Humanized antibody and method for using the same
The present invention aims to provide a humanized antibody or an antigen-binding fragment thereof having stable physical property, superior in tumor accumulation, and capable of binding to mucin subtype 5AC. The above-mentioned problem is solved by the present invention that provides a humanized antibody or an antigen-binding fragment thereof having a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1-4 or a mutated amino acid sequence thereof, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5-8 or a mutated amino acid sequence thereof, and capable of binding to mucin subtype 5AC.
1 . A non-conjugated humanized antibody capable of binding to mucin subtype 5AC, comprising:
a heavy chain variable region consisting of
(1) an amino acid sequence having not less than 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1,
(2) an amino acid sequence having not less than 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2,
(3) an amino acid sequence having not less than 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, or
(4) an amino acid sequence having not less than 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; and
a light chain variable region consisting of
(5) an amino acid sequence having not less than 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 5,
(6) an amino acid sequence having not less than 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 6,
(7) an amino acid sequence having not less than 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, or
(8) an amino acid sequence having not less than 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 8,
or an antigen-binding fragment thereof,
wherein the sequence of the CDR regions of the heavy and light chains are not modified.
2 . The humanized antibody or an antigen-binding fragment thereof according to claim 1 , wherein the heavy chain variable region consists of
(1) an amino acid sequence having not less than 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1,
(3) an amino acid sequence having not less than 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, or
(4) an amino acid sequence having not less than 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 and a light chain variable region consisting of
(5) an amino acid sequence having not less than 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 5,
(6) an amino acid sequence having not less than 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 6,
(7) an amino acid sequence having not less than 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, or,
(8) an amino acid sequence having not less than 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 8,
or an antigen-binding fragment thereof.
3 . The humanized antibody or an antigen-binding fragment thereof according to claim 1 , comprising:
a heavy chain variable region consisting of
(1) an amino acid sequence having not less than 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1,
(3) an amino acid sequence having not less than 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, or
(4) an amino acid sequence having not less than 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and
a light chain variable region consisting of
(5) an amino acid sequence having not less than 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 5,
(6) an amino acid sequence having not less than 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 6,
(7) an amino acid sequence having not less than 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, or
(8) an amino acid sequence having not less than 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8.
4 . The humanized antibody or an antigen-binding fragment thereof according to claim 1 , comprising:
(1) a heavy chain variable region consisting of an amino acid sequence having not less than 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and
(7) a light chain variable region consisting of an amino acid sequence having not less than 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 7.
5 . The humanized antibody, or an antigen-binding fragment thereof according to claim 1 , comprising:
a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7.
6 . The humanized antibody according to claim 1 , having an in vitro binding activity to mucin subtype 5AC comparable to or greater than that of a chimeric antibody.
7 . A composition comprising:
the humanized antibody according to claim 1 , or an antigen-binding fragment thereof.
8 . A nucleic acid encoding the humanized antibody according to claim 1 .
9 . An expression vector, comprising:
the nucleic acid according to claim 8 .
10 . A host cell comprising:
the expression vector according to claim 9 .
11 . A method for producing a non-conjugated humanized antibody or an antigen-binding fragment thereof in the host cell according to claim 10 , comprising:
inserting a nucleic acid encoding the humanized antibody or an antigen-binding fragment thereof into an expression vector,
introducing the nucleic acid containing expression vector into a host cell; culturing the host cell containing the expression vector, and
isolating the humanized antibody or an antigen-binding fragment thereof from a culture supernatant of the host cell by chromatographic purification.
12 . An in vitro method for evaluating an expression level of mucin subtype 5AC in a cell or a tissue sample, comprising:
contacting the cell or tissue sample with a humanized antibody or an antigen-binding fragment thereof of claim 1 , wherein the antibody or fragment thereof is labeled;
detecting the labeled humanized antibody or an antigen-binding fragment in the cell or tissue sample, thereby generating a signal; and
evaluating an expression level of mucin subtype 5AC expressed in the cell or tissue sample based on the signal.
13 . An in vitro method for measuring a volume of a cancer overexpressing mucin subtype 5AC in a subject, comprising:
contacting a tissue sample of the subject and a tissue sample of a healthy subject with a humanized antibody or an antigen-binding fragment thereof of claim 1 , wherein the antibody or fragment thereof is labeled;
detecting the labeled humanized antibody or an antigen-binding fragment in the tissue sample of the subject, thereby generating a first signal;
detecting the labeled humanized antibody or an antigen-binding fragment in the tissue sample of the healthy subject, thereby generating a second signal;
comparing the first signal and the second signal, wherein the comparison correlates to a volume of the cancer present in the subject.
14 . An in vitro method for measuring a volume of a cancerous tissue overexpressing mucin subtype 5AC in a subject, comprising:
contacting a tissue sample of the subject with a humanized antibody or an antigen-binding fragment thereof of claim 1 , wherein the antibody or fragment thereof is labeled;
detecting the labeled humanized antibody or an antigen-binding fragment in the tissue sample of the subject, thereby generating a signal; and
calculating the volume of a cancerous tissue volume based on the signal intensity.
15 . A kit for the in vitro diagnosis of a cancer overexpressing mucin subtype 5AC comprising the humanized antibody or an antigen-binding fragment thereof of claim 1 , and a label for detecting the antibody or an antigen-binding antibody fragment thereof.