Antibodies binding to vista at acidic pH
The present application relates to antibodies specifically binding to the V-domain immunoglobulin-containing suppressor of T-cell activation (VISTA) at acidic pH and their use in cancer treatment. In some embodiments, the antibodies bind specifically to human VISTA at acidic pH, but do not significantly bind to human VISTA at neutral or physiological pH.
1 . An antibody that specifically binds to human VISTA (hVISTA), wherein the antibody comprises a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDR1), CDR2 and CDR3 comprising the amino acid sequence of residues 26-35, 50-56, and 99-110, respectively, of SEQ ID NO: 570, 574, 578, 582, 586, 590, 594, 598, 602, 606, 610, 614, 618, 622, 626, 630, 634, 638, 642, 646, 650, 654, 658, 662, 666, 670, 674, 678, 682, or 686, and a light chain variable region (VL) comprising the amino acid sequence of residues 24-35, 51-57, and 90-98, respectively, of SEQ ID NO: 96.
2 . The antibody of claim 1 , wherein the antibody comprises a VH comprising an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 570, 574, 578, 582, 586, 590, 594, 598, 602, 606, 610, 614, 618, 622, 626, 630, 634, 638, 642, 646, 650, 654, 658, 662, 666, 670, 674, 678, 682, or 686.
3 . The antibody of claim 1 , wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 570, 574, 578, 582, 586, 590, 594, 598, 602, 606, 610, 614, 618, 622, 626, 630, 634, 638, 642, 646, 650, 654, 658, 662, 666, 670, 674, 678, 682, or 686.
4 . The antibody of claim 1 , wherein the antibody comprises a VL comprising an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 96.
5 . The antibody of claim 1 , wherein the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 96 or of SEQ ID NO: 96 modified by a T85V substitution.
6 . The antibody of claim 1 , wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 182, 183, or 184.
7 . The antibody of claim 1 , wherein the antibody comprises a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 98, or of SEQ ID NO: 98 modified by a T85V substitution.
8 . The antibody of claim 1 , wherein the antibody binds near the histidine rich region of h VISTA.
9 . The antibody of claim 1 , wherein the antibody binds near the histidine rich region of h VISTA in conditions having a pH of 6.0-6.5.
10 . The antibody of claim 1 , wherein the antibody competes or cross-competes for binding to hVISTA with an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 570, 574, 578, 582, 586, 590, 594, 598, 602, 606, 610, 614, 618, 622, 626, 630, 634, 638, 642, 646, 650, 654, 658, 662, 666, 670, 674, 678, 682, or 686; and a VL comprising the amino acid sequence of SEQ ID NO: 96.
11 . The antibody of claim 1 , wherein the antibody does not bind significantly to a h VISTA modified in that one or more of the following amino acid residues have been mutated: T35, Y37, K38, T39, Y41, R54, T61, F62, Q63, L65, H66, L67, H68, H69, F97, L115, V117, I119, H121, H122, S124, E125, R127, as determined by yeast mutational analysis.
12 . The antibody of claim 1 , wherein the antibody specifically binds to hVISTA under acidic conditions, and wherein the antibody:
inhibits the interaction between hVISTA and (a) T cells, (b) PSGL-1, or (c) VSIG-3;
enhances T cell activation by enhancing T cell proliferation; enhancing IFN-γ production from T cells; or stimulating T cell receptor mediated NF-kB signaling;
contacts h VISTA through one or more residues Y37, T39, R54, F62, H66, V117, I119 or S124, as determined using a yeast surface display and NGS assay;
and wherein numbering is that of mature hVISTA (SEQ ID NO: 2);
binds to the histidine-rich β-sheet extension of h VISTA;
contacts H121, H122 and/or H123 of mature h VISTA;
binds to Region 1: 57 LGPVDKGHDVTF 68 (SEQ ID NO: 566); Region 2: 86 RRPIRNLTFQDL 97 (SEQ ID NO: 567); and Region 3: 148 VVEIRHHHSEHRHGAME 165 (SEQ ID NO: 568) of h VISTA having SEQ ID NO: 1;
competes for binding to h VISTA with an antibody comprising VH and VL amino acid sequences as follows:
a VH comprising the amino acid sequence of SEQ ID NO: 95 and a VL comprising the amino acid sequence of SEQ ID NO: 96;
a VH comprising the amino acid sequence of SEQ ID NO: 103 and a VL comprising the amino acid sequence of SEQ ID NO: 104;
a VH comprising the amino acid sequence of SEQ ID NO: 99 and a VL comprising the amino acid sequence of SEQ ID NO: 100;
a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 68;
a VH comprising the amino acid sequence of SEQ ID NO: 53 and a VL comprising the amino acid sequence of SEQ ID NO: 54;
a VH comprising the amino acid sequence of SEQ ID NO: 55 and a VL comprising the amino acid sequence of SEQ ID NO: 56; or
a VH comprising the amino acid sequence of SEQ ID NO: 551 and a VL comprising the amino acid sequence of SEQ ID NO: 552;
contacts hVISTA through at least one glutamic acid, aspartic acid or histidine residue that is located in VH CDR1, CDR2 or CDR3;
has a mean residence time (MRT) of at least 100, 200, 300, 400, 500, 600 or 700 hours;
accumulates preferably in tumor tissues in comparison to lung, liver, and spleen in hVISTA knock-in mice harboring MC38 tumors; or
accumulates preferably in tumor tissues in comparison to lung, liver, and spleen in a subject to which the antibody was administered.
13 . The antibody of claim 1 , wherein the antibody specifically binds to hVISTA under acidic conditions with a K D (or koff) that is at least 10 fold, 100 fold or 1000 fold lower than its K D or koff of binding to hVISTA under neutral or physiological pH, and wherein the antibody:
specifically binds to hVISTA at a histidine rich region of the hVISTA extracellular domain (ECD) or a polypeptide comprising amino acid residues 35-127 of SEQ ID NO: 2;
lacks significant binding to hVISTA at a histidine rich region of the ECD or a polypeptide comprising amino acid residues 35-127 of SEQ ID NO: 2, at physiological pH or neutral pH;
specifically binds to cynomolgus (cyno) VISTA at a histidine rich region of the ECD, at acidic pH;
lacks significant binding to cyno VISTA at a histidine rich region of the ECD, at physiological pH or neutral pH;
has reduced binding to hVISTA-ECD having a substitution at one or more of the following amino acids: T35, Y37, K38, T39, Y41, R54, T61, F62, Q63, L65, H66, L67, H68, H69, F97, L115, V117, I119, H121, H122, S124, E125, R127 relative to hVISTA ECD having SEQ ID NO: 2;
cross-competes for binding to hVISTA with an antibody comprising VH and VL amino acid sequences as follows: a VH comprising the amino acid sequence of SEQ ID NO: 95 and a VL comprising the amino acid sequence of SEQ ID NO: 96; a VH comprising the amino acid sequence of SEQ ID NO: 103 and a VL comprising the amino acid sequence of SEQ ID NO: 104; or a VH comprising the amino acid sequence of SEQ ID NO: 99 and a VL comprising the amino acid sequence of SEQ ID NO: 100;
inhibits binding of hVISTA to human T cells expressing VISTA at acidic pH;
inhibits binding of hVISTA to PSGL-1 at acidic pH, wherein PSGL-1 is with or without siayl lewis X;
has a mean residence time (MRT) of at least 100, 200, 300, 350, 400, 450, 500, 600 or 700 hours in cynomolgus monkeys;
stimulates T cell activation by enhancing T cell proliferation; enhancing IFN-γ production from T cells; or stimulating T cell receptor mediated NF-kB signaling;
inhibits VISTA mediated cell: cell adhesion;
specifically binds to hVISTA in samples of human tumor cells or samples of inflamed human tissue that express VISTA;
contacts hVISTA through one or more residues Y37, T39, R54, F62, H66, V117, I119 or S124, as determined using a yeast surface display and NGS assay, and wherein numbering is that of mature hVISTA (SEQ ID NO: 2);
binds to Region 1 of hVISTA: 57 LGPVDKGHDVTF 68 (SEQ ID NO: 566); Region 2: 86 RRPIRNLTFQDL 97 (SEQ ID NO: 567); and Region 3: 148 VVEIRHHHSEHRVHGAME 165 (SEQ ID NO: 568) having SEQ ID NO: 1, and optionally wherein the binding is strongest to Region 2, as determined by MS-HDX;
binds to the histidine-rich β-sheet extension of hVISTA, as determined by crystallography;
contacts H121, H122 and/or H123 of mature hVISTA (distance of 4.0 Ångströms (Å) or less), as determined by crystallography; or
contacts hVISTA through at least one glutamic acid, aspartic acid or histidine residue that is located in VH CDR1, CDR2 or CDR3.
14 . The antibody of claim 1 , wherein the antibody has an isoelectric point (pI) between 6.5 and 6.8, as measured by icIEF.
15 . The antibody of claim 1 , which is an IgG1, IgG2 or IgG4 antibody.
16 . An isolated nucleic acid encoding an antibody of claim 1 .
17 . A cell comprising the isolated nucleic acid of claim 16 .
18 . A method of preparing an antibody, comprising culturing the cell of claim 17 in conditions under which the antibody is expressed.
19 . A composition comprising an isolated antibody of claim 1 and a pharmaceutically acceptable carrier.
20 . A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the antibody of claim 1 , wherein the antibody stimulates an immune response and/or is a VISTA antagonist.
21 . A method of treating an infectious disease in a subject, comprising administering to the subject a therapeutically effective amount of the antibody of claim 1 , wherein the antibody stimulates an immune response and/or is a VISTA antagonist.
22 . The antibody of claim 1 , wherein the antibody comprises a VH comprising the amino acid sequence SEQ ID NO: 570, 574, 578, 582, 586, 590, 594, 598, 602, 606, 610, 614, 618, 622, 626, 630, 634, 638, 642, 646, 650, 654, 658, 662, 666, 670, 674, 678, 682, or 686, modified by 1, 2, 3, 4, or 5 amino acid substitutions in the framework regions.
23 . The antibody of claim 1 , wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 570, 574, 578, 582, 586, 590, 594, 598, 602, 606, 610, 614, 618, 622, 626, 630, 634, 638, 642, 646, 650, 654, 658, 662, 666, 670, 674, 678, 682, or 686, and a VL comprising the amino acid sequence of SEQ ID NO: 96.