Humanized antibody targeting the tumor associated antigen IL13Ra2
The present invention provides humanized antibodies that bind to IL13Rα2, an interleukin-13 receptor that is overexpressed by the majority of glioblastoma tumors and not expressed at significant levels in normal brain tissue. Also provided are bispecific T cell engagers that bind to both IL13Rα2 and to the T cell co-receptor CD3 as well as methods for treating cancer, in which these humanized antibodies are used to target tumors.
1 . A humanized antibody that binds to IL13Rα2 comprising:
a variable light domain (V L ) comprising an amino acid sequence of SEQ ID NO:53; and
a variable heavy domain (V H ) comprising an amino acid sequence of SEQ ID NO:54.
2 . The humanized antibody of claim 1 , wherein (a) X 1 in V L is F; (b) X 2 in V H is E, (c) X 3 in V H is A; or (d) combinations of (a), (b) and (c).
3 . The humanized antibody of claim 1 , further comprising:
an agent selected from a therapeutic agent and a detection agent.
4 . The humanized antibody of claim 1 , wherein the variable heavy domain (V H ) and the variable light domain (V L ) are linked by a flexible linker, wherein the linker is an amino acid sequence of about 4-25 amino acids in length and comprising glycine and serine.
5 . The humanized antibody of claim 1 , wherein the antibody is a single-chain variable fragment antibody.
6 . The humanized antibody of claim 1 , wherein the antibody comprises a signal sequence N-terminal to the variable heavy domain (V H ).
7 . The humanized antibody of claim 6 , wherein the signal sequence is SEQ ID NO:29.
8 . A method of treating an IL13Rα2-expressing cancer in a subject, the method comprising: administering a therapeutically effective amount of the humanized antibody of claim 1 to treat the cancer.
9 . A humanized antibody that binds to IL13Rα2, comprising:
(a) a heavy chain comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12; and
(b) a light chain comprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.
10 . The humanized antibody of claim 9 , wherein the heavy chain is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, and SEQ ID NO:12, and comprises one or more mutations selected from M34L, M34A, M34I, M34V, D52E, P53A, D55E, and G56A; and
wherein the light chain is selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, and comprises one or more mutations selected from M37L, M37I, M37V, Q58R, Q58A, Q94E, Q94R, Q94A, W100F, and W100Y.
11 . The humanized antibody of claim 9 , wherein the antibody cannot isomerize and comprises:
(i) a heavy chain selected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO: 12, wherein the heavy chain comprises G56A, and a light chain selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16; or
(ii) a heavy chain selected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, wherein the V H comprises D55E, and a light chain selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.
12 . The humanized antibody of claim 9 , wherein the antibody comprises:
(i) SEQ ID NO:1 and SEQ ID NO:5;
(ii) SEQ ID NO:1 and SEQ ID NO:6;
(iii) SEQ ID NO:1 and SEQ ID NO:7;
(iv) SEQ ID NO:1 and SEQ ID NO:8;
(v) SEQ ID NO:2 and SEQ ID NO:5;
(vi) SEQ ID NO:2 and SEQ ID NO:6;
(vii) SEQ ID NO:2 and SEQ ID NO:7;
(viii) SEQ ID NO:2 and SEQ ID NO:8;
(ix) SEQ ID NO:3 and SEQ ID NO:5
(x) SEQ ID NO:3 and SEQ ID NO:6;
(xi) SEQ ID NO:3 and SEQ ID NO:7;
(xii) SEQ ID NO:3 and SEQ ID NO:8;
(xiii) SEQ ID NO:4 and SEQ ID NO:5;
(xiv) SEQ ID NO:4 and SEQ ID NO:6;
(xv) SEQ ID NO:4 and SEQ ID NO:7; or
(xvi) SEQ ID NO:4 and SEQ ID NO:8.
13 . An engineered bispecific antibody comprising a first single-chain variable fragment (scFv) that binds to CD3 and a second scFv that binds to IL13Rα2, wherein the first scFv comprises:
(a) a variable light domain (V H ) comprising an amino acid sequence of SEQ ID NO:51; a
(b) a first flexible linker; and
(c) a variable heavy domain (V L ) comprising an amino acid sequence of SEQ ID NO:52, and
wherein the second scFv comprises:
(d) a variable light domain (V L ) comprising an amino acid sequence of SEQ ID NO:53;
(e) a second linker; and
(f) a variable heavy domain (V H ) comprising an amino acid sequence of SEQ ID NO:54; and
wherein the bispecific antibody comprises from N terminal to C terminal: the V H of the first scFv, the V L of the first scFv, the V L of the second scFv, and the V H of the second scFv.
14 . The engineered bispecific antibody of claim 13 , wherein the first single-chain variable fragment (scFv) that binds to CD3 and second scFv that binds to IL13Rα2 are linked via a third flexible linker.
15 . The engineered bispecific antibody of claim 13 , wherein (a) the first and second linker are an amino acid sequence of about 10-20 amino acids consisting of glycine and serine, (b) the third linker is an amino acid sequence of about 20-30 amino acids consisting of glycine and serine, or (c) both (a) and (b).
16 . The engineered bispecific antibody of claim 15 , wherein the first and second linker are SEQ ID NO:55, and the third linker is SEQ ID NO:56.
17 . The engineered bispecific antibody of claim 13 , wherein amino acid sequence further comprises a signal peptide comprising SEQ ID NO:50 on the N terminal end.
18 . The engineered bispecific antibody of claim 13 , wherein the V H of the second scFv comprises a mutation, wherein the mutation is at least one of X 2 is E and X 3 is A.
19 . The engineered bispecific antibody of claim 13 , wherein the engineered bispecific T cell engager comprises:
an amino acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60.
20 . A method of treating an IL13Rα2-expressing cancer in a subject, the method comprising: administering a therapeutically effective amount of the bispecific antibody of claim 13 to the subject to treat the cancer.