXCR3 peptides can be achieved by expressing GST-fused CXCR3 peptides in cells carrying a vector capable of expressing a fusion gene in which a polynucleotide encoding a CXCR3 peptide is fused in-frame with a polynucleotide encoding GST, followed by purification using a glutathione column. The resulting signals are suitably analyzed by fitting them to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0042] One of the substances to be observed for interaction (CXCR3 ligand) is immobilized on a gold film on a sensor chip. When light is shone from the back of the sensor chip so that it is totally reflected at the interface between the gold film and the glass, a portion of the reflected light exhibits a reduced reflection intensity (SPR signal). The other substance to be observed for interaction (analyte; when a CXCR3 ligand is immobilized, full-length CXCR3 or the CXCR3 peptide described above can be used as the analyte) is poured over the surface of the sensor chip. Upon binding of the CXCR3 ligand and the analyte, the mass of the immobilized CXCR3 ligand molecule increases, changing the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, the signal returns to its original position upon dissociation). The Biacore system plots the amount of this shift, i.e., the change in mass at the sensor chip surface, on the vertical axis, and displays the change in mass over time as measurement data (sensorgram). The kinetics (association rate constant (ka) and dissociation rate constant (kd)) can be calculated from the sensorgram curve, and the dissociation constant (KD) can be calculated from the ratio of these constants. Inhibition assays and equilibrium value analysis are also suitable for use in the BIACORE method. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010, and examples of equilibrium value analysis are described in Methods Enzymol. 2000;323:325-40. Using a similar method, full-length CXCR3 or the aforementioned CXCR3 peptide can be immobilized on a gold film of a sensor chip, and a CXCR3 ligand can be injected as an analyte to measure the binding between CXCR3 and CXCR3. The full-length CXCR3 or the aforementioned CXCR3 peptide immobilized on the gold film of the sensor chip may be purified, or may be derived from cells expressing the full-length CXCR3 or the aforementioned CXCR3 peptide, or a cell membrane fraction thereof.The CXCR3 ligands of the present disclosure can specifically bind to full-length CXCR3 or the CXCR3 peptides with a dissociation constant (KD) of 100 μM, 10 μM, 1 μM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 25 pM, 10 pM, 5 pM, 1 pM, 0.5 pM, or 0.1 pM or less.

[0043] Alternatively, the binding activity of a CXCR3 ligand to immobilized full-length CXCR3 or the CXCR3 peptide can be evaluated based on the principles of ELISA. For example, full-length CXCR3 or the CXCR3 peptide is immobilized in the wells of an ELISA plate. A CXCR3 ligand solution is contacted with the immobilized full-length CXCR3 or the CXCR3 peptide in the wells, and CXCR3 ligands that bind to the immobilized full-length CXCR3 or the CXCR3 peptide are detected with an antibody that binds to the CXCR3 ligand. Alternatively, a CXCR3 ligand is immobilized in the wells of an ELISA plate, a solution of full-length CXCR3 or the CXCR3 peptide is contacted with the immobilized CXCR3 ligand in the wells, and full-length CXCR3 or the CXCR3 peptides that bind to the immobilized CXCR3 ligand are detected with an antibody that binds to full-length CXCR3 or the CXCR3 peptide.

[0044] One method for measuring the binding affinity of CXCR3 ligands to CXCR3 is to label the CXCR3 ligand using a radioisotope. Specifically, a radioisotope-labeled CXCR3 ligand is prepared and added to CXCR3-expressing cells and incubated. After incubation, the sample is passed through a filter, allowing the CXCR3-bound CXCR3 ligand to be adsorbed onto the filter along with the cells. The filter is then dried and the amount of CXCR3 ligand adsorbed to the filter can be measured by measuring the radiation dose. This method has been reported to be applied to CXCL10 (MOLECULAR AND CELLULAR BIOLOGY, Aug. 2006, pp. 5838-5849, Vol. 26, No. 15; THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 278, No. 19, Issue of May 9, pp. 17066-17074, 2003). In addition, by measuring the 15N-1H HSQC NMR spectrum and 2D NOESY NMR spectrum of a 15N-labeled CXCR3 ligand and comparing the NMR spectra obtained before and after the addition of a CXCR3 peptide, it is also possible to analyze the residues in the CXCR3 ligand involved in CXCR3 binding and the strength of the binding (Biochemistry, 2002, 41, 10418-10425).

[0045] CXCR3 Ligand As used herein, the term "CXCR3 ligand" refers to a molecule capable of binding to CXCR3. For example, a protein comprising a site that interacts with the ligand-binding site of CXCR3 is included in the CXCR3 ligand of the present disclosure. This term does not refer to a "full-length" unprocessed CXCR3 ligand, but rather to a mature CXCR3 ligand that is secreted extracellularly as a result of intracellular processing. As used herein, unprocessed CXCR3 ligands are referred to as CXCR3 ligand precursors. The CXCR3 ligand of the present disclosure may also exist in a fused form with other proteins or polypeptides, such as antibodies, antibody Fc regions, and albumin. As used herein, CXCR3 ligands include not only naturally occurring CXCR3 ligands but also recombinant polypeptides and recombinant proteins modified based on them. In a specific embodiment, the CXCR3 ligand is a polypeptide capable of binding to CXCR3.

[0046] In certain embodiments, the CXCR3 ligands provided herein have the activity of inducing migration of cells expressing CXCR3. In certain embodiments, the CXCR3 ligands provided herein have the activity of inducing migration of cells expressing CXCR3, such as activated T helper type 1 (Th1) lymphocytes, cytotoxic T cells, natural killer cells, macrophages, dendritic cells, B lymphocyte subsets, some epithelial cells, endothelial cells, and Ba / F3 cells.

[0047] The activity of a target protein to induce migration of cells expressing CXCR3 can be measured using CXCR3-expressing transfectants or cells isolated from living organisms. As a specific example, Ba / F3 transfectant cells (hereinafter referred to as BaF3 / mCXCR3) expressing mouse CXCR3 (mCXCR3) and HTS Transwell TM-96 Permeable Supports with 5.0 μm Pore Polycarbonate Membrane (Cat. 3387, Corning) were used, and the target protein was used as the analyte. The final concentration of each analyte in the solution was adjusted to 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, or 1000 nM, and 236 μL of each solution was transferred to the lower chamber. 2.0 × 10 BaF3 / mCXCR3 cells were then transferred to the upper chamber. 5 The cells were seeded at 75 μL / well to achieve a 5-hour incubation at 37°C under 5% carbon dioxide. After the 5-hour incubation, 100 μL of the solution in the lower chamber was transferred to a 96-well Assay Plate with a White Lid and Flat Bottom (Cat. 3917, Corning) and incubated with CellTiter-Glo 2.0. TM Add 100 μL of Luminescent Cell Viability Assay solution (Cat. G9242, Promega). After 10 minutes of incubation at room temperature, luminescence is measured using Envision (PerkinElmer) to assess the degree of cell migration into the lower chamber. The amount of cells that have migrated into the lower chamber is reflected by luminescence intensity. The activity of the target protein to induce migration of CXCR3-expressing cells is measured by the migration and invasion of cells into a Boyden chamber (transwell) or tissue, i.e., the cell migration rate.

[0048] In one aspect, the CXCR3 ligand provided herein is a CXCR3 ligand in which at least one or both of (i) the introduction of a disulfide bond and (ii) an amino acid modification in the N-terminal region has been added to the amino acid sequence of a parent CXCR3 ligand, thereby enhancing the CXCR3-expressing cell migration activity compared to the parent CXCR3 ligand. In a specific aspect, the parent CXCR3 ligand has a CXC motif. In a specific aspect, the parent CXCR3 ligand has a CXC motif of CTC (Cys-Thr-Cys), CLC (Cys-Leu-Cys), or CSC (Cys-Ser-Cys). In a specific aspect, the amino acid sequence of the parent CXCR3 ligand comprises at least a portion of the amino acid sequence of a native human CXCR3 ligand. In a specific aspect, the amino acid sequence of the parent CXCR3 ligand comprises at least a portion of the amino acid sequence of native human CXCL10.

[0049] In certain embodiments, the CXCR3 ligands provided herein are variants of a parent CXCR3 ligand comprising a portion of the amino acid sequence of native human CXCL10. The CXCR3 ligands provided herein comprise a portion of the amino acid sequence of native human CXCL10. In certain embodiments, the CXCR3 ligands provided herein are variants of native human CXCL10. The CXCR3 ligands provided herein are obtained by further modifying a parent CXCR3 ligand, which is a variant of native human CXCL10. For example, the CXCR3 ligands provided herein are obtained by further modifying a parent CXCR3 ligand, which is a variant of native human CXCL10 that confers Furin protease resistance. For example, the CXCR3 ligands provided herein are obtained by further modifying the amino acid sequence of a parent CXCR3 ligand, which is a variant of native human CXCL10 that has been modified with an R75A mutation to confer Furin protease resistance, and by further modifying the amino acid sequence to enhance the CXCR3-expressing cell migration activity compared to the parent CXCR3 ligand. In this case, the portion of the amino acid sequence of native human CXCL10 refers to an amino acid sequence comprising the 75th amino acid from the N-terminus of the amino acid sequence of native human CXCL10. In a specific embodiment, the CXCR3 ligand provided herein is a CXCR3 ligand that has enhanced CXCR3-expressing cell migration activity compared to a parent CXCR3 ligand comprising a portion of the amino acid sequence of native human CXCL10, wherein the amino acids at positions corresponding to the 18th and 60th amino acids in the amino acid sequence of native human CXCL10 are substituted with Cys. In a specific embodiment, the CXCR3 ligand further comprises a CXCR3 ligand in which the amino acid at position corresponding to the 1st amino acid in the amino acid sequence of native human CXCL10 is substituted with Tyr. Furthermore, the CXCR3 ligand may also comprise a CXCR3 ligand in which the amino acid at position corresponding to the 2nd amino acid in the amino acid sequence of native human CXCL10 is substituted with Val.In certain embodiments, the CXCR3 ligand provided herein has enhanced CXCR3-expressing cell migration activity compared to a parent CXCR3 ligand comprising a portion of the amino acid sequence of native human CXCL10, wherein the amino acid at position 1 in the amino acid sequence of native human CXCL10 is substituted with Tyr. Furthermore, the CXCR3 ligand may have the amino acid at position 2 in the amino acid sequence of native human CXCL10 substituted with Val. The CXCR3-expressing cell migration activity of the CXCL10 variants provided herein is higher than that of the parent CXCR3 ligand. That is, the CXCR3-expressing cell migration activity of the CXCL10 variants provided herein is enhanced compared to the parent CXCR3 ligand. For example, the CXCR3-expressing cell migration activity of the CXCL10 variants provided herein is 110% or more of the CXCR3-expressing cell migration activity of the parent CXCR3 ligand. To confirm that the CXCR3 ligand of the present invention has enhanced CXCR3-expressing cell migration activity compared to the parent CXCR3 ligand, it is preferable to include the parent CXCR3 ligand as a control in an assay system for CXCR3-expressing cell migration activity and compare the luminescence intensities obtained from the same experiment between the CXCR3 ligand to be compared and the control, in order to ensure uniformity of the CXCR3 cells used in the assay. The CXCR3-expressing cell migration activity of a CXCR3 ligand is not limited to any particular analyte concentration in the assay system for CXCR3-expressing cell migration activity, but can be 110% or more of the CXCR3-expressing cell migration activity of the parent CXCR3 ligand at at least one of the following concentrations: 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, and 1000 nM. When a fusion protein containing a CXCR3 ligand is used as an analyte for analyzing the migration activity of CXCR3-expressing cells, it is preferable that the parent CXCR3 ligand used as a control has the same molecular format as the fusion protein containing the CXCR3 ligand (i.e., a control fusion protein is prepared using the parent CXCR3 ligand instead of the CXCR3 ligand provided in this specification).In certain embodiments, the CXCR3 ligands provided herein exhibit chemotactic activity for CXCR3-expressing cells that is comparable to or greater than that of native human CXCR3 ligands. In certain embodiments, the CXCR3 ligands provided herein exhibit chemotactic activity for CXCR3-expressing cells that is comparable to or greater than that of native human CXCL10. Comparison between native human CXCR3 ligands and native human CXCL10 is performed as described above.

[0050] Enhancement of the CXCR3-expressing cell migration activity can be confirmed, for example, by an increase in the number of cells that migrate in an assay system or a decrease in the EC50 value. Enhancement of the CXCR3-expressing cell migration activity of the CXCR3 ligand of the present invention can be confirmed, for example, by a greater number of cells that migrate in an assay system or a lower EC50 value compared to the parent CXCR3 ligand. A CXCR3 ligand of the present invention that has a higher CXCR3-expressing cell migration activity than the parent CXCR3 ligand can be confirmed, for example, by a greater number of cells that migrate in an assay system with the CXCR3 ligand than with the parent CXCR3 ligand, or a lower EC50 value with the CXCR3 ligand than with the parent CXCR3 ligand. In a specific embodiment, the CXCR3 ligand concentration at which the CXCR3-expressing cell migration activity of the CXCR3 ligand provided herein is maximized is lower than the concentration of the protein at which the CXCR3-expressing cell migration activity of the parent CXCR3 ligand is maximized. The CXCR3-expressing cell migration activity of the CXCR3 ligand of the present invention is equivalent to or higher than that of native human CXCL10 for CXCR3-expressing cells, for example, if the number of cells moving in an assay system is equivalent to or higher in the case of the CXCR3 ligand than in the case of native human CXCL10, or if the EC50 value is equivalent to or lower in the case of the CXCR3 ligand than in the case of native human CXCL10. In a specific embodiment, the CXCR3 ligand concentration at which the CXCR3-expressing cell migration activity of the CXCR3 ligand provided herein is maximized is lower than the concentration of the protein at which the CXCR3-expressing cell migration activity of native human CXCL10 is maximized.

[0051] Amino acid modifications in the N-terminal region that enhance the migratory activity of CXCR3-expressing cells include, for example, substituting amino acid residues in the N-terminal region with amino acid residues that stabilize insertion into the transmembrane region of CXCR3-expressing cells and subsequent interaction. Potential amino acid modifications in the N-terminal region that increase the migratory activity of CXCR3 ligands compared to the parent CXCR3 ligand include, for example, substituting the first amino acid residue from the N-terminus of native human CXCL10 with Y, F, H, T, or M when the parent CXCR3 ligand is native human CXCL10 or a CXCL10 variant obtained by modifying the amino acid sequence of native human CXCL10. In certain embodiments, the first amino acid from the N-terminus of the CXCR3 ligands and polypeptides provided herein is not V. In certain embodiments, the second amino acid from the N-terminus of the CXCR3 ligands and polypeptides provided herein is not P. In certain embodiments, the first amino acid from the N-terminus of the CXCR3 ligands and polypeptides provided herein is Y. In certain embodiments, the CXCR3 ligands and polypeptides provided herein have a V at the second amino acid from the N-terminus. In certain embodiments, the CXCR3 ligands and polypeptides provided herein have a Y substituted for the V at the first amino acid from the N-terminus of the parent CXCR3 ligand sequence. In certain embodiments, the parent CXCR3 ligand comprises at least a portion of the sequence of native human CXCL10 (SEQ ID NO: 25), and the CXCR3 ligands and polypeptides provided herein have a Y substituted for the V at least at the first amino acid from the N-terminus of the native human CXCL10 sequence (SEQ ID NO: 25) or the human CXCL10 variant (SEQ ID NO: 1). In this case, the at least a portion of the sequence of native human CXCL10 (SEQ ID NO: 25) refers to a sequence comprising the first and second amino acids from the N-terminus of native human CXCL10. In preferred embodiments, the CXCR3 ligands and polypeptides comprising these amino acid sequences have CXCR3-expressing cell migration activity that is equivalent to or greater than that of native human CXCL10.In certain embodiments, the CXCR3 ligands and polypeptides provided herein have at least the second N-terminal P substituted with V relative to the sequence of native human CXCL10 (SEQ ID NO: 25) or a human CXCL10 variant (SEQ ID NO: 1).

[0052] In certain embodiments, the CXCR3 ligands provided herein comprise an amino acid substitution N-terminal to the CXC motif of said parent CXCR3 ligand. In certain embodiments, when a parent CXCR3 ligand comprises at least a portion of the amino acid sequence of native human CXCL10, the CXCR3 ligands provided herein comprise an amino acid substitution at an amino acid position corresponding to positions 1 to 8 in the amino acid sequence of said native human CXCL10. In certain embodiments, the CXCR3 ligands provided herein comprise a substitution of the amino acid at position 1 in the amino acid sequence of the parent CXCR3 ligand.

[0053] In certain embodiments, the CXCR3 ligands and polypeptides provided herein have the sequence YPL or YVL at their N-terminus.

[0054] In certain embodiments, the CXCR3 ligands and polypeptides provided herein have the sequence YPLSRT (SEQ ID NO: 13) or YVLSRT (SEQ ID NO: 14) at their N-terminus.

[0055] The site for inserting a disulfide bond that enhances the migration activity of CXCR3-expressing cells is identified by predicting the physical properties of the variant using MD simulation, protein structure modeling, or the like. Combinations of positions for altering cysteine ​​residues are identified as candidates for sites at which the stability of the variant is increased compared to the parent CXCR3 ligand. The positions for altering cysteine ​​residues that are candidates for sites at which the stability of the variant is increased compared to the parent CXCR3 ligand are outside the β-sheet region of the parent CXCR3 ligand. In a specific embodiment, a disulfide bond is introduced by substituting an amino acid in the docking domain of the CXCR3 ligand with cysteine. In a specific embodiment, the parent CXCR3 ligand comprises three β-strands, and the disulfide bond is formed by substituting at least one amino acid in any two regions selected from the following in the amino acid sequence of the parent CXCR3 ligand: (i) the first loop between the CXC motif and the first β-strand located most N-terminally; (ii) the second loop between the first β-strand and the second β-strand located second most N-terminally; and (iii) the third loop between the second β-strand and the third β-strand located third most N-terminally. For example, at least one amino acid in the first loop and at least one amino acid in the second loop are substituted with cysteine ​​to introduce a disulfide bond between the first loop and the second loop. Alternatively, for example, at least one amino acid in the first loop and at least one amino acid in the third loop are substituted with cysteine ​​to introduce a disulfide bond between the first loop and the third loop. Alternatively, for example, at least one amino acid in the second loop and at least one amino acid in the third loop may be substituted with cysteine ​​to introduce a disulfide bond between the second loop and the third loop.Potential positions for cysteine ​​modification at which the stability of the variant is increased compared to that of the parent CXCR3 ligand include, for example, positions 14, 18, 21, 25, 41, 46, 55, 56, 60, and 67 from the N-terminus of native human CXCL10 when the parent CXCR3 ligand is native human CXCL10 or a CXCL10 variant obtained by modifying the amino acid sequence of native human CXCL10. Potential combinations of positions for cysteine ​​modification are, for example, combinations of amino acids selected from positions 14 and 55, positions 18 and 60, positions 21 and 67, positions 25 and 46, and positions 41 and 56 from the N-terminus of native human CXCL10 when the parent CXCR3 ligand is native human CXCL10 or a CXCL10 variant obtained by modifying the amino acid sequence of native human CXCL10. In certain embodiments, the CXCR3 ligands provided herein have C at at least one amino acid selected from the 14th, 18th, 21st, 25th, 41st, 46th, 55th, 56th, 60th, and 67th amino acids from the N-terminus. In certain embodiments, the CXCR3 ligands provided herein have C at any combination of amino acids selected from the 14th and 55th, 18th and 60th, 21st and 67th, 25th and 46th, and 41st and 56th amino acids from the N-terminus. That is, amino acid sequences in which any combination selected from the above combinations is substituted with C are preferred for the CXCR3 ligands provided herein. In certain embodiments, at least one set of substitutions is introduced, and any of 1 to 5 sets of substitutions can be selected. In preferred embodiments, CXCR3 ligands and polypeptides consisting of these amino acid sequences have CXCR3-expressing cell migration activity equivalent to or higher than that of native human CXCL10.In certain embodiments, the CXCR3 ligands provided herein have amino acid substitutions with C for any combination of amino acids selected from the 14th and 55th, 18th and 60th, 21st and 67th, 25th and 46th, and 41st and 56th amino acids from the N-terminus of the sequence of native human CXCL10 (SEQ ID NO: 25) or the human CXCL10 variant (SEQ ID NO: 1).

[0056] In certain embodiments, the CXCR3 ligands and polypeptides provided herein have a C at the 18th amino acid from the N-terminus. In certain embodiments, the CXCR3 ligands provided herein have a C at the 60th amino acid from the N-terminus. In this case, at least a portion of the sequence of native human CXCL10 (SEQ ID NO: 25) refers to a sequence comprising the 18th and 60th amino acids from the N-terminus of native human CXCL10. In preferred embodiments, CXCR3 ligands and polypeptides consisting of these amino acid sequences have a CXCR3-expressing cell migration activity that is equivalent to or greater than that of native human CXCL10.

[0057] In certain embodiments, the CXCR3 ligands and polypeptides provided herein have an amino acid 18 from the N-terminus that is not P. In certain embodiments, the CXCR3 ligands provided herein have an amino acid 60 from the N-terminus that is not A.

[0058] In certain embodiments, the CXCR3 ligands and polypeptides provided herein have at least a substitution of P at position 18 from the N-terminus with C and A at position 60 from the N-terminus with C relative to the sequence of native human CXCL10 (SEQ ID NO: 25) or a human CXCL10 variant (SEQ ID NO: 1). In certain embodiments, the CXCR3 ligands and polypeptides provided herein have a substitution of P at position 18 from the N-terminus with V relative to the sequence of the parent CXCR3 ligand. In certain embodiments, the parent CXCR3 ligand comprises at least a portion of the sequence of native human CXCL10 (SEQ ID NO: 25), and the CXCR3 ligands and polypeptides provided herein have at least a substitution of P at position 18 from the N-terminus and A at position 60 from the N-terminus with C, respectively, relative to the sequence of native human CXCL10 (SEQ ID NO: 25) or a human CXCL10 variant (SEQ ID NO: 1). In this case, the amino acid sequence comprising at least a portion of the sequence of native human CXCL10 (SEQ ID NO: 25) may be an amino acid sequence comprising the 18th and 60th amino acids from the N-terminus. In a preferred embodiment, a CXCR3 ligand and a polypeptide comprising these amino acid sequences have a CXCR3-expressing cell migration activity equivalent to or higher than that of native human CXCL10. In a specific embodiment, the CXCR3 ligand provided herein is a polypeptide having the following amino acid sequence: (X1)(X2)LSRTVRCT CISISNQ(X3)VN PRSLEKLEII PASQFCPRVE IIATMKKKG EKRCLNPESK(X4) IKNLLKAVSK ERSK(X5)SP (SEQ ID NO: 53), wherein (X1) is V or Y, (X2) is P or V, (X3) and (X4) are C, and (X5) is R or A. In certain embodiments, the CXCR3 ligand provided herein is a polypeptide having the following amino acid sequence: (X1)(X2)LSRTVRCT CISISNQ(X3)VN PRSLEKLEII PASQFCPRVE IIATMKKKG EKRCLNPESK(X4) IKNLLKAVSK ERSK(X5)SP (SEQ ID NO: 54), wherein (X1) is Y, (X2) is P or V, (X3) is P or C, (X4) is A or C, and (X5) is R or A.

[0059] In certain embodiments, the CXCR3 ligands provided herein have a CXC motif, wherein two cysteines in the CXC motif can form disulfide bonds with cysteines other than the CXC motif in the CXCR3 ligand. The CXC motif in the CXCR3 ligand can be selected from CTC (Cys-Thr-Cys), CLC (Cys-Leu-Cys), and CSC (Cys-Ser-Cys).

[0060] In certain aspects, the CXCR3 ligands provided herein are any of CXCL10 variants, CXCL11 variants, CXCL9 variants, hITIP variants, and chimeric proteins made from these variants.

[0061] In certain embodiments, the CXCR3 ligands and polypeptides provided herein have any of the following (b1) to (b7): (b1) a sequence set forth in any one of SEQ ID NOs: 4 to 12; (b2) a sequence exhibiting 90% or greater sequence identity to SEQ ID NO: 1; (b3) a sequence exhibiting 90% or greater sequence identity to SEQ ID NO: 25; (b4) a sequence exhibiting 90% or greater sequence identity to SEQ ID NO: 26; (b5) a sequence exhibiting 90% or greater sequence identity to SEQ ID NO: 27; (b6) a sequence exhibiting 90% or greater sequence identity to SEQ ID NO: 28; or (b7) a sequence containing 10 or less amino acid substitutions, insertions, or deletions relative to a sequence selected from SEQ ID NOs: 1, 4 to 12, and 25 to 28. In preferred embodiments, these CXCR3 ligands and polypeptides have CXCR3-expressing cell migration activity that is equivalent to or greater than that of native human CXCL10.

[0062] In certain embodiments, the CXCR3 ligands and polypeptides provided herein have a sequence that exhibits 90% or greater sequence identity, 95% or greater sequence identity, 96% or greater sequence identity, 97% or greater sequence identity, 98% or greater sequence identity, or 99% or greater sequence identity to any one of SEQ ID NOs: 1, 4-12, 25-28.

[0063] In one embodiment, the CXCR3 ligand provided herein is a CXCR3 ligand in which the amino acid corresponding to amino acid position 7, where the N-terminal amino acid in the amino acid sequence of the parent CXCR3 ligand is considered to be position 1, is substituted with Pro, thereby improving blood stability compared to the parent CXCR3 ligand. In a specific embodiment, the amino acid sequence of the parent CXCR3 ligand comprises at least a portion of the amino acid sequence of a native human CXCR3 ligand. In a specific embodiment, the amino acid sequence of the parent CXCR3 ligand comprises at least a portion of the amino acid sequence of native human CXCL10. In a specific embodiment, the CXCR3 ligand provided herein is a variant of the parent CXCR3 ligand comprising a portion of the amino acid sequence of native human CXCL10. The CXCR3 ligand provided herein comprises a portion of the amino acid sequence of native human CXCL10. In a specific embodiment, the CXCR3 ligand provided herein is a variant of native human CXCL10. The CXCR3 ligand provided herein is a parent CXCR3 ligand, which is a variant of native human CXCL10, further modified. For example, a CXCR3 ligand provided herein that has improved blood stability compared to a parent CXCR3 ligand is a parent CXCR3 ligand that is a variant of native human CXCL10 with amino acid modifications to enhance the chemotactic activity of CXCR3-expressing cells, and that further comprises modifications to the amino acid sequence of the parent CXCR3 ligand, which is a variant of native human CXCL10 with at least one or both of the following amino acid modifications to enhance the chemotactic activity of CXCR3-expressing cells: (i) introduction of a disulfide bond and (ii) amino acid modifications in the N-terminal region. For example, a CXCR3 ligand provided herein that has improved blood stability compared to a parent CXCR3 ligand is a parent CXCR3 ligand that is a variant of native human CXCL10 with resistance to DPPIV cleavage, and that further comprises modifications to enhance blood stability compared to the parent CXCR3 ligand.For example, a CXCR3 ligand provided herein that has improved blood stability compared to its parent CXCR3 ligand comprises a parent CXCR3 ligand that is a variant of native human CXCL10 that has undergone a P2V modification to confer resistance to DPPIV cleavage, and further comprises an amino acid modification that improves blood stability compared to the parent CXCR3 ligand. For example, a CXCR3 ligand provided herein that has improved blood stability compared to its parent CXCR3 ligand comprises a parent CXCR3 ligand that is a variant of native human CXCL10 that has undergone a further modification that confers resistance to Furin protease. For example, a CXCR3 ligand provided herein that has improved blood stability compared to its parent CXCR3 ligand comprises a parent CXCR3 ligand that is a variant of native human CXCL10 that has undergone a R75A modification to confer resistance to Furin protease, and further comprises an amino acid modification that improves blood stability compared to the parent CXCR3 ligand. In certain embodiments, the CXCR3 ligand of the present disclosure is a CXCR3 ligand that includes one or more of the following amino acid modifications: amino acid modifications for improving blood stability compared to the parent CXCR3 ligand, amino acid modifications for enhancing CXCR3-expressing cell migration activity, amino acid modifications for conferring resistance to DPPIV cleavage, and amino acid modifications for conferring resistance to Furin protease. In certain embodiments, the CXCR3 ligand provided herein is a CXCR3 ligand, and is a polypeptide having the sequence of YPLSRTP (SEQ ID NO: 67), YVLSRTP (SEQ ID NO: 68), VPLSRTP (SEQ ID NO: 70), or VVLSRTP (SEQ ID NO: 71) at its N-terminus. In certain embodiments, the CXCR3 ligand provided herein is a polypeptide having the following amino acid sequence: (X1)(X2)LSRT(X6)RCT CISISNQ(X3)VN PRSLEKLEII PASQFCPRVE IIATMKKKG EKRCLNPESK(X4) IKNLLKAVSK ERSK(X5)SP (SEQ ID NO: 69), wherein (X1) is V or Y, (X2) is P or V, (X3) is P or C, (X4) is A or C, (X5) is R or A, and (X6) is P.

[0064] CXCR3 Ligand Variants In certain embodiments, amino acid sequence variants of the CXCR3 ligands provided herein are also contemplated. Amino acid sequence variants of the CXCR3 ligands may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the CXCR3 ligand or by peptide synthesis. Such modifications include, for example, deletions from the amino acid sequence of the CXCR3 ligand, insertions into the amino acid sequence of the CXCR3 ligand, and / or substitutions of residues within the amino acid sequence of the CXCR3 ligand. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., the ability to induce migration of CXCR3-expressing cells).

[0065] Substitution, Insertion, and Deletion Mutants In certain embodiments, CXCR3 ligand mutants are provided that have one or more amino acid substitutions. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading "Exemplary Substitutions" and are detailed below with reference to classes of amino acid side chains. Amino acid substitutions may be introduced into a CXCR3 ligand of interest, and the products may be screened for a desired activity, such as, for example, retained / improved activity in inducing migration of CXCR3-expressing cells.

[0066]

[0067] Amino acids can be divided into groups based on shared side chain properties: (1) hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) acidic: aspartic acid (Asp), glutamic acid (Glu); (4) basic: histidine (His), lysine (Lys), arginine (Arg); (5) residues that affect chain orientation: glycine (Gly), proline (Pro); (6) aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Nonconservative substitutions involve exchanging a member of one of these classes for another.

[0068] In certain embodiments, one or more substitutions, insertions, or deletions may be made as long as such modifications do not substantially reduce the potency of the CXCR3 ligand. In certain embodiments of the CXCR ligand variants described above, the variants contain one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications. When modifications are made to a CXCR3 ligand, the amino acid positions identified in the present disclosure are preferably adjusted by aligning the amino acid sequences before and after modification to identify homologous amino acid residues. For example, the positions corresponding to the 18th and 60th amino acid residues from the N-terminus of native human CXCL10 refer to the positions of the variants assuming no changes or deviations in amino acid positions due to modifications other than the substitution of the 18th and 60th amino acids. For example, if an amino acid insertion results in an amino acid position value greater than 18, the amino acid position is subtracted by the number of inserted amino acids. Furthermore, for example, when the value of an amino acid position is an amino acid position lower than 18 or 60 due to the removal of amino acids, the number of amino acid positions is increased by the number of removed amino acids. Note that when a CXCR3 ligand is modified, the amino acid positions specified in the present disclosure may be specified in the amino acid sequence of the variant.

[0069] Amino Acid Sequence Identity "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared.

[0070] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, along with user documentation, has been submitted to the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it can be stated that a given amino acid sequence A has or contains a certain percent amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y. where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0071] The present disclosure also relates to a method for enhancing the CXCR3-expressing cell migration activity of a CXCR3 ligand. The method for enhancing the CXCR3-expressing cell migration activity of a CXCR3 ligand provided herein comprises modifying the sequence of a parent CXCR3 ligand. In certain embodiments, the method for enhancing the CXCR3-expressing cell migration activity of a CXCR3 ligand provided herein comprises modifying amino acids in the N-terminal region of the sequence of the parent CXCR3 ligand. In addition, in certain embodiments, the method for enhancing the CXCR3-expressing cell migration activity of a CXCR3 ligand provided herein comprises introducing a disulfide bond into the sequence of the parent CXCR3 ligand.

[0072] In certain embodiments, the parent CXCR3 ligand used in the method for enhancing the CXCR3-expressing cell migratory activity of a CXCR3 ligand provided herein has a CXC motif. In certain embodiments, the CXC motif is CTC (Cys-Thr-Cys), CLC (Cys-Leu-Cys), or CSC (Cys-Ser-Cys). In certain embodiments, the parent CXCR3 ligand used in the method for enhancing the CXCR3-expressing cell migratory activity of a CXCR3 ligand provided herein comprises a portion of the amino acid sequence of a native human CXCR3 ligand. In certain embodiments, the native human CXCR3 ligand is native human CXCL10. In certain embodiments, the first amino acid from the N-terminus of the parent CXCR3 ligand used in the method for enhancing the CXCR3-expressing cell migratory activity of a CXCR3 ligand provided herein is V. In certain embodiments, the second amino acid from the N-terminus of the parent CXCR3 ligand used in the method for enhancing the CXCR3-expressing cell migratory activity of a CXCR3 ligand provided herein is P. In a more specific embodiment, the N-terminal sequence of the parent CXCR3 ligand used in the method for enhancing the CXCR3-expressing cell migratory activity of a CXCR3 ligand provided herein is VPL or VVL. In an even more specific embodiment, the N-terminal sequence of the parent CXCR3 ligand used in the method for enhancing the CXCR3-expressing cell migratory activity of a CXCR3 ligand provided herein is VPLSRT (SEQ ID NO: 19) or VVLSRT (SEQ ID NO: 20).

[0073] In certain embodiments, the parent CXCR3 ligand used in the methods for enhancing the chemotactic activity of CXCR3-expressing cells provided herein has a CXC motif. Each of the two cysteines in the CXC motif can form a disulfide bond with a cysteine ​​other than the CXC motif in the parent CXCR3 ligand. The CXC motif in the parent CXCR3 ligand can be selected from CTC (Cys-Thr-Cys), CLC (Cys-Leu-Cys), and CSC (Cys-Ser-Cys). In specific embodiments, the CXC motif in the parent CXCR3 ligand can be located adjacent to the C-terminus of the N-terminal sequence VPLSRTVR (SEQ ID NO: 21) or VVLSRTVR (SEQ ID NO: 22).

[0074] In certain embodiments, the parent CXCR3 ligand used in the method for enhancing the CXCR3-expressing cell migration activity of a CXCR3 ligand provided herein may further have any of the following (b1) to (b6) at the C-terminus of the CXC motif: (b1) the sequence from amino acid 12 to amino acid 77 of any of SEQ ID NOs: 4 to 12; (b2) the sequence from amino acid 12 to amino acid 77 of SEQ ID NO: 25; (b3) the sequence from amino acid 12 to amino acid 73 of SEQ ID NO: 26; (b4) the sequence from amino acid 12 to amino acid 103 of SEQ ID NO: 27; (b5) the sequence from amino acid 12 to amino acid 77 of SEQ ID NO: 1; (b6) the sequence from amino acid 12 to amino acid 77 of SEQ ID NO: 28.

[0075] In certain embodiments, the parent CXCR3 ligand used in the method for enhancing the migratory activity of CXCR3-expressing cells of a CXCR3 ligand provided herein is selected from native CXCL10, native CXCL11, native CXCL9, a CXCL10 variant, a CXCL11 variant, a CXCL9 variant, and chimeric proteins prepared therefrom. In certain embodiments, the parent CXCR3 ligand used in the method for enhancing the migratory activity of CXCR3-expressing cells of a CXCR3 ligand provided herein is resistant to Furin protease. In certain embodiments, the parent CXCR3 ligand used in the method for enhancing the migratory activity of CXCR3-expressing cells of a CXCR3 ligand provided herein is a CXCL10 variant with an R75A mutation in the amino acid sequence of native human CXCL10.

[0076] In certain embodiments, the method for enhancing the CXCR3-expressing cell migration activity of a CXCR3 ligand provided herein comprises modifying the amino acid sequence of the parent CXCR3 ligand by at least one or both of (i) introducing a disulfide bond and (ii) modifying an amino acid in the N-terminal region. In certain embodiments, the method for enhancing the CXCR3-expressing cell migration activity of a CXCR3 ligand provided herein comprises introducing at least one amino acid substitution, V1Y, or P18C and A60C, into the amino acid sequence of the parent CXCR3 ligand. In other words, the present specification provides use of any of the following to enhance the CXCR3-expressing cell migration activity of a CXCR3 ligand: (1) substituting V for Y at the first amino acid of the parent CXCR3 ligand; or (2) substituting P for C at the 18th amino acid from the N-terminus of the parent CXCR3 ligand and A for C at the 60th amino acid. The method for enhancing the CXCR3-expressing cell migration activity of a CXCR3 ligand provided herein may further comprise substituting P with V at the second amino acid from the N-terminus of the parent CXCR3 ligand, in addition to the above.

[0077]

[0001] The present disclosure also relates to a method for improving the blood stability of a CXCR3 ligand. The method for improving the blood stability of a CXCR3 ligand provided herein comprises modifying the sequence of a parent CXCR3 ligand. In certain embodiments, the method for improving the blood stability of a CXCR3 ligand provided herein comprises substituting Pro for the amino acid corresponding to amino acid position 7, where the N-terminal amino acid in the amino acid sequence of the parent CXCR3 ligand is position 1. In certain embodiments, the 7th amino acid from the N-terminus of the parent CXCR3 ligand used in the method for improving the blood stability of a CXCR3 ligand provided herein is V. In certain embodiments, the parent CXCR3 ligand used in the method for improving the blood stability of a CXCR3 ligand provided herein comprises a portion of the amino acid sequence of a native human CXCR3 ligand. In certain embodiments, the native human CXCR3 ligand is native human CXCL10.

[0078] In certain embodiments, the parent CXCR3 ligand used in the method for improving the blood stability of a CXCR3 ligand provided herein is selected from native CXCL10, native CXCL11, native CXCL9, a CXCL10 variant, a CXCL11 variant, a CXCL9 variant, and chimeric proteins prepared therefrom. In certain embodiments, the parent CXCR3 ligand used in the method for improving the blood stability of a CXCR3 ligand provided herein is resistant to Furin protease. In certain embodiments, the parent CXCR3 ligand used in the method for improving the blood stability of a CXCR3 ligand provided herein is a CXCL10 variant with an R75A modification added to the amino acid sequence of native human CXCL10.

[0079] In certain embodiments, the method for improving the blood stability of a CXCR3 ligand provided herein further comprises modifying the amino acid sequence of the parent CXCR3 ligand by at least one or both of (i) introducing a disulfide bond and (ii) modifying an amino acid in the N-terminal region. In certain embodiments, the method for improving the blood stability of a CXCR3 ligand provided herein comprises introducing at least one amino acid substitution of V1Y, or P18C and A60C, into the amino acid sequence of the parent CXCR3 ligand. In addition to the above, the method for improving the blood stability of a CXCR3 ligand provided herein may further comprise substituting P for V at the second amino acid from the N-terminus of the parent CXCR3 ligand.

[0080] The present disclosure also relates to a method for producing a CXCR3 ligand with enhanced blood stability compared to its parent ligand. The method for producing a CXCR3 ligand with enhanced blood stability compared to its parent ligand, as provided herein, comprises modifying the sequence of the parent CXCR3 ligand. In a specific embodiment, the method for producing a CXCR3 ligand with enhanced blood stability compared to its parent ligand, as provided herein, comprises substituting Pro for the amino acid at position 7 of the amino acid sequence of the parent CXCR3 ligand, where the N-terminal amino acid is position 1. In a specific embodiment, the seventh amino acid from the N-terminus of the parent CXCR3 ligand used in the method for producing a CXCR3 ligand with enhanced blood stability compared to its parent ligand, as provided herein, is V. In a specific embodiment, the parent CXCR3 ligand used in the method for producing a CXCR3 ligand with enhanced blood stability compared to its parent ligand, as provided herein, comprises a portion of the amino acid sequence of a native human CXCR3 ligand. In a specific embodiment, the native human CXCR3 ligand is native human CXCL10.

[0081] In a specific embodiment, the parent CXCR3 ligand used in the method provided herein for producing a CXCR3 ligand having enhanced blood stability relative to the parent ligand is selected from native CXCL10, native CXCL11, native CXCL9, a CXCL10 variant, a CXCL11 variant, a CXCL9 variant, and chimeric proteins prepared therefrom. In a specific embodiment, the parent CXCR3 ligand used in the method provided herein for producing a CXCR3 ligand having enhanced blood stability relative to the parent ligand is resistant to Furin protease. In a specific embodiment, the parent CXCR3 ligand used in the method provided herein for producing a CXCR3 ligand having enhanced blood stability relative to the parent ligand is a CXCL10 variant obtained by adding an R75A alteration to the amino acid sequence of native human CXCL10.

[0082] In certain embodiments, the method provided herein for producing a CXCR3 ligand having enhanced blood stability compared to the parent ligand further comprises the step of modifying the amino acid sequence of the parent CXCR3 ligand by at least one or both of (i) introducing a disulfide bond and (ii) modifying an amino acid in the N-terminal region. In certain embodiments, the method provided herein for producing a CXCR3 ligand having enhanced blood stability compared to the parent ligand comprises introducing at least one amino acid substitution of V1Y, or P18C and A60C, into the amino acid sequence of the parent CXCR3 ligand. In addition to the above, the method provided herein for producing a CXCR3 ligand having enhanced blood stability compared to the parent ligand may further comprise substituting P with V at the second amino acid from the N-terminus of the parent CXCR3 ligand.

[0083] Fusion Protein Comprising a CXCR3 Ligand One embodiment of the present disclosure relates to a fusion protein comprising a CXCR3 ligand. In a specific embodiment, the fusion protein of the present disclosure relates to a fusion protein comprising a CXCR3 ligand at the N-terminus. The fusion protein of the present disclosure may be a fusion protein in which a CXCR3 ligand is fused to an antibody, or a fusion protein in which a CXCR3 ligand is fused to another protein or polypeptide, such as an antibody Fc region or albumin. Herein, a molecule that binds to a CXCR3 ligand may be referred to as a ligand-binding molecule. Specific examples include fusion proteins in which an antibody (including a complete antibody, a full-length antibody, or an antibody fragment), an antibody Fc region, or another protein or polypeptide, such as albumin, is fused to the C-terminus of a CXCR3 ligand. Fusion proteins comprising a CXCR3 ligand can be purified using a substance that binds to the fused protein. For example, when fused to an antibody Fc region, the fusion protein comprising a CXCR3 ligand can be recovered by adsorption to immobilized protein A.

[0084] The ligand-binding molecule contained in the fusion protein of the present disclosure may be a polypeptide having at least one cleavage site. That is, the fusion protein of the present disclosure may be a fusion protein in which an antibody (including a complete antibody, a full-length antibody, an antibody fragment, etc.) having at least one cleavage site, an antibody Fc region, albumin, or other protein or polypeptide is fused to the C-terminus of a CXCR3 ligand. Ligand-binding molecules containing a cleavage site are described, for example, in WO2018 / 097308 and WO2019 / 107384. The cleavage site may be located anywhere in the polypeptide as long as cleavage attenuates the binding of the ligand-binding molecule to the ligand. Furthermore, the polypeptide may contain one or more cleavage sites. For example, the ligand-binding molecule may be a complete antibody or antibody fragment having at least one cleavage site, and when the complete antibody or antibody fragment is cleaved at at least one cleavage site, its binding to a CXCR3 ligand or a polypeptide having a sequence disclosed herein is attenuated. For example, when the cleavage site is cleaved, a CXCR3 ligand or a polypeptide having a sequence disclosed herein is released from the intact antibody or antibody fragment. The cleavage site may include, for example, a protease cleavage sequence. The protease may be, for example, a target tissue-specific protease, such as a cancer tissue-specific protease or an inflammatory tissue-specific protease. Examples of proteases include metalloproteases, serine proteases, aspartic acid proteases, cysteine ​​proteases, and threonine proteases, including, but not limited to, matriptase and urokinase (uPA). Flexible linkers may be added to one or both ends of the cleavage site or protease cleavage sequence. For example, the intact antibody or antibody fragment has an antibody constant region, an antibody VH, and an antibody VL, and the cleavage site or protease cleavage sequence is located near the boundary between the antibody constant region and the antibody VH and / or near the boundary between the antibody constant region and the antibody VL.The cleavage site or the protease cleavage sequence may be inserted into at least one of the following: (i) any position in the sequence from amino acid 109 (Kabat numbering) of the antibody VH to amino acid 122 (EU numbering) of the antibody heavy chain constant region; and (ii) any position in the sequence from amino acid 104 (Kabat numbering) of the antibody VL to amino acid 113 (EU numbering) of the antibody light chain constant region. For example, the antibody VL and antibody VH of a complete antibody or antibody fragment are associated, and this association is disrupted by cleavage of the cleavage site or the protease cleavage sequence with a protease. Furthermore, a CXCR3 ligand or a polypeptide having a sequence disclosed herein may be fused to an antibody Fc region, or a CXCR3 ligand or a polypeptide having a sequence disclosed herein may be fused to a complete antibody or antibody fragment via a linker.

[0085] In the fusion protein disclosed herein, the CXCR3 ligand and the fusion partner can be fused via a linker. The linker used to fuse the CXCR3 ligand and the fusion partner can be any peptide linker that can be introduced by genetic engineering or a synthetic compound linker (e.g., the linker disclosed in Protein Engineering, 9 (3), 299-305, 1996). The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose.By way of example, and not limitation, for peptide linkers: SerGly.Ser(GS) SerGly(SG) Gly.Gly.Ser(GGS) Gly.Ser.Gly(GSG) Ser.Gly.Gly(SGG) Gly.Ser.Ser(GSS) Ser.Ser.Gly(SSG) Ser.Gly.Ser(SGS) Gly.Gly.Gly.Ser(GGGS, SEQ ID NO:29) Gly.Gly.Ser.Gly(GGSG, SEQ ID NO:30) Gly.Ser.Gly.Gly(GSGG, SEQ ID NO:31) Ser.Gly.Gly.Gly(SGGG, SEQ ID NO:32) Gly.Ser.Ser.Gly(GSSG, SEQ ID NO:33) Gly.Gly.Gly.Gly.Ser(GGGGS, SEQ ID NO:34) Gly.Gly.Gly.Ser.Gly(GGGSG, SEQ ID NO:35) Gly.Gly.Ser.Gly.Gly.Gly (GGSGG, SEQ ID NO: 36), Gly.Ser.Gly.Gly.Gly (GSGGG, SEQ ID NO: 37), Gly.Ser.Gly.Gly.Gly.Ser (GSGGS, SEQ ID NO: 38), Ser.Gly.Gly.Gly.Gly (SGGGG, SEQ ID NO: 39), Gly.Ser.Ser.Gly.Gly (GSSGG, SEQ ID NO: 40), Gly.Ser.Gly.Ser.Gly (GSGSG, SEQ ID NO: 41), Ser.Gly.Gly.Ser.Gly (SGGSG, SEQ ID NO: 42), Gly.Ser.Ser.Ser.Gly (GSSSG, SEQ ID NO: 43), Gly.Gly.Gly.Gly.Gly.Ser (GGGGGS, SEQ ID NO: 44), Ser.Gly.Gly.Gly.Gly.Gly (SGGGGG, SEQ ID NO: 45). Examples include Gly.Gly.Gly.Gly.Gly.Gly.Gly.Ser (GGGGGGS, SEQ ID NO: 46), Ser.Gly.Gly.Gly.Gly.Gly.Gly (SGGGGGG, SEQ ID NO: 47), (Gly.Gly.Gly.Gly.Ser (GGGGS, SEQ ID NO: 34))n (Ser.Gly.Gly.Gly.Gly (SGGGG, SEQ ID NO: 39))n [n is an integer of 1 or more].However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0086] The synthetic compound linkers (chemical cross-linkers) are cross-linkers commonly used for cross-linking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), and these cross-linkers are commercially available.

[0087] Antibodies The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0088] Antibody Fragment An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0089] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0090] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of an antibody (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FR) and three complementarity-determining regions (CDR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). One VH or VL domain may be sufficient to confer antigen-binding specificity.

[0091] Fc Region: The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residues of the Fc region may be present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991. In the present invention, the Fc region may include various modifications. For example, modifications to increase the yield of Fc hetero-associated molecules and modifications to suppress binding to FcγR are known.

[0092] Nucleic Acid / Polynucleotide An "isolated" nucleic acid / polynucleotide refers to a nucleic acid / polynucleotide molecule that has been separated from a component of its original environment. An isolated nucleic acid / polynucleotide includes a nucleic acid / polynucleotide molecule contained in a cell that normally contains the nucleic acid / polynucleotide molecule, but where the nucleic acid / polynucleotide molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0093] The present disclosure also relates to nucleic acids / polynucleotides encoding CXCR3 ligands or nucleic acids / polynucleotides encoding fusion proteins containing the CXCR3 ligands. A "nucleic acid / polynucleotide encoding a CXCR3 ligand" refers to one or more nucleic acid molecules encoding a CXCR3 ligand, including nucleic acid molecules carried on a single vector or separate vectors and nucleic acid molecules present at one or more locations in a host cell. A "nucleic acid / polynucleotide encoding a CXCR3 ligand" can be operably linked to an expression control region. Expression control regions include promoters, enhancers, terminators, etc. The expression control region linked to the nucleic acid / polynucleotide is generally of heterologous origin. Alternatively, if necessary, the expression control region of a gene originally encoding a natural CXCR3 ligand can be combined.

[0094] Vectors. The present disclosure also relates to vectors containing a nucleic acid / polynucleotide encoding a CXCR3 ligand or a nucleic acid / polynucleotide encoding a fusion protein containing the CXCR3 ligand. As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which it is introduced. A vector can mediate the expression of a nucleic acid to which it is operatively linked. Such vectors are also referred to herein as "expression vectors." In an expression vector, the "nucleic acid / polynucleotide encoding a CXCR3 ligand" can be operably linked to an expression control region. Expression control regions include promoters, enhancers, terminators, etc. The expression control region linked to the nucleic acid / polynucleotide is generally of heterologous origin. Alternatively, the expression control region of a gene encoding a natural CXCR3 ligand can be combined, if necessary.

[0095] Host Cells The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0096] The present disclosure also relates to host cells comprising a nucleic acid / polynucleotide encoding a CXCR3 ligand or a nucleic acid / polynucleotide encoding a fusion protein comprising said CXCR3 ligand.

[0097] Method for Producing CXCR3 Ligands The polynucleotides disclosed herein are typically carried (inserted) into an appropriate vector and then introduced into host cells. The vector is not particularly limited as long as it stably retains the inserted nucleic acid. For example, if Escherichia coli is used as the host, a cloning vector such as the pBluescript vector (Stratagene) is preferred, although various commercially available vectors can also be used. Expression vectors are particularly useful when used to produce the CXCR3 ligands of the present disclosure or fusion proteins containing the CXCR3 ligands. Expression vectors are not particularly limited as long as they express the ligand-binding molecules in vitro, in Escherichia coli, cultured cells, or individual organisms. For example, the pBEST vector (Promega) for in vitro expression, the pET vector (Invitrogen) for Escherichia coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) for individual organisms are preferred. Insertion of the DNA of the present disclosure into a vector can be performed by standard methods, such as ligation using a restriction enzyme site (Current Protocols in Molecular Biology, ed., Ausubel et al. (1987) Published by John Wiley & Sons, Sections 11.4-11.11). The host cell is not particularly limited, and various host cells can be used depending on the purpose. Examples of cells for expressing a CXCR3 ligand or fusion protein include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (e.g., yeast, Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells), and plant cells.Vectors can be introduced into host cells by known methods, such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology, ed., Ausubel et al. (1987) Publish. John Wiley & Sons, Sections 9.1-9.9), lipofectamine (GIBCO-BRL), or microinjection. To secrete a CXCR3 ligand or fusion protein expressed in host cells into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, an appropriate secretion signal can be incorporated into the target ligand-binding molecule or fusion protein. These signals may be endogenous or heterologous to the target ligand-binding molecule or fusion protein. Specific examples of signal sequences that can be used include MNQTAILICCLIFLTLSGIQG (SEQ ID NO: 48), MKKSGVLFLLGIILLVLIGVQG (SEQ ID NO: 49), MSVKGMAIALAVILCATVVQG (SEQ ID NO: 50), and MGWSCIILFLVATATGVHS (SEQ ID NO: 52). Among these signal sequences, SEQ ID NOS: 48-50 are derived from human CXCL10, human CXCL9, and human CXCL11, respectively. Regarding recovery of the CXCR3 ligand or fusion protein in the above production method, if the CXCR3 ligand or fusion protein of the present disclosure is secreted into the culture medium, the culture medium is recovered. If the CXCR3 ligand or fusion protein of the present disclosure is produced intracellularly, the cells are first lysed, and then the CXCR3 ligand or fusion protein is recovered. To recover and purify the CXCR3 ligand or fusion protein of the present disclosure from recombinant cell culture, known methods can be used, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography.

[0098] The CXCR3 ligands provided herein can also be produced by enhancing the CXCR3-expressing cell migration activity of a parent CXCR3 ligand. In certain embodiments, the methods for producing CXCR3 ligands provided herein include modifying the sequence of the parent CXCR3 ligand. In certain embodiments, the methods for producing CXCR3 ligands provided herein include modifying the amino acid sequence of the parent CXCR3 ligand by at least one or both of (i) introducing a disulfide bond and (ii) substituting an amino acid in the N-terminal region.

[0099] In certain embodiments, the first amino acid from the N-terminus of a parent CXCR3 ligand used in the methods for producing a CXCR3 ligand provided herein is V. In certain embodiments, the second amino acid from the N-terminus of a parent CXCR3 ligand used in the methods for producing a CXCR3 ligand provided herein is P. In more specific embodiments, the N-terminal sequence of a parent CXCR3 ligand used in the methods for producing a CXCR3 ligand provided herein is VPL or VVL. In an even more specific embodiment, the N-terminal sequence of a parent CXCR3 ligand used in the methods for producing a CXCR3 ligand provided herein is VPLSRT (SEQ ID NO: 19) or YVLSRT (SEQ ID NO: 14).

[0100] In certain embodiments, the parent CXCR3 ligand used in the methods for producing CXCR3 ligands provided herein has a CXC motif. Each of the two cysteines in the CXC motif can form a disulfide bond with a cysteine ​​other than the CXC motif in the parent CXCR3 ligand. The CXC motif in the parent CXCR3 ligand can be selected from CTC (Cys-Thr-Cys), CLC (Cys-Leu-Cys), and CSC (Cys-Ser-Cys). In specific embodiments, the CXC motif in the parent CXCR3 ligand can be located adjacent to the C-terminus of the N-terminal sequence VPLSRTVR (SEQ ID NO: 21) or VVLSRTVR (SEQ ID NO: 22). In certain embodiments, the parent CXCR3 ligand used in the methods for producing CXCR3 ligands provided herein comprises a portion of the amino acid sequence of a native human CXCR3 ligand. In certain embodiments, the parent CXCR3 ligand used in the methods for producing CXCR3 ligands provided herein comprises a portion of the amino acid sequence of human CXCL10.

[0101] In certain embodiments, the parent CXCR3 ligand used in the methods for producing CXCR3 ligands provided herein is selected from native CXCL10, native CXCL11, native CXCL9, CXCL10 variants, CXCL11 variants, CXCL9 variants, and chimeric proteins made therefrom.

[0102] In the present invention, when a CXCR3 ligand is obtained by expression of a polynucleotide encoding it, an "amino acid substitution" can be introduced by substituting a codon in the polynucleotide. Alternatively, when a CXCR3 ligand is synthesized by linking amino acid residues, a CXCR3 ligand containing an amino acid substitution can be obtained by linking the amino acid residues for substitution. In a specific embodiment, the method for producing a CXCR3 ligand provided herein can further additionally include a step of recovering or isolating the CXCR3 ligand containing the modification.

[0103] Treatment As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the CXCR3 ligands of the present disclosure are used to delay disease onset or slow disease progression.

[0104] Pharmaceutical Composition The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredients contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0105] Pharmaceutically acceptable carriers: A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0106] Pharmaceutical compositions comprising a CXCR3 ligand or a fusion protein comprising a CXCR3 ligand The present disclosure also relates to pharmaceutical compositions (medicines) comprising a CXCR3 ligand of the present disclosure and a pharmaceutically acceptable carrier, and pharmaceutical compositions (medicines) comprising a fusion protein comprising a CXCR3 ligand of the present disclosure and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical compositions of the present disclosure are for treating diseases, including, but not limited to, cancer, tumors, and inflammatory diseases.

[0107] In the present disclosure, the term "pharmaceutical composition comprising a CXCR3 ligand" can be rephrased as "a method for treating a disease, comprising administering a CXCR3 ligand to a subject," or as "use of a CXCR3 ligand in the manufacture of a medicament for treating a disease." The term "pharmaceutical composition comprising a CXCR3 ligand" can be rephrased as "use of a CXCR3 ligand for treating a disease," or as "use of a CXCR3 ligand in the treatment of a disease." The CXCR3 ligands of the present disclosure, fusion proteins comprising a CXCR3 ligand of the present disclosure, and pharmaceutical compositions of the present disclosure can be used to treat diseases, including, but not limited to, cancer, tumors, and inflammatory diseases. The term "pharmaceutical composition comprising a fusion protein comprising a CXCR3 ligand" can be rephrased as "a method for treating a disease, comprising administering a fusion protein comprising a CXCR3 ligand to a subject," or as "use of a fusion protein comprising a CXCR3 ligand in the manufacture of a medicament for treating a disease." The term "pharmaceutical composition comprising a fusion protein comprising a CXCR3 ligand" can also be rephrased as "use of a fusion protein comprising a CXCR3 ligand for treating a disease" or "use of a fusion protein comprising a CXCR3 ligand in treating a disease." Furthermore, the present invention relates to a "method for producing a pharmaceutical composition for treating a disease, comprising the step of combining (or mixing) a CXCR3 ligand or a fusion protein comprising the same with a pharmaceutically acceptable carrier." In a specific embodiment, the disease may be one that requires administration of a CXCR3 ligand or a fusion protein comprising the same for its treatment.

[0108] In some embodiments of the present disclosure, a composition comprising a CXCR3 ligand can be administered to an individual. In some embodiments of the present disclosure, a fusion protein comprising a CXCR3 ligand can be administered to an individual. When a CXCR3 ligand or a fusion protein comprising the same is administered to a subject for the treatment of a disease, these active ingredients can be administered in a therapeutically effective amount. That is, the present invention can include a step of administering a therapeutically effective amount of a CXCR3 ligand or a fusion protein comprising the same to a subject in need of disease treatment.

[0109] The pharmaceutical compositions of the present disclosure can be formulated using methods known to those skilled in the art. For example, they can be used parenterally in the form of injections of sterile solutions or suspensions in water or other pharmaceutically acceptable liquids. For example, they can be formulated by appropriately combining them with pharmacologically acceptable carriers or vehicles, specifically, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., and blending them into unit dosage forms required for generally accepted pharmaceutical practice. The amount of active ingredient in these preparations is set so that an appropriate volume within the indicated range is obtained.

[0110] Sterile compositions for injection can be formulated according to standard pharmaceutical practice using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose, or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Suitable solubilizers, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., Polysorbate 80™, HCO-50), can be used in combination. Oily solutions include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol can also be used in combination as solubilizers. The compositions can also be formulated with buffers (e.g., phosphate buffer and sodium acetate buffer), soothing agents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solutions are usually filled into appropriate ampoules.

[0111] The pharmaceutical compositions of the present disclosure are preferably administered parenterally. For example, compositions may be administered in the form of injections, intranasal administration, pulmonary administration, or transdermal administration. Systemic or local administration may be achieved, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. The administration method may be appropriately selected depending on the patient's age and symptoms. The dosage of a pharmaceutical composition containing a CXCR3 ligand may be set, for example, in the range of 0.0001 mg to 1,000 mg per kg of body weight per administration. Alternatively, a dosage of 0.001 to 100,000 mg per patient may be set, although the present disclosure is not necessarily limited to these values. The dosage and administration method vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can determine an appropriate dosage and administration method taking these conditions into consideration.

[0112] The following are examples of the methods and compositions of the present disclosure. In light of the above general description, it will be understood that various other embodiments may be practiced.

[0113] Example 1. Construction of human CXCL10 (hCXCL10) variants and hCXCL10 variant Fc fusions. Human CXCL10 (hCXCL10, Refseq: NP_001556.2, Uniprot ID: P02778) was mutated to confer resistance to Furin protease, resulting in the hCXCL10 variant hCXCL10R75A (SEQ ID NO: 1). Human CXCL10 variants with modifications in the N-loop, C-terminal α-helix region, and / or N-terminal region were also constructed. The names, modifications, and SEQ ID NOs of the constructed hCXCL10 variants are shown in Table 2. When wild-type CXCL10 without the R75A modification is expressed in mammalian cells, this region is cleaved by cellular proteases, resulting in cleavage of the protein fused to the C-terminal region. Therefore, in order to express a CXCL10-protein fusion in which wild-type CXCL10 or a variant thereof is fused to a protein at the C-terminus, it is desirable to add the R75A modification.

[0114] In Table 2, the N-terminal sequence is any one of YPLSRTVR (SEQ ID NO: 15), YVLSRTVR (SEQ ID NO: 16), VPLSRTVR (SEQ ID NO: 21), and VVLSRTVR (SEQ ID NO: 22).

[0115] As previously mentioned, modifications contributing to the receptor binding step were investigated to enhance CXCR3 binding and activation of CXCR3-expressing cells. Modifications that stabilize the CXCR3-bound state were identified through analysis using the Molecular Operating Environment (MOE 2020.0901 win64) integrated computational science system. Specifically, the structural data for hCXCL10 (PDB ID: 1O7Y) was loaded into MOE, and structural stabilization due to modifications was calculated. Modifications that are thought to stabilize the structure around the docking domain were identified (Figure 1A). Potential disulfide bond introduction sites that would increase the stability of the modified variants compared to the parent CXCR3 ligand were identified as combinations of amino acids selected from positions 14 and 55, 18 and 60, 21 and 67, 25 and 46, and 41 and 56 from the N-terminus. Among these, we examined variants in which positions 18 and 60 were replaced with cysteines. Regarding the modification of the N-terminal region, which contributes to the activation step, we investigated substitution of amino acid residues in the N-terminal region, considering that the interaction site on the receptor side is the transmembrane domain, and that the interaction can be stabilized by adjusting the hydrophobicity and side chain size of the N-terminal region. Specifically, we investigated variants with V1Y substitutions in the N-terminal amino acids.

[0116] To facilitate the purification of various hCXCL10 variants, we constructed hCXCL10 variant Fc fusions by fusing the hCXCL10 variant with the Fc domain of human IgG1 antibody. A schematic diagram of the hCXCL10 variant Fc fusion is shown in Figure 1B.

[0117] A hCXCL10 variant Fc fusion was prepared by fusing the above-mentioned hCXCL10 variant with G1T4k.one / / VHn-G1T4h.one.H435R (G1T4k.one (sequence number: 2), VHn-G1T4h.one.H435R (sequence number: 3)), a variant of the Fc domain of human IgG1 antibody (hIgG1). Specifically, expression vectors encoding the genes for peptide chains linking the C-terminus of each hCXCL10 variant with the N-terminus of G1T4k.one in G1T4k.one / / VHn-G1T4h.one.H435R were prepared by methods known to those skilled in the art, and these peptide chains were combined with VHn-G1T4h.one.H435R. hCXCL10 variant Fc fusions in which one hCXCL10 variant was linked to an hIgG1 Fc domain variant were expressed by transient expression using Expi 293 (Life Technologies) by methods known to those skilled in the art, and purified by methods known to those skilled in the art using protein A. The Fc domain variants used in this study contained mutations to increase the yield of Fc hetero-associated molecules and mutations to suppress FcγR binding.

[0118] Example 2. Evaluation of cell migration activity of hCXCL10 variant Fc fusion The hCXCL10 variant Fc fusion prepared in Example 1 was evaluated to determine whether it induces migration of cells expressing the CXCL10 receptor, i.e., the chemotactic activity of CXCR3-expressing cells. The cell migration activity was evaluated using Ba / F3 transfectant cells (hereinafter referred to as BaF3 / mCXCR3) expressing mouse CXCR3 (mCXCR3) and HTS Transwell TM -96 Permeable Supports with 5.0 μm Pore Polycarbonate Membrane (Cat. 3387, Corning) was used for evaluation.

[0119] The following hCXCL10 variant Fc fusions prepared in Example 1 were used as analytes: hCXCL10R75A-G1T4k.one / / VHn-G1T4h.one.H435R, hCXCL10R75A.0639-G1T4k.one / / VHn-G1T4h.one.H435R, hCXCL10R75A.0640-G1T4k.one / / VHn-G1T4h.one.H435R, hCXCL10R75A.0641-G1T4k.one / / VHn-G1T4h.one.H435R, hCXCL10R75A.0642-G1T4k.one / / VHn-G1T4h.one.H435R, hCXCL10R75A.0016-G1T4k.one / / VHn-G1T4h.one.H435R, hCXCL10R75A.0643-G1T4k.one / / VHn-G1T4h.one.H435R, hCXCL10R75A.0644-G1T4k.one / / VHn-G1T4h.one.H435R, hCXCL10R75A.0669-G1T4k.one / / VHn-G1T4h.one.H435R, hCXCL10R75A.0670-G1T4k.one / / VHn-G1T4h.one.H435R.

[0120] The final concentrations of each analyte in the solution were adjusted to 3 nM, 10 nM, 100 nM, 300 nM, and 1000 nM, and 235 μL of each solution was transferred to the lower chamber. Then, 2.0 × 10 BaF3 / mCXCR3 cells were placed in the upper chamber. 5 The cells were seeded at 75 μL / well to achieve a total of 100 cells / well, and the reaction was carried out for 5 hours. The reaction was carried out under 5% carbon dioxide gas at 37°C. After 3 or 6 hours of reaction, 100 μL of the solution in the lower chamber was transferred to an OptiPlate-96 (Cat. 6005299, PerkinElmer) and incubated with CellTiter-Glo. TM100 μL of Luminescent Cell Viability Assay solution (Cat. G7571, Promega) was added. After 10 minutes of incubation at room temperature, luminescence was measured using a SpectraMax M3 multimode microplate reader (Molecular Devices) to assess the degree of cell migration to the lower chamber. The cell migration rate for each well was calculated as the relative value of the luminescence in the lower chamber to the luminescence obtained from the total number of cells loaded in the upper chamber (luminescence value / luminescence value for all cells).

[0121] The amount of cells that migrated to the lower chamber was reflected by the luminescence intensity. Both unmodified hCXCL10 and hCXCL10R75A-G1T4k.one / / VHn-G1T4h.one.H435R exhibited concentration-dependent cell migration activity, as described in WO2020116498A1.

[0122] (1) The cell migration activity of the P18C, A60C variant hCXCL10R75A-G1T4k.one / / VHn-G1T4h.one.H435R was compared with that of other hCXCL10 variant Fc fusions after 5 hours of incubation. The results are shown in Figure 2 and Table 3. Compared to hCXCL10R75A-G1T4k.one / / VHn-G1T4h.one.H435R, hCXCL10R75A.0642-G1T4k.one / / VHn-G1T4h.one.H435R, which contains the P18C / A60C variant, exhibited higher cell migration activity at concentrations of 3 nM, 10 nM, and 300 nM than hCXCL10R75A-G1T4k.one / / VHn-G1T4h.one.H435R at the same concentrations. On the other hand, the P18C and A60C modifications alone did not enhance activity, strongly suggesting that the enhancement of activity by the P18C / A60C modification is due to the introduction of an SS bond between amino acid positions 18 and 60 and the resulting stabilization of the surrounding region.

[0123]

[0124] (2) P18C, A60C, and P2V Modifications P2V has been reported as a modification introduced into hCXCL10 to confer resistance to DPPIV cleavage ( WO2020116498A1 ). Here, we confirmed the activity-enhancing effect of combining the P2V and P18C / A60C modifications. Compared to hCXCL10R75A-G1T4k.one / / VHn-G1T4h.one.H435R, hCXCL10R75A.0644-G1T4k.one / / VHn-G1T4h.one.H435R, which contained the P2V / P18C / A60C alterations, exhibited higher cell migration activity at concentrations of 3 nM, 10 nM, 300 nM, and 1000 nM than hCXCL10R75A-G1T4k.one / / VHn P18C / A60C alteration-G1T4h.one.H435R at the same concentrations (Fig. 3, Table 4). This indicates that the combined P18C / A60C and P2V alterations enhance cell migration activity. Furthermore, by combining the P18C / A60C and P2V modifications, a CXCL10 variant was created that was resistant to DPPIV cleavage and had an activity-enhancing effect.

[0125]

[0126] (3) V1Y variant hCXCL10R75A-G1T4k.one / / VHn-G1T4h.one.H435R, which has improved N-terminal hydrophobicity due to V1Y, exhibited higher cell migration activity at concentrations of 3 nM, 30 nM, 100 nM, and 300 nM than hCXCL10R75A-G1T4k.one / / VHn-G1T4h.one.H435R at the same concentrations. As mentioned above, P2V has been reported as a modification introduced into hCXCL10 to confer resistance to DPPIV cleavage. hCXCL10R75A.0643-G1T4k.one / / VHn-G1T4h.one.H435R, which contained the V1Y / P2V alterations, exhibited higher cell migration activity at concentrations of 3 nM, 30 nM, 100 nM, and 300 nM than hCXCL10R75A-G1T4k.one / / VHn-G1T4h.one.H435R at the same concentrations (Figure 4, Table 5). This indicates that the combination of the V1Y and P2V alterations enhances CXCL10 activity. Furthermore, the combination of the V1Y and P2V alterations produced a CXCL10 variant that was resistant to DPPIV cleavage and had enhanced activity.

[0127]

[0128] (4) V1Y, P18C, A60C variants. hCXCL10R75A.0642-G1T4k.one / / VHn-G1T4h.one.H435R, which combines the P18C / A60C and V1Y variants discovered in this study to introduce the V1Y / P18C / A60C variant, and hCXCL10R75A.0670-G1T4k.one / / VHn-G1T4h.one.H435R, which further combines this with the P2V variant to introduce the V1Y / P2V / P18C / A60C variant, exhibited potency of 3 nM, 10 nM, 30 nM, 100 nM, and 300 nM. At each nM concentration, it exhibited higher cell migration activity than hCXCL10R75A-G1T4k.one / / VHn-G1T4h.one.H435R at the same concentration (Figure 5, Table 6).

[0129]

[0130] hCXCL10R75A.0639-G1T4k.one / / VHn-G1T4h.one.H435R tended to exhibit higher cell migration activity than hCXCL10R75A-G1T4k.one / / VHn-G1T4h.one.H435R at low concentrations, and hCXCL10R75A.0642-G1T4k.one / / VHn-G1T4h.one.H435R at medium concentrations, whereas hCXCL10R75A.0670-G1T4k.one / / VHn-G1T4h.one.H435R exhibited an activity-enhancing effect over a wide concentration range, from low to medium.

[0131] These results suggest that the human CXCL10 variants contained in these human CXCL10 variant Fc fusions have higher activity than the unaltered parent CXCR3 ligand.

[0132] Example 3. Construction of human CXCL10 (hCXCL10) variants and hCXCL10 variant-antibody fusions for improved blood stability (3-1) Human CXCL10 (hCXCL10) variants for improved resistance to protease cleavage The N-terminus of hCXCL10 is known to be cleaved by various proteases, including DPP4, DPP8, DPP9, chathepsin K, chathepsin S, chathepsin L, MMP-9, and MMP-2, and such cleavage is known to reduce its activity as a CXCR3 agonist (Non-patent document: Bronger, H., Magdolen, V., Goettig, P. et al. Proteolytic chemokine cleavage as a regulator of lymphocytic infiltration in solid tumors. Cancer Metastasis Rev 38, 417-430 (2019)). Therefore, to maintain the activity of CXCL10, it is preferable to prevent unwanted cleavage of the hCXCL10 N-terminal region by proteases present in the blood, local tumors, inflammatory sites, etc. To improve the resistance of the hCXCL10 N-terminal region to proteases, we constructed hCXCL10R75A.0303 (SEQ ID NO: 55) by adding the V7P modification to hCXCL10R75A.0016.

[0133] (3-2) Construction of hCXCL10 variant-antibody fusions To enable the CXCL10 variants of the present invention to exert their effects at specific sites in the body (e.g., tumors), we attempted to construct fusions between the CXCL10 variants (ligands) and ligand-binding molecules with tunable binding activity. Ligand-binding molecules with tunable binding activity have already been reported (e.g., WO2019 / 107380, WO2019 / 107384). Using the DNA sequence encoding MabCXCL10_G7 (heavy chain: G7HFR0039H-G1T4h (SEQ ID NO: 56), light chain: G7L-LT0 (SEQ ID NO: 57)), a neutralizing antibody against human CXCL10, as a template, a DNA sequence encoding the anti-CXCL10 antibody heavy chain G7HFR0039H.12aa0054-G1T4h (SEQ ID NO: 58) was prepared by a method known to those skilled in the art, in which sequences cleaved by urokinase (uPA) and matriptase (MT-SP1), which are expressed in a cancer-specific manner, were inserted near the boundary between the variable and constant regions of G7HFR0039H-G1T4h. Furthermore, a linker sequence (SEQ ID NO: 66) consisting of a glycine-serine polymer was inserted at the boundary between the variable regions of the hCXCL10 mutants (hCXCL10R75A (SEQ ID NO: 1), hCXCL10R75A.0016 (SEQ ID NO: 8), and hCXCL10R75A.0303 (SEQ ID NO: 55)) and the anti-CXCL10 antibody heavy chain G7HFR0039H.12aa0054-G1T4h (hCXCL10R75A.G4SGGGG.G7HFR0039H.12aa0054-G1T4h (SEQ ID NO: 59), hCXCL10R75A.0016.G4SGGGG.G7HFR0039H.12aa0054-G1T4h) Expression vectors encoding hCXCL10R75A.0303.G4SGGGG.G7HFR0039H.12aa0054-G1T4h (SEQ ID NO: 60), hCXCL10R75A.0303.G4SGGGG.G7HFR0039H.12aa0054-G1T4h (SEQ ID NO: 61)) were constructed by methods known to those skilled in the art.Furthermore, to express hCXCL10-fused anti-hCXCL10 antibodies in which one hCXCL10 molecule is bound to each molecule, an expression vector encoding the heavy chain of an anti-keyhole limpet hemocyanin antibody (IC17HdK-G1T4k.H435R (SEQ ID NO: 62)) was constructed by a method known to those skilled in the art. The Fc variants used in the study contained mutations to increase the yield of Fc hetero-associated molecules and to suppress FcγR binding. These heavy chain variants and light chains are combined to form hCXCL10-fused anti-hCXCL10 antibodies: hCXCL10R75A.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0 (SEQ ID NO: 59, SEQ ID NO: 57, SEQ ID NO: 62) and hCXCL10R75A.0016.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0 (SEQ ID NO: 60, SEQ ID NO: 57, SEQ ID NO: 62). hCXCL10R75A.0303.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0 (SEQ ID NO: 61, SEQ ID NO: 57, SEQ ID NO: 62) were transiently expressed using Expi293 (Life Technologies) by a method known to those skilled in the art, and purified using Protein A by a method known to those skilled in the art.

[0134] Example 4. Evaluation of blood stability of hCXCL10 variant-antibody fusions (4.1) (P2V, V7P) A mouse PK study was performed to evaluate the blood stability of CXCL10. The following hCXCL10 fusion anti-hCXCL10 antibodies prepared in Example 3 were used as analytes: hCXCL10R75A.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0, hCXCL10R75A.0016.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0, hCXCL10R75A.0303.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0. Each antibody molecule was intravenously administered at 10 mg / kg to 6-week-old male C57BL / 6J mice (Jackson Laboratories Japan, Ltd.) in triplicate. Blood samples were collected from the jugular vein at 5 minutes, 7 hours, 1 day, 3 days, and 7 days, or at 5 minutes, 30 minutes, 1 hour, 4 hours, 1 day, and 5 days after administration. The collected blood was centrifuged at 4000 g for 10 minutes at 4°C, and the supernatant was collected as plasma. Plasma antibody concentrations were measured by LC / ESI-MS / MS. Standard curve samples were prepared using mouse plasma at concentrations of 0.5, 1.0, 2.0, 4.0, 8.0, 16, and 32 μg / mL or 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16, and 32 μg / mL. 3 μL of the standard curve samples or mouse plasma samples was mixed with 50 μL of magnetic beads (prepared in-house) containing immobilized anti-human Fc region antibodies suspended in 50 mmol / L ammonium bicarbonate or LowCross-buffer (Candor Bioscience GmbH, 100 500). The beads were washed three times with PBS containing 0.05% Tween-20 and once with PBS, mixed with 25 μL of protein denaturing solution (8 mmol / L dithiothreitol, 7.5 mol / L urea, 99 ng / mL lysozyme in 50 mmol / L ammonium bicarbonate), and then incubated at 56°C for 45 minutes.After adding 2 μL of 500 mmol / L iodoacetamide and incubating in the dark at 37°C for 30 minutes, 160 μL of 0.621 μg / mL sequencing-grade modified trypsin (Promega, V5117) in 50 mmol / L ammonium bicarbonate was added and incubated overnight at 37°C for trypsin digestion. The reaction was stopped by adding 5 μL of 10% TFA, and an 80 μL sample was collected and subjected to LC / ESI-MS / MS analysis. LC / ESI-MS / MS was performed using a Xevo TQ-S tandem quadrupole mass spectrometer (Waters) equipped with an Acquity I-class or I-class plus 2D high-performance liquid chromatograph (Waters). Antibody-derived peptides produced by trypsin digestion were quantified by selected reaction monitoring (SRM). The peptide sequences and SRMs used for quantification are shown in Table 7. Using Masslynx Ver. 4.2 (Waters) software, the peak area was weighted 1 / x relative to the antibody concentration. 2 Sample quantification was performed using a calibration curve obtained by linear regression using the formula: The quantified peptide concentration was divided by the peptide concentration of the antibody CH region to calculate the remaining rate of each sequence site.

[0135] The results are shown in Figure 7 and Table 8. Compared with hCXCL10R75A.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0, hCXCL10R75A.0016.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0, which had P2V modifications, showed a significantly higher mean cell death rate on Day 1 and Day 2 compared with hCXCL10R75A.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0. At each time point on Day 5, hCXCL10R75A.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0 showed a higher hCXCL10 N-terminal residual rate. Furthermore, hCXCL10R75A.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0, which contained the P2V / V7P alteration, showed a significantly higher mean ... At each time point on Day 5, hCXCL10R75A.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0 showed a higher hCXCL10 N-terminal residual rate than hCXCL10R75A.0016.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0.

[0136] These results demonstrate that hCXCL10 containing P2V and / or V7P modifications has improved stability in the blood compared with hCXCL10 lacking these modifications. This is particularly effective when hCXCL10 is fused to a peptide or antigen-binding molecule having a cleavage site that can be cleaved by a tumor-specific or inflammation-specific protease. Specifically, hCXCL10 containing P2V and / or V7P modifications has improved stability in the blood compared with hCXCL10 lacking these modifications. These modifications prevent hCXCL10 from being cleaved by nonspecific proteases in the blood. This allows, for example, hCXCL10 variant-antibody fusions to reach the target organ, lesion, tumor, or other site without cleavage of the hCXCL10 variant in the blood, allowing the hCXCL10 variant to exert its effects at the target site (e.g., after the antibody fused to the hCXCL10 variant is cleaved by a site-specific protease and released from the antibody).

[0137]

[0138]

[0139] Example 5. Evaluation of cell migration activity of hCXCL10 variant Fc fusions. In Example 4 and above, V7P was identified as a modification to be introduced into hCXCL10 for the purpose of improving blood stability. Here, the effect of combining the V7P modification with the V1Y / P2V / P18C / A60C modification to enhance cell migration activity was examined. hCXCL10R75A.0670, in which the DPPIV-resistant P2V modification and the activity-enhancing V1Y / P18C / A60A modification were introduced into hCXCL10R75A, was compared with hCXCL10R75A.0613 (SEQ ID NO: 63), in which the V7P modification was further introduced. A hCXCL10 variant Fc fusion was prepared by fusing this hCXCL10 variant with G1T4k.one / / VHn-G1T4h.one.H435R (G1T4k.one (sequence number: 2), VHn-G1T4h.one.H435R (sequence number: 3)), a variant of the Fc domain of human IgG1 antibody (hIgG1). Specifically, expression vectors encoding genes for peptide chains linking the C-terminus of each hCXCL10 variant with the N-terminus of G1T4k.one in G1T4k.one / / VHn-G1T4h.one.H435R were prepared by methods known to those skilled in the art, and these peptide chains were combined with VHn-G1T4h.one.H435R to produce hCXCL10 variant Fc fusions in which one hCXCL10 variant was linked to an hIgG1 Fc domain variant. These were expressed by transient expression using Expi 293 (Life Technologies) by methods known to those skilled in the art, and purified by methods known to those skilled in the art using protein A. The Fc domain variants used in this study contained mutations to increase the yield of Fc hetero-associated molecules and mutations to suppress FcγR binding.

[0140] hCXCL10R75A.0613-G1T4k.one / / VHn-G1T4h.one.H435R exhibited cell migration activity at concentrations of 3 nM, 10 nM, 30 nM, 100 nM, and 300 nM that was comparable to or superior to that of hCXCL10R75A.0670-G1T4k.one / / VHn-G1T4h.one.H435R at the same concentrations (Fig. 8, Table 9). This indicates that the V7P modification does not inhibit the activity-enhancing effect even when combined with the DPPIV-resistant P2V modification and the activity-enhancing V1Y / P18C / A60A modification.

[0141]

[0142] Example 6 Evaluation of Blood Stability of hCXCL10 Variant-Antibody Fusion Proteins (6.1) Using the DNA sequence encoding hCXCL10R75A.0303.G4SGGGG.G7HFR0039H.12aa0054-G1T4h (SEQ ID NO: 61) as a template, a DNA sequence encoding hCXCL10R75A.0659.G4SGGGG.G7HFR0039H.0004.N0222-G1T4h (SEQ ID NO: 64) was prepared by methods known to those skilled in the art. Compared to hCXCL10R75A.0303.G4SGGGG.G7HFR0039H.0004.N0222-G1T4h, hCXCL10R75A.0659.G4SGGGG.G7HFR0039H.0004.N0222-G1T4h, the activity-enhancing modifications P18C / A60C have been added to the hCXCL10 portion. Modifications have also been made to the protease recognition sequence inserted between the variable and constant regions and to the heavy chain variable region of MabCXCL10_G7. However, these modifications are intended to improve the cleavage efficiency of the protease recognition sequence and the release efficiency of hCXCL10 after cleavage, respectively, and do not contribute to the stability of the hCXCL10 N-terminus. Light chain: Using the DNA sequence encoding G7L-LT0 (SEQ ID NO: 57) as a template, a DNA sequence encoding G7L.R38E-LT0 (SEQ ID NO: 65) was prepared by methods known to those skilled in the art. The modifications introduced into the MabCXCL10_G7 light chain were also intended to improve the efficiency of hCXCL10 release after heavy chain cleavage and do not contribute to the stability of the hCXCL10 N-terminus. These heavy chain variants were combined with the light chain and IC17HdK-G1T4k.H435R (sequence number: 62) to obtain the hCXCL10-fused anti-hCXCL10 antibody: hCXCL10R75A.0659.G4SGGGG.G7HFR0039H.0004.N0222-G1T4h / G7L.R38E-LT0 / / IC17HdK-G1T4k.H435R / G7L.R38E-LT0 (sequence number: 63). This antibody was transiently expressed using Expi293 (Life Technologies) by a method known to those skilled in the art, and purified using protein A by a method known to those skilled in the art.

[0143] (6.2) A mouse PK study was conducted to evaluate the blood stability of CXCL10. The experiment was performed as described in 4.1. Blood samples were collected 5 minutes, 1 hour, 7 hours, 1 day, and 5 days after administration. The following hCXCL10 fusion anti-hCXCL10 antibody prepared in Example 6.1 was used as the analyte: hCXCL10R75A.0659.G4SGGGG.G7HFR0039H.0004.N0222-G1T4h / G7L.R38E-LT0 / / IC17HdK-G1T4k.H435R / G7L.R38E-LT0.

[0144] The results are shown in Figure 9 and Table 10. hCXCL10R75A.0659.G4SGGGG.G7HFR0039H.0004.N0222-G1T4h / G7L.R38E-LT0 / / IC17HdK-G1T4k.H435R / G7L.R38E-LT0 showed a similar hCXCL10 N-terminal retention rate to hCXCL10R75A.0303.G4SGGGG.G7HFR0039H.12aa0054-G1T4h / G7L-LT0 / / IC17HdK-G1T4k.H435R / G7L-LT0 at both Day 1 and Day 5. This indicates that hCXCL10 N-terminal stabilization by the P2V and / or V7P modifications can be combined with the P18C / A60C modification.

[0145]

[0146] While the foregoing invention has been described in detail by way of illustration and illustration for purposes of clarity of understanding, the descriptions and illustrations herein should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties.

[0147] The present disclosure provides a CXCR3 ligand having chemotactic activity for CXCR3-expressing cells, which is useful for treating / preventing diseases by inducing the migration of CXCR3-expressing cells.

Claims

1. A CXCR3 ligand having enhanced CXCR3-expressing cell migration activity compared to the parent CXCR3 ligand, wherein the amino acid sequence of the parent CXCR3 ligand is modified by at least one of (i) introducing a disulfide bond and (ii) modifying an amino acid in the N-terminal region, or both of these modifications; A CXCR3 ligand, wherein the amino acid modification in the N-terminal region comprises substituting the amino acid at the first position from the N-terminus with Tyr.

2. The CXCR3 ligand described in claim 1, wherein the parent CXCR3 ligand comprises at least a portion of the amino acid sequence of a natural human CXCR3 ligand.

3. The CXCR3 ligand according to claim 2, wherein the natural human CXCR3 ligand is natural human CXCL10.

4. The CXCR3 ligand according to claim 2, wherein the disulfide bond is introduced by substituting Cys at amino acids corresponding to the following amino acid positions (i) and (ii) in the amino acid sequence of the native human CXCR3 ligand: (i) at least one selected from the group consisting of positions 18, 14, 21, 25, and 41; (ii) at least one selected from the group consisting of positions 60, 55, 67, 46, and 56; 5. The CXCR3 ligand of claim 2, wherein the disulfide bond is introduced at an amino acid position corresponding to at least one selected from the group consisting of combinations of the following amino acid positions in the amino acid sequence of the native human CXCR3 ligand: (i) 18th and 60th places; (ii) 14th and 55th positions; (iii) positions 21 and 67; (iv) positions 25 and 46; and (v) 41st and 56th places.

6. The CXCR3 ligand according to claim 2, wherein the amino acid at the position corresponding to the 18th amino acid from the N-terminus in the amino acid sequence of the native human CXCR3 ligand has been substituted with Cys, and the amino acid at the position corresponding to the 60th amino acid has been substituted with Cys.

7. A CXCR3 ligand as described in claim 2, further comprising a substitution of Val for the amino acid at the second amino acid position from the N-terminus in the amino acid sequence of the natural human CXCR3 ligand.

8. A CXCR3 ligand according to claims 2 and 7, further comprising a substitution of Ala for the amino acid at the position corresponding to the 75th amino acid from the N-terminus in the amino acid sequence of the native human CXCR3 ligand.

9. The CXCR3 ligand described in claim 8, further comprising a substitution of Pro for the amino acid at the seventh position from the N-terminus in the amino acid sequence of the natural human CXCR3 ligand.

10. The CXCR3 ligand according to claim 1, further comprising at least one modification selected from amino acid substitution, deletion, and insertion.

11. A polypeptide in which the amino acids at positions 18 and 60 of the amino acid sequence of native human CXCL10 or a CXCL10 variant, where the N-terminal amino acid of native human CXCL10 is defined as position 1, are substituted with Cys.

12. The polypeptide according to claim 11, wherein the N-terminal amino acid (position 1) of the amino acid sequence of native human CXCL10 or a CXCL10 variant has been substituted with Tyr.

13. A polypeptide described in claim 12, in which the amino acid corresponding to amino acid position 2 of the amino acid sequence of native human CXCL10 or a CXCL10 variant, when the N-terminal amino acid of native human CXCL10 is positioned 1, is further replaced with Val.

14. A polypeptide described in any of claims 11, 12, or 13, in which the amino acid corresponding to amino acid position 75, when the N-terminal amino acid of native human CXCL10 is positioned as 1, of the amino acid sequence of native human CXCL10 or a CXCL10 variant, has been further substituted with Ala.

15. A polypeptide described in claim 14, in which the amino acid sequence of native human CXCL10 or a CXCL10 variant further includes a substitution of Pro for the amino acid corresponding to amino acid position 7, where the N-terminal amino acid of native human CXCL10 is considered to be position 1.

16. A fusion protein comprising the CXCR3 ligand described in claim 10.

17. A fusion protein comprising the polypeptide described in claim 15.

18. A pharmaceutical composition comprising the CXCR3 ligand described in claim 10.

19. A pharmaceutical composition comprising the polypeptide described in claim 15.

20. A pharmaceutical composition comprising the fusion protein described in claim 16.

21. A pharmaceutical composition comprising the fusion protein described in claim 17.

22. A method for enhancing the CXCR3-expressing cell migration activity of a CXCR3 ligand, comprising the step of making at least one or both of the following modifications to the amino acid sequence of a parent CXCR3 ligand: (i) introducing a disulfide bond, and (ii) modifying an amino acid in the N-terminal region.