EA044968B1 - HUMANIZED ANTIBODY AND METHOD OF ITS APPLICATION - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to a humanized antibody that specifically binds to mucin subtype 5AC (MUC5AC), or an antigen-binding fragment thereof, and a method of using it.

State of the art

Mucin is a major component of mucus secreted by epithelial cells etc. of animals, and is a glycoprotein containing large amounts of sugar with a molecular weight of 1 to 10 million. Mucin includes secretory mucin produced by an epithelial cell and the like, and membrane-bound mucin which has a hydrophobic transmembrane region and exists associated with a cell membrane. Mucin core proteins are collectively called MUCs, and there are at least 20 types of core protein-encoding genes known to exist. One of them, MUC5AC, is a secretory mucin.

MUC5AC is expressed in the stomach and trachea in normal tissues and has been reported to be overexpressed in pancreatic cancer. Overexpression has also been reported in thyroid cancer, liver cancer, colorectal cancer, gastric cancer, urothelial cancer, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, and bile duct cancer. As antibodies to MUC5AC, a mouse antibody obtained using a pancreatic cancer mucin fraction purified from a xenograft of the human pancreatic cancer cell line SW1990 and chimeric antibodies (Patent Documents 1, 2, Non-Patent Documents 1, 2) as the antigen, and Humanized antibodies obtained from them have been reported (Patent Document 3).

In recent years, antibody-drug complexes have been actively developed, in which a drug with an antitumor effect is conjugated to an antibody that has the ability to target cancer cells. For example, when an antibody is contemplated to be used as a delivery vehicle for an antibody-drug complex, since the antibody must be further subjected to a production step (eg, a conjugation step), denaturation and aggregation of the antibody during the production step must be considered. Therefore, antibodies used in production must have more stable properties as antibodies than conventional antibody preparations. On the other hand, when an antibody is intended to be used as a delivery vehicle for an antibody-drug complex, since the distribution of a drug with an extremely potent cell-killing effect into normal tissues results in significant side effects, the antibodies used for the antibody-drug complex must have more stable physical properties as antibodies and higher accumulation in tumor tissues than conventional antibody preparations.

The chimeric antibodies disclosed in Non-Patent Documents 1 and 2 have been confirmed to exhibit tumor accumulation in vivo as well as in patients with pancreatic cancer. Since accumulation in the liver and kidneys was also observed, application in pharmaceuticals requires further improvement of tumor accumulation. Non-patent documents 1, 2 do not disclose any information about the physical properties (denaturation, aggregation) of the antibody.

In addition, various physical properties of the chimeric antibodies disclosed in non-patent documents 1, 2 were evaluated. As a result, it was found that the antibodies easily denature and aggregate when heated (low temperature of the midpoint of denaturation and low temperature of the onset of aggregation), and the advisability of their use in the quality, for example, of a delivery vehicle for an antibody-drug complex is low. Needless to say, when an antibody is used in a subsequent manufacturing step (such as a conjugation step), it is better to use an antibody that has as good physical properties as possible (an antibody that does not cause denaturation or aggregation).

Patent Document 3 discloses a humanized antibody; however, it focuses on the production of a humanized antibody with high binding activity to MUC5AC and does not disclose or imply the stability of the antibody. In addition, various physical properties of the humanized antibody disclosed in Patent Document 3 were evaluated. As a result, it was found that antibodies easily aggregate when heated (the onset of aggregation temperature is low), and it is advisable to use them, for example, as a delivery vehicle for an antibody-drug complex. low. It is advisable to avoid denaturation and aggregation of antibodies as much as possible, since there is an expected risk of reducing the effectiveness of the compound and causing side effects. It is known that the below average denaturation temperature and onset of aggregation temperature also correlate with long-term stability upon cooling and the like, and from the point of view of stability not only at the production stage but also during storage, it is desirable to develop an antibody with good physical properties that do not contribute to mild denaturation or aggregation of the antibody.

- 1 044968

List of documents

Patent documents.

Patent Document 1: JP-A-H7-203974.

Patent Document 2: JP-A-H11-5749.

Patent Document 3: WO 2013/157102.

Non-patent documents.

Non-patent document 1: Japanese Journal of Clinical Medicine, vol. 64, extra issue 1, 2006, p. 274-278.

Non-patent document 2: Japanese Journal of Cancer Research, 90, 1179-1186, 1999.

The essence of the invention

Technical problem.

Accordingly, the problem of the present invention is to obtain a humanized antibody or antigen-binding fragment thereof that has stable physical properties, is superior to analogues in its ability to accumulate in a tumor, and is capable of binding to mucin of the 5AC subtype. More specifically, a humanized antibody or antigen-binding fragment thereof that is not easily denatured or aggregated by heat and is capable of binding to mucin subtype 5AC, as well as a humanized antibody or antigen-binding fragment thereof that exhibits higher accumulation in tumor tissue than in normal tissue, and capable of binding to mucin of the 5AC subtype.

Solution to the problem.

The inventors of the present invention have conducted intensive research in an attempt to solve the above problems and have found that the problem can be solved in the following ways.

That is, the present invention relates to the following:

1. A humanized antibody capable of binding to mucin subtype 5AC and containing or consisting of a heavy chain variable region consisting of:

(1) an amino acid sequence of SEQ ID NO: 1, an amino acid sequence having at least 90% or at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence of SEQ ID NO: 1 in which no more than 10 or no more than 5 amino acids were deleted, replaced or added;

(2) an amino acid sequence of SEQ ID NO: 2, an amino acid sequence having at least 90% or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence of SEQ ID NO: 2 in which the amino acids have been deleted, replaced or no more than 10 or no more than 5 amino acids are added;

(3) an amino acid sequence of SEQ ID NO: 3, an amino acid sequence having at least 90% or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence of SEQ ID NO: 3 in which the amino acids have been deleted, replaced or no more than 10 or no more than 5 amino acids are added; or (4) an amino acid sequence of SEQ ID NO: 4, an amino acid sequence having at least 90% or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence of SEQ ID NO: 4 in which, no more than 10 or no more than 5 amino acids are replaced or added, and the light chain variable region, consisting of:

(5) an amino acid sequence of SEQ ID NO: 5, an amino acid sequence having at least 90% or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence of SEQ ID NO: 5 in which the amino acids have been deleted, replaced or no more than 10 or no more than 5 amino acids are added;

(6) an amino acid sequence of SEQ ID NO: 6, an amino acid sequence having at least 90% or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence of SEQ ID NO: 6 in which the amino acids have been deleted, replaced or no more than 10 or no more than 5 amino acids are added;

(7) an amino acid sequence of SEQ ID NO: 7, an amino acid sequence having at least 90% or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence of SEQ ID NO: 7 in which the amino acids have been deleted, replaced or no more than 10 or no more than 5 amino acids are added; or (8) an amino acid sequence of SEQ ID NO: 8, an amino acid sequence having at least 90% or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence of SEQ ID NO: 8 in which, no more than 10 or no more than 5 amino acids or its antigen-binding fragment are replaced or added.

2. A humanized antibody or antigen-binding fragment thereof according to claim 1, where the heavy chain variable region consists of:

(1) amino acid sequence SEQ ID NO: 1, an amino acid sequence having at least 90% or at least 95% sequence identity with the amino acid

- 2 044968 sequence SEQ ID NO: 1, or the amino acid sequence SEQ ID NO: 1, in which no more than 10 or no more than 5 amino acids are deleted, replaced or added;

(3) an amino acid sequence of SEQ ID NO: 3, an amino acid sequence having at least 90% or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence of SEQ ID NO: 3 in which the amino acids have been deleted, replaced or no more than 10 or no more than 5 amino acids are added; or (4) an amino acid sequence of SEQ ID NO: 4, an amino acid sequence having at least 90% or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence of SEQ ID NO: 4 in which, no more than 10 or no more than 5 amino acids are replaced or added.

3. A humanized antibody or antigen-binding fragment thereof according to claim 1 or 2, containing or consisting of:

(1) a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 1, an amino acid sequence having at least 90% or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1, in which no more than 10 or no more than 5 amino acids are removed, replaced or added; and (7) a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 7, an amino acid sequence having at least 90% or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence of SEQ ID NO: : 7, in which no more than 10 or no more than 5 amino acids are deleted, replaced or added.

4. A humanized antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, comprising or consisting of a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 5.

5. A humanized antibody or antigen binding fragment thereof as claimed in claim 1 or 2, comprising or consisting of a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 6.

6. A humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, containing or consisting of a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 7.

7. A humanized antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, comprising or consisting of a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

8. A humanized antibody or antigen binding fragment thereof as claimed in claim 1 or 2, comprising or consisting of a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 3, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 5.

9. A humanized antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, comprising or consisting of a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 3, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 6.

10. A humanized antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, comprising or consisting of a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 3, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 7.

11. A humanized antibody or antigen binding fragment thereof as claimed in claim 1 or 2, comprising or consisting of a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 3, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

12. A humanized antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, comprising or consisting of a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 5.

13. A humanized antibody or antigen-binding fragment thereof according to claim 1 or claim 2, comprising or consisting of a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 6.

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14. A humanized antibody or antigen binding fragment thereof as claimed in claim 1 or 2, comprising or consisting of a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 7.

15. A humanized antibody or antigen binding fragment thereof as claimed in claim 1 or 2, comprising or consisting of a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

16. The humanized antibody or antigen binding fragment thereof according to any one of claims 1 to 15, wherein the antibody or antigen binding fragment is an isolated antibody or isolated antigen binding fragment.

17. A humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

18. A humanized antibody according to any one of claims 1 to 17, wherein the antibody has a most common particle size (Pk1 mode diameter) of no more than 20 nm, no more than 15 nm, no more than 12 nm, no more than 11 nm, or no more than 10 nm.

19. A humanized antibody according to any one of claims 1 to 18, where the antibody has an average denaturation temperature (Tm1) of at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58° C, not less than 59°C or not less than 60°C.

20. A humanized antibody according to any one of claims 1 to 19, where the antibody has an onset of aggregation temperature (Tagg) of at least 50°C, at least 55°C, at least 60°C, at least 61°C, at least 62° C, not less than 63°C, not less than 64°C or not less than 65°C.

21. A humanized antibody according to any one of claims 1 to 20, having in vitro binding activity to mucin subtype 5AC comparable to or greater than that of the chimeric antibody.

22. A humanized antibody according to any one of claims 1 to 21, where its accumulation in tumor tissue expressing mucin of the 5AC subtype is no less than 10 times, no less than 11 times, no less than 12 times, no less than 13 times, no less than 14 times, no less than 15 times, no less than 16 times, no less than 17 times, no less than 18 times, no less than 19 times or no less than 20 times higher than the accumulation in liver tissue 7 days after administration of the antibody.

23. A humanized antibody according to any one of claims 1 to 22, where its accumulation in tumor tissue expressing mucin of the 5AC subtype is no less than 10 times, no less than 11 times, no less than 12 times, no less than 13 times, no less than 14 times, no less than 15 times, no less than 16 times, or no less than 17 times the accumulation in kidney tissue 7 days after antibody administration.

24. A composition containing a humanized antibody according to any one of claims 1 to 23 or an antigen-binding fragment thereof.

25. A pharmaceutical composition containing a humanized antibody according to any one of claims 1 to 23 or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

26. A pharmaceutical composition for the treatment of cancer overexpressing mucin subtype 5AC, containing a humanized antibody according to any one of claims 1 to 23 or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition according to claim 26, wherein the cancer is pancreatic cancer, thyroid cancer, liver cancer, colorectal cancer, gastric cancer, urothelial cancer, breast cancer, cervical cancer, ovarian cancer, endometrial carcinoma or bile duct cancer .

28. The pharmaceutical composition according to claim 27, wherein the cancer is pancreatic cancer.

29. An antibody-drug conjugate comprising a humanized antibody according to any one of claims 1 to 23 or an antigen-binding fragment thereof, and a drug used for diagnosis and/or treatment that is conjugated thereto.

30. The antibody-drug conjugate of claim 29, wherein the drug is a toxin, fluorescent labeling agent, nucleic acid drug, viral vector, nanoparticle, small molecule drug, or cytokine.

31. The antibody-drug conjugate of claim 29 or 30, wherein the humanized antibody or antigen-binding fragment thereof and the drug are connected by a linker.

32. The antibody-drug conjugate of claim 31, wherein the linker is one or more linkers selected from the group consisting of a PEG linker, a maleimide linker, a PASylated linker, a HESylated linker, a bis(sulfosuccinimidyl)suberate linker , a nucleic acid linker, a peptide linker, a silane linker, a polysaccharide linker, a linker that is a heat-sensitive or radiation-sensitive (IR, near-IR, UV) bond, a linker that is a pH-sensitive bond, a hydrolytic linker, and a linker, obtained by covalent coupling, amide coupling, carbon-carbon multiple bond addition, Husgen cycloaddition to azidoalkyne, Diels-Alder reaction, disulfide coupling, Michael addition, silane coupling, nucleophilic ring opening reaction of urethane, epoxide, non-aldol carbonyl and

- 4 044968 1,3-dipolar addition reaction or cycloaddition reaction such as tosylation.

33. A pharmaceutical composition containing the antibody-drug conjugate according to any one of claims 29-32 and a pharmaceutically acceptable carrier.

34. Nucleic acid encoding a humanized antibody according to any one of claims 1 to 23.

35. An expression vector containing the nucleic acid according to claim 34.

36. A host cell containing the expression vector of claim 35.

37. A method for producing a humanized antibody according to any one of claims 1-23 or an antigen-binding fragment thereof, including:

(1) the step of inserting a nucleic acid encoding a humanized antibody or an antigen-binding fragment thereof into an expression vector, (2) the step of introducing the above-mentioned nucleic acid into a host cell using an expression vector containing the nucleic acid, (3) the step of culturing the host cell, containing the expression vector, and (4) the step of isolating the humanized antibody or antigen-binding fragment thereof from the host cell culture supernatant by purification by chromatography.

38. The use of a humanized antibody according to any one of claims 1 to 23 or an antigen-binding fragment thereof in the preparation of a composition according to claim 24, a pharmaceutical composition according to any of claims 25 to 28 and 33 or an antibody-drug conjugate according to any of claims 29 to 32.

39. A method for assessing the level of expression of mucin subtype 5AC in a cell or tissue, including:

(1) the step of obtaining a labeled humanized antibody or an antigen-binding fragment thereof by attaching a label to the humanized antibody according to any one of claims 1 to 23 or an antigen-binding fragment thereof, which allows detection of the antibody or an antigen-binding fragment thereof, (2) the step of contacting a cell or tissue with the labeled a humanized antibody or an antigen-binding fragment thereof; and (3) the step of measuring the amount of label associated with the cell or tissue and estimating therefrom the level of expression of the 5AC subtype mucin expressed in the cell or tissue.

40. A therapeutic agent for the treatment of cancer overexpressing mucin subtype 5AC, comprising an effective amount of a pharmaceutical composition comprising a humanized antibody according to any one of claims 1 to 23 or an antigen binding fragment thereof, or an antibody-drug conjugate according to any one of claims 29 to 32 and pharmaceutically acceptable carrier.

41. The therapeutic agent for treating cancer according to claim 40, wherein the drug reduces the number of cancer cells and/or reduces the size of the tumor.

42. A method for measuring the volume of a cancer overexpressing mucin subtype 5AC in a subject, comprising:

(1) the step of obtaining a labeled humanized antibody or antigen-binding fragment thereof by attaching a label to the humanized antibody according to any one of claims 1 to 23 or an antigen-binding fragment thereof, where the label allows detection of the antibody or antigen-binding fragment thereof, (2) the step of contacting the subject's tissue and normal tissue with the labeled humanized antibody or antigen binding fragment thereof, (3) the step of measuring the amount of label in the subject tissue and normal tissue, and (4) the step of comparing the amount of label measured in the subject tissue with a standard amount, which is the amount of label measured in normal tissue, and the step of measuring the volume of cancerous tissue in a portion where the amount measured in the subject's tissue exceeds a standard amount.

43. A method for diagnosing whether a subject is affected by a cancer overexpressing mucin subtype 5AC, comprising:

(1) the step of producing a labeled humanized antibody or antigen-binding fragment thereof by attaching a label to the humanized antibody of any one of claims 1 to 23 or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is detectable, (2) the step of contacting a cell or tissue of a subject and normal cell or tissue with a labeled humanized antibody or antigen binding fragment thereof, (3) the step of measuring the amount of label in the cell or tissue of the subject and the normal tissue, and (4) the step of comparing the amount of label measured in the cell or tissue of the subject with a standard amount that represents is the amount of label measured in a normal cell or tissue, and when the amount of label measured in a cell or tissue of a subject is significantly greater than the standard amount, the subject is diagnosed as having a cancer overexpressing mucin subtype 5AC.

44. The method according to claim 42 or 43, where the contact step is carried out in vitro.

45. A method for measuring the volume of a cancer overexpressing mucin subtype 5AC in a subject,

- 5 044968 including:

(1) the step of obtaining a labeled humanized antibody or antigen-binding fragment thereof by attaching a label to the humanized antibody according to any one of claims 1 to 23 or an antigen-binding fragment thereof, where the label allows detection of the antibody or antigen-binding fragment thereof, (2) the step of detecting the label in the tissue of the subject to which the labeled humanized antibody or antigen-binding fragment thereof is administered, and (3) the step of measuring the volume of the part in which accumulation of the labeled humanized antibody or antigen-binding fragment thereof was detected in the above-mentioned step (2).

46. A method for diagnosing whether a subject is affected by a cancer overexpressing mucin subtype 5AC, comprising:

(1) the step of obtaining a labeled humanized antibody or antigen binding fragment thereof by attaching a label to the humanized antibody according to any one of claims 1 to 23 or an antigen binding fragment thereof, where the label allows detection of the antibody or antigen binding fragment thereof, (2) the step of detecting the label in the tissue of the subject to which the labeled humanized antibody or antigen-binding fragment thereof is administered, and (3) the step of diagnosing that the subject is affected by cancer overexpressing mucin subtype 5AC upon detecting accumulation of the labeled humanized antibody or antigen-binding fragment thereof in the tissue of the subject in the above-mentioned step (2).

47. The method according to claim 45 or 46, characterized in that the detection step is carried out in vitro.

48. A method of treating cancer overexpressing mucin subtype 5AC, comprising administering an effective amount of a pharmaceutical composition comprising a humanized antibody according to any one of claims 1 to 23 or an antigen binding fragment thereof and a pharmaceutically acceptable carrier to a subject affected by cancer.

49. The method according to claim 48, which reduces the number of cancer cells and/or reduces the size of the tumor.

50. A method for diagnosing whether a subject is affected by a cancer overexpressing mucin subtype 5AC, comprising:

(1) the step of obtaining a labeled humanized antibody or antigen-binding fragment thereof by attaching a label to the humanized antibody according to any one of claims 1 to 23 or an antigen-binding fragment thereof, where the label allows detection of the antibody or antigen-binding fragment thereof, (2) the step of collecting cells or tissues from subject and a healthy subject, (3) the step of contacting the cell or tissue with the labeled humanized antibody or antigen binding fragment thereof, (4) the step of measuring the amount of label in the cell or tissue, and (5) the step of diagnosing that the subject is affected by a cancer overexpressing a mucin subtype 5AC, when the amount of label measured in a cell or tissue taken from a subject is significantly greater than the amount of label measured in a cell or tissue taken from a healthy person.

51. A method for diagnosing whether a subject is affected by a cancer overexpressing mucin subtype 5AC, comprising:

(1) the step of obtaining a labeled humanized antibody or antigen binding fragment thereof by attaching a label to the humanized antibody according to any one of claims 1 to 23 or an antigen binding fragment thereof, where the label allows detection of the antibody or antigen binding fragment thereof, (2) the step of administering the labeled humanized antibody to the subject or an antigen-binding fragment thereof, (3) the step of detecting the label in the tissue of the subject, and (4) the step of diagnosing that the subject is affected by cancer overexpressing mucin subtype 5AC upon detecting accumulation of the labeled humanized antibody or antigen-binding fragment thereof in the tissue of the subject in the above-mentioned step ( 3).

52. A method of treating cancer in a subject diagnosed with cancer overexpressing mucin subtype 5AC by the method of claim 50 or 51, comprising the step of administering to the subject an effective amount of a pharmaceutical composition comprising a humanized antibody of any one of claims 123 or an antigen binding fragment thereof and a pharmaceutically acceptable carrier.

53. A kit for the treatment and/or diagnosis of cancer overexpressing mucin subtype 5AC, containing a humanized antibody according to any one of claims 1 to 23 or an antigen-binding fragment thereof with a label that allows detection of the antibody or an antigen-binding antibody fragment thereof.

Beneficial effect of the invention.

The inventors of the present invention have conducted intensive research in an attempt to solve the problems and have successfully found a humanized antibody that has the ability to bind to 5AC subtype mucin and does not undergo easy denaturation or aggregation even when heated, i.e.

- 6 044968 humanized antibody which has the above-mentioned binding activity and also has a high average denaturation temperature and a high onset of aggregation temperature and shows very high accumulation in tumor tissues, higher than in normal tissues, which has led to the completion of the present invention.

Brief description of drawings

In fig. 1 shows the amino acid sequences of the heavy chain variable regions 1-4 (H01-H04) of the humanized antibody of the present invention.

In fig. 2 shows the amino acid sequences of the light chain variable regions 1-4 (L01-L04) of the humanized antibody of the present invention.

In fig. 3 shows the amino acid sequences of heavy chain variable regions 5-6 (H05-H06) and light chain variable regions 5-7 (L05-L07) of the humanized antibody from Patent Document 3.

In fig. 4 shows the amino acid sequences of the heavy chain variable region 7 (H07) and the light chain variable region 8 (L08) of the chimeric antibody of Patent Document 1.

Fig. 5 is a photograph showing in vivo tumor accumulation of the compound of Example 3-1 (antibody 3) over time. Top row of images of Fig. 5 is a photograph of the first mouse, and the bottom row of images is a photograph of the second mouse. Photographs of mice before administration, 1 day after administration, 2 days after administration, 3 days after administration, and 7 days after administration are shown in that order from left to right.

Description of Embodiments (1) The humanized antibody of the present invention.

The present invention relates to a humanized antibody having a specific heavy chain variable region and a specific light chain variable region and capable of binding to 5AC subtype mucin, and an antigen binding fragment thereof. The humanized antibody of the present invention is characterized in that it has stable physical properties and is better accumulated in the tumor. The humanized antibody of the present invention may further have a suitable heavy chain constant region and a suitable light chain constant region in addition to a specific heavy chain variable region and a specific light chain variable region.

As used herein, the term humanized antibody refers to an antibody having a complementarity determining region (CDR) derived from a non-human animal in which at least a portion of the heavy chain variable region and/or light chain variable region is a region other than the CDR, has been modified to be more human-like, namely more similar to the human germline variable sequence. Complementarity determining regions exist in each variable region of the heavy chain and light chain of an antibody, and they are called complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) on the N-terminal side. The framework region is the region adjacent to the complementarity determining region in the variable region, and they are called framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3) and framework region 4 (FR4) on the N-terminal side .

As used herein, the term antigen binding fragment means an antibody fragment consisting of a portion of a humanized antibody of the present invention and having the ability to bind to subtype 5AC mucin. The number of amino acids contained in the polypeptide constituting the antigen-binding fragment is not particularly limited, provided that it has the ability to bind to subtype 5AC mucin.

In fig. 1 shows the amino acid sequence of the heavy chain variable region of the present invention. Heavy chain variable region 1 (H01), heavy chain variable region 2 (H02), heavy chain variable region 3 (H03), and heavy chain variable region 4 (H04) in FIG. 1 respectively correspond to SEQ ID NO: 1-4 in the sequence listing appended to this specification. The underlined part in FIG. 1 is a CDR site.

In fig. 2 shows the amino acid sequence of the light chain variable region of the present invention. Light chain variable region 1 (L01), light chain variable region 2 (L02), light chain variable region 3 (L03), and light chain variable region 4 (L04) in FIG. 2 respectively correspond to SEQ ID NO: 5-8 in the sequence listing appended to this specification. The underlined part in FIG. 2 is a CDR site.

In other words, the heavy chain variable region of the humanized antibody of the present invention consists of the amino acid sequence of any of the sequences of SEQ ID NO: 1 to SEQ ID NO: 4, and the light chain variable region consists of the amino acid sequence of any of the sequences of SEQ ID NO: 5 to SEQ ID NO: 8. That is, the humanized antibody of the present invention consists of a combination of the above four heavy chain variable regions (H01-H04) and four light chain variable regions

- 7 044968 chains (L01-L04).

The four heavy chain variable regions (H01-H04) and four light chain variable regions (L01-L04) of the present invention are obtained by humanizing the variable region of a mucin subtype 5AC antibody based on a chimeric antibody and engineering the antibody to improve thermostability, aggregation properties and accumulation in tumors. The CDR site required for antigen binding remains unchanged. The term chimeric antibody as used herein means the chimeric antibody disclosed in Patent Document 1 unless otherwise specified.

A preferred humanized antibody of the present invention has the heavy chain variable region H01, H03 or H04 and any of L01 to L04 as the light chain variable region.

The most preferred humanized antibody of the present invention has a heavy chain variable region H01 and a light chain variable region L03.

However, the heavy chain variable region of the humanized antibody of the present invention is not limited to the regions defined by the amino acid sequence of SEQ ID NO: 1 SEQ ID NO: 4, and also includes variants retaining function. That is, a mutant heavy chain variable region consisting of an amino acid sequence having at least 90%, preferably at least 95%, even more preferably at least 98%, most preferably at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 1 - SEQ ID NO: 4 is also covered by the heavy chain variable region of the present invention if it can bind to a 5AC subtype mucin in combination with the light chain variable region of the present invention.

As used herein, the term amino acid sequence identity refers to the identity of amino acid sequences between two proteins being analyzed and is expressed as the percentage (%) of amino acid residues that match in an optimal amino acid sequence alignment obtained using mathematical algorithms known in the art. Amino acid sequence identity can be determined by visual inspection and mathematical calculations, and can also be calculated using a homology search program (eg, BLAST, FASTA) or sequence alignment program (eg, ClustalW) known to those skilled in the art or genetic processing software. information (for example, GENETYX [registered trademark]), etc. In particular, the identity of the amino acid sequence in the present description can be determined using the ClustalW systematic analysis program (http://clustalw.ddbj.nig.ac.jp/index.php?lang=ja) published on the DDBJ website (Data Bank Japan DNA) with initial setting conditions (Version 2.1, Alignment Type: Slow, DNA Weight Matrix: Gonnet, GAP OPEN: 10, GAP EXTENSION: 0.1).

In addition, as the heavy chain variable region of the humanized antibody of the present invention, a mutated heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 4, where not more than 10, preferably not more than 8, even more preferably not more than 5, most preferably no more than 3 amino acids removed, replaced or added is also covered by the heavy chain variable region of the present invention if it can bind to a 5AC mucin in combination with the light chain variable region of the present invention.

The light chain variable region of the humanized antibody of the present invention is not limited to the amino acid sequence of SEQ ID NO: 5 to SEQ ID NO: 8, and also includes variants retaining function. That is, a mutated light chain variable region consisting of an amino acid sequence having at least 90%, preferably at least 95%, even more preferably at least 98%, most preferably at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 5 - SEQ ID NO: 8 is also covered by the light chain variable region of the present invention if it can bind to a 5AC subtype mucin in combination with the heavy chain variable region of the present invention.

In addition, as a light chain variable region of the humanized antibody of the present invention, a mutated light chain variable region consisting of the amino acid sequence of SEQ ID NO: 5 to SEQ ID NO: 8, where not more than 10, preferably not more than 8, even more preferably not more than 5, most preferably no more than 3 amino acids removed, replaced or added is also covered by the light chain variable region of the present invention if it can bind to a 5AC subtype mucin in combination with the heavy chain variable region of the present invention.

The humanized antibody or antigen binding fragment of the present invention may be an isolated antibody or isolated antigen binding fragment. That is, the humanized antibody of the present invention is isolated, for example, from the supernatant of a host cell culture into which a nucleic acid encoding the humanized antibody or antigen binding fragment of the present invention has been introduced. Such nucleic acid and host cell

- 8 044968 are described in detail below.

Moreover, the humanized antibody of the present invention may be a polyclonal antibody or a monoclonal antibody.

In addition, the humanized antibody of the present invention has stable physical properties and is not easy to aggregate. The humanized antibody particles of the present invention most often have a small size, which is specifically no more than 20 nm, no more than 15 nm, no more than 12 nm, no more than 11 nm, or no more than 10 nm. When antibody aggregation occurs, many antibodies are found having a particle size greater than 100 nm. As used herein, the term most common particle size refers to the particle size most frequently detected when measuring the particle size distribution of the humanized antibody of the present invention.

One of the characteristics of the humanized antibody of the present invention is that it does not denature easily when heated. Specifically, the midpoint temperature of denaturation of the humanized antibody of the present invention is not less than 50°C, not less than 55°C, not less than 56°C, not less than 57°C, not less than 58°C, not less than 59°C, or not less than 60 °C. When the humanized antibody of the present invention is heated and the antibody is denatured, the center of gravity of the fluorescence spectrum peak of the amino acids originally present in the antibody is shifted. The term denaturation midpoint temperature as used herein refers to the temperature at the midpoint of the peak offset (the temperature at which the normal state and the degenerated state reach a 1:1 ratio).

One of the characteristics of the humanized antibody of the present invention is that it does not easily aggregate when heated. Specifically, the onset of aggregation temperature (Tagg) of the humanized antibody of the present invention is at least 50°C, at least 55°C, at least 60°C, at least 61°C, at least at least 62°C, at least 63°C , not less than 64°C or not less than 65°C. When the humanized antibody of the present invention is heated and the antibody aggregates, the scattering intensity in the static light scattering measurement becomes high. As used herein, the term aggregation onset temperature refers to the temperature at which scattered light begins to increase. In the following example, scattered light was measured at a wavelength of 266 nm.

In addition, the humanized antibody of the present invention is characterized by high in vitro binding activity to mucin subtype 5AC. Specifically, the in vitro binding activity of the humanized antibody of the present invention to the 5AC subtype mucin is comparable to or no less than that of the chimeric antibody disclosed in Patent Document 1. As used herein, comparable to may mean the same activity, and may also mean a difference of not more than about 2. 0 times, no more than 1.9 times, no more than 1.8 times, no more than 1.7 times, no more than 1.6 times, no more than 1.5 times, no more more than 1.4 times, no more than 1.3 times, no more than 1.2 times, or no more than 1.1 times, or no less than 1.0 times, no less than 0, 9 times, no less than 0.8 times, no less than 0.7 times, no less than 0.6 times or no less than 0.5 times.

In addition, since the humanized antibody of the present invention has high binding activity against 5AC mucin, it exhibits high accumulation in tumor tissues expressing 5AC mucin in vivo when administered to a living body. As used herein, the term accumulation in tumor tissues expressing 5AC mucin subtype refers to how many times more the humanized antibody of the present invention accumulates in tumor tissues expressing 5AC mucin subtype compared to normal tissue.

Accumulation in tumor tissue expressing mucin subtype 5AC is no less than 10 times, no less than 11 times, no less than 12 times, no less than 13 times, no less than 14 times, no less than 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, or at least 20 times greater than in liver tissue 7 days after administration of the humanized antibody of the present invention. Accumulation in tumor tissue expressing mucin subtype 5AC is no less than 10 times, no less than 11 times, no less than 12 times, no less than 13 times, no less than 14 times, no less than 15 times, no less than 16 times, or no less than 17 times more than in kidney tissue 7 days after administration of the humanized antibody of the present invention.

(2) A composition containing the humanized antibody of the present invention.

The present invention relates to a composition containing the humanized antibody of the present invention described in (1), or an antigen binding fragment thereof. The composition containing the humanized antibody of the present invention is useful for the treatment and/or diagnosis of cancer overexpressing mucin subtype 5AC.

That is, the present invention is a pharmaceutical composition containing a humanized antibody of the present invention and a pharmaceutically acceptable carrier. The humanized antibody or antigen binding fragment thereof of the present invention specifically binds to mucin subtype 5AC and inhibits the proliferation of cancer cells expressing it. Thus, the pharmaceutical composition of the present invention

- 9 044968 is applicable for the treatment of cancer overexpressing mucin subtype 5AC.

Examples of cancer to be treated in accordance with the present invention include pancreatic cancer, thyroid cancer, liver cancer, colorectal cancer, gastric cancer, urothelial cancer, breast cancer, cervical cancer, ovarian cancer and endometrial carcinoma and, in particular, pancreatic cancer can be effectively treated.

Examples of cancer to be treated in accordance with the present invention also include bile duct cancer.

There are multiple reports that subtype 5AC mucin carries antigen for CA19-9 (PLoS ONE (Dec 2011, vol. 6, issue 12, e29180, p. 1-10)). Therefore, examples of cancer to be treated according to the present invention also include biliary tract cancer, uterine cancer, lung cancer and esophageal cancer overexpressing CA19-9, and these cancers can be effectively treated.

The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and, for example, it can be obtained in the form of oral preparations such as tablet, powder, granule, suspension, emulsion, capsule and the like, or parenteral preparations such as injection, suppository , external liquid and the like. The pharmaceutically acceptable carrier used in the present invention may be one commonly used in the art, and may be appropriately selected by those skilled in the art based on the dosage form and route of administration.

As the pharmaceutically acceptable carrier used in the present invention for oral preparations such as tablet and the like, for example, an excipient, a binder, a lubricant, a surfactant, a diluent, a preservative, a stabilizer, a corrective a humectant, a preservative, an antioxidant and the like, and the pharmaceutical preparation can be formulated in a conventional manner. At the same time, those skilled in the art can select, for example, a suitable surfactant, diluent, preservative, stabilizer, flavoring agent, wetting agent, preservative and antioxidant based on general technical knowledge in the relevant art.

In the case of parenteral administration, injections are the typical preparations. The pharmaceutically acceptable carrier used in the present invention for injection may be, for example, a water-soluble solvent such as saline solution, Ringer's solution and the like, or a water-insoluble solvent such as vegetable oil or fatty acid ester and the like, and the drug can be prepared by dissolving in it. At this time, for example, an isotonic agent, a solubilizing agent, a stabilizer, a preservative, a suspending agent, an emulsifier, and the like may optionally be added to prepare the preparation.

(3) An antibody-drug conjugate, in which the drug is combined with a humanized antibody of the present invention.

The present invention relates to an antibody-drug conjugate in which a drug used for diagnosis and/or treatment is associated with a humanized antibody or an antigen-binding fragment thereof described in (1). Specific examples of the drug include a toxin, a fluorescent labeling agent, a nucleic acid drug, a viral vector, a nanoparticle, a small molecule drug, and a cytokine. Because the humanized antibody and antigen binding fragment thereof of the present invention bind to subtype 5AC mucin, they can aid in the diagnosis or treatment of cancer overexpressing subtype 5AC mucin by forming an antibody-bound drug.

When the above-mentioned drug is a label capable of detecting the antibody of the present invention, for example, a fluorescent labeling agent, the combined body of the antibody of the present invention and the label can be used as a means for detecting 5AC subtype mucin. For example, the antibody of the present invention coupled with a fluorescent labeling agent is introduced into a living body, and then, based on the label, an organ overexpressing 5AC subtype mucin can be detected, thereby facilitating the diagnosis of cancer. Even when not administered to a living body, expression of the 5AC subtype mucin in a tissue or cell taken from a living body can be detected by contacting the tissue or cell in vitro with the antibody of the present invention coupled to a fluorescent labeling agent. A method for diagnosing cancer using the antibody of the present invention is described in detail below.

The fluorescent labeling agent to be associated with the antibody of the present invention includes, but is not limited to, a fluorescent protein, a fluorescent dye, and the like. Specific examples of the fluorescent protein include, but are not limited to, green fluorescent protein, red fluorescent protein, and yellow fluorescent protein. Specific examples of the fluorescent dye include, but are not limited to, fluorescein, rhodamine, Cy dye, Alexa Fluor (registered trademark), HiLyte Fluor™, phycoerythrin (PE), and allophycocyanin (APC).

- 10 044968

When the above-mentioned drug is a therapeutic agent, a drug having the effects of both the therapeutic agent and the humanized antibody of the present invention can be produced by forming a bound body. A synergistic increase in the effectiveness of a drug can be achieved by combining the humanized antibody of the present invention with, for example, an anticancer toxin, a nucleic acid drug, a viral vector, nanoparticles, a small molecule drug, or a cytokine.

The term toxin as used herein is a concept that includes a cytotoxin or cytotoxic agent and refers to any drug that is harmful to cell growth and proliferation and can act to reduce, inhibit or destroy cells or malignant tumors. Specific examples include, but are not limited to, ricin, saporin, diphtheria toxin, Pseudomonas toxin and the like.

The term nucleic acid drug as used herein refers to a drug having a natural nucleotide or a chemically modified nucleotide as its backbone, and refers to any drug that can act directly on a living organism without gene expression. Specific examples include, but are not limited to, siRNA, microRNA, antisense nucleic acid, decoy nucleic acid, aptamer, and CpG oligonucleotide.

The term viral vector as used herein refers to any vector having the ability to efficiently introduce and express a target gene by incorporating a foreign gene into a virus in which the ability to replicate and proliferate has been removed by genetic engineering, or a virus that retains part of the ability to replication and proliferation. Specific examples include, but are not limited to, adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, and herpes simplex virus.

The term nanoparticle as used herein refers to any nanometer-sized delivery vehicle capable of transporting a drug to a given cell or tissue. Specific examples include, but are not limited to, liposomes, nanomicelles, and PLGA nanoparticles.

The term small molecule drug as used herein is a concept that includes a cytotoxic agent, a chemotherapeutic agent, a targeted anticancer agent, and an immunotherapeutic agent, and refers to any drug that can be used to treat cell proliferation disorders such as cancer and so on. Specific examples include, but are not limited to, MMAE, MMAF, DM-1, DM-4, 5-fluorouracil, doxorubicin, irinotecan, calicheamicin, and the like.

The term cytokine as used herein refers to any physiologically active substance that is a low molecular weight protein secreted by cells that participates in cell-cell interactions and affects surrounding cells. Specific examples include, but are not limited to, TNF-α, interferon-α, interferon-β, interferon-γ, interleukin-2, and the like.

In addition, by mixing an antibody-drug conjugate and a pharmaceutically acceptable carrier, a pharmaceutical composition for treating cancer overexpressing 5AC mucin subtype or a therapeutic agent for treating cancer overexpressing mucin subtype 5AC can be prepared. An embodiment of such a pharmaceutical composition and therapeutic agent is described for the pharmaceutical composition in (2). Such a pharmaceutical composition or therapeutic agent may reduce the number of cancer cells and/or reduce the size of the tumor.

In a preferred embodiment of the present invention, the humanized antibody or antigen binding fragment thereof and the drug are connected by a linker. The linker used here is preferably a linker capable of effectively linking the antibody of the present invention to a drug under mild conditions. Specific examples of a linker that can be used in the present invention include, but are not limited to, a linker; PEG linker, maleimide linker, PAS-ylated linker, HES-ylated linker, bis-(sulfosuccinimidyl)suberate linker, nucleic acid-based linker, peptide linker, silane linker, polysaccharide linker, linker that is heat sensitive or radiation sensitive (IR, near-IR, UV) bond, linker which is a pH-sensitive bond, hydrolytic linker and linker obtained by covalent bonding, amide bonding, carbon-carbon multiple bond addition, Husgen cycloaddition to azidoalkyne, Diels-Alder reaction, disulfide bonding, Michael addition, silane reaction, nucleophilic ring opening reaction of urethane, epoxide, non-aldol carbonyl and 1,3-dipolar addition reaction or cycloaddition reaction such as tosylation.

(4) The nucleic acid encoding the humanized antibody of the present invention is

- 11 044968 vector containing nucleic acid, host cell containing the vector.

The present invention relates to a nucleic acid encoding a humanized antibody or antigen binding fragment thereof described in (1). Since the amino acid sequences of the variable heavy chain region and the variable light chain region of the humanized antibody of the present invention are disclosed in SEQ ID NO: 1 to SEQ ID NO: 8, those skilled in the art can obtain a nucleic acid encoding a humanized antibody having these amino acid sequences. When the humanized antibody of the present invention is intended to be expressed using a host cell as described below, a codon optimized for expression in that host is preferably selected as the codon of the nucleic acid encoding it.

The mutated heavy chain variable region described in (1) can be obtained by subjecting the nucleic acid encoding the heavy chain variable region shown in SEQ ID NO: 1-4, for example, to site-directed mutagenesis (for example, Nucleic Acid Research, Vol. 10, No. 20, p.64876500, 1982, the contents of which are incorporated by reference in their entirety, which is a well known method. The mutated light chain variable region can also be obtained by subjecting the nucleic acid encoding the light chain variable region shown in SEQ ID NO: 5-8, for example, to site-directed mutagenesis.

An expression vector containing such a nucleic acid can be prepared. The vector may optionally contain, in addition to the nucleic acid encoding the humanized antibody of the present invention, a Kozak sequence to enhance translation efficiency, a signal sequence that promotes secretion of the humanized antibody of the present invention into the medium upon introduction into a host, a promoter sequence, and the like. The vector that can be used in the present invention can be selected from those commonly used in the relevant art, and plasmid vectors, especially pcDNA3.4 used in the following example, are preferred.

Moreover, the present invention relates to a host cell containing the above-mentioned expression vector. Since the host cell thus obtained contains a nucleic acid encoding a humanized antibody or an antigen-binding fragment thereof described in (1), it can be used in the method for producing a humanized antibody to be described later in (5). As the method for introducing the gene into a cell, a method commonly used in the relevant art, for example, a method known to those skilled in the art, such as the calcium phosphate method, the electroporation method, the lipofection method and the DEAE-dextran method, can be used. The administration method using the lipofection method is particularly preferred, as shown in the following example.

The host cell used for this purpose may be those cells which are commonly used in the relevant art. Examples of such host cells include CHO cells, 293 cells, Escherichia coli, Pichia yeast, Sf9 cells and the like. An expression system kit for expressing the target protein is also now commercially available. The ExpiCHO system (Thermo Fisher Scientific), used in the following example, is particularly preferred for rapid and reliable expression of the target protein.

(5) A method for producing the humanized antibody of the present invention.

The present invention also relates to a method for producing a humanized antibody or an antigen-binding fragment thereof as described in (1). The production method includes inserting the nucleic acid encoding the humanized antibody of the present invention, or an antigen-binding fragment thereof, described in paragraph (4) above into an expression vector, introducing this nucleic acid into a host cell using an expression vector containing the nucleic acid, culturing the cell -host after administration of the nucleic acid and obtaining the humanized antibody of the present invention from the host cell culture supernatant using purification methods such as chromatography and the like.

Conveniently and preferably, the humanized antibody or antigen binding fragment thereof of the present invention is secreted into a culture supernatant by culturing a host cell. Therefore, as described in (4), it is desirable to construct a vector and select a host cell such that the host cell effectively secretes the antibody of the present invention into the culture supernatant.

The humanized antibody or antigen binding fragment thereof of the present invention can be obtained from the culture supernatant using purification methods such as chromatography and the like. As means for chromatography, various means known in the relevant art can be used, such as affinity chromatography, ion exchange chromatography, size exclusion chromatography and the like. Particularly preferred is affinity chromatography using a Protein A column, as used in the following example.

- 12 044968 (6) Method of treating cancer.

The present invention relates to a method of treating cancer using a humanized antibody or antigen binding fragment thereof described in (1), or an antibody-drug conjugate. That is, cancer can be treated by administering an effective amount of a pharmaceutical composition comprising a humanized antibody or antigen binding fragment thereof of the present invention and a pharmaceutically acceptable carrier to a subject affected by cancer overexpressing mucin subtype 5AC. Since the humanized antibody or antigen-binding fragment thereof or antibody-drug conjugate of the present invention binds to 5AC subtype mucin and inhibits the proliferation of cancer cells overexpressing 5AC subtype mucin, treatment of cancer can be expected. The dosage form and method of administration of the pharmaceutical composition in this case are the same as those described for the pharmaceutical composition in (2).

Examples of cancer to be treated in accordance with the present invention include pancreatic cancer, thyroid cancer, liver cancer, colorectal cancer, gastric cancer, urothelial cancer, breast cancer, cervical cancer, ovarian cancer and endometrial carcinoma and, in In particular, pancreatic cancer can be effectively treated.

Examples of cancer to be treated in accordance with the present invention also include bile duct cancer.

There are numerous reports suggesting that mucin subtype 5AC is an antigen carrier for CA19-9 (PLoS ONE (December 2011, vol. 6, issue 12, e29180, p. 1-10)). Therefore, examples of cancer to be treated according to the present invention also include biliary tract cancer, uterine cancer, lung cancer and esophageal cancer overexpressing CA19-9, and can be effectively treated.

As used herein, the term subject refers to a human or animal such as a mouse, rat, monkey, guinea pig, chimpanzee, sheep, goat, dog, cat, pig, cattle, horse or the like, preferably human, but not specifically limited to.

As used herein, the term effective amount is an amount that can provide beneficial effects of treating cancer in a subject. The effective amount administered to a subject varies depending on the type of subject, the body weight of the subject, the dosage form (tablet, injection, etc.) and route of administration (oral administration, parenteral administration, etc.), the severity of the cancer, and the like. similar. Doctors and veterinarians can consider these factors and determine the appropriate effective amount.

When treating cancer by administering a humanized antibody or an antigen binding fragment or antibody-drug conjugate thereof of the present invention, the number of cancer cells is reduced and/or the size of the tumor is reduced.

(7) Method for diagnosing cancer.

The present invention relates to a method for diagnosing cancer using a humanized antibody or antigen-binding fragment thereof described in (1), or an antibody-drug conjugate.

In one embodiment, a humanized antibody or antigen binding fragment thereof or an antibody-drug conjugate of the present invention is labeled so that it can be detected, cells or tissues are collected from a subject and a healthy subject, the collected cells or tissues are contacted with the labeled humanized antibody of the present invention, measuring the amount of label in the collected cells or tissues and comparing the amount of labeled antibody bound to cells or tissues taken from a subject with the amount of labeled antibody bound to cells or tissues taken from a healthy subject, whereby cancer diagnosis can be made. When the amount of label found in a cell or tissue taken from a subject is greater than the amount of label found in a cell or tissue taken from a healthy subject, the subject may be diagnosed with a cancer overexpressing mucin subtype 5AC.

As used herein, the term healthy subject refers to a healthy person who is not clearly suffering from cancer, as opposed to the subject diagnosed with cancer in the present invention. In such healthy subjects, overexpression of the 5AC mucin subtype is not observed and the subject can be used as a control.

As used herein, the term label means any label that binds to the humanized antibody or antigen binding fragment thereof of the present invention and enables its detection. Specific examples of the label include fluorescent labeling agents.

In another embodiment of the invention, a label is attached to a humanized antibody or an antigen binding fragment thereof of the present invention so that it can be detected by a cell or tissue of a subject and a cell or tissue of a healthy subject comes into contact with the labeled humanized antibody or an antigen binding fragment thereof of the present invention,

- 13 044968 measure the amount of label in a cell or tissue of a subject and a cell or tissue of a healthy subject, and the amount of label measured in a cell or tissue of a subject is compared with a standard amount, which is the amount of label measured in a cell or tissue of a healthy subject. When the amount measured in a tissue or cell of a subject is significantly higher than the standard amount, the subject may be diagnosed with a cancer overexpressing mucin subtype 5AC.

As used herein, the term standard amount refers to the amount of label measured in a healthy subject and indicates the overall level of expression of the 5AC subtype mucin in non-malignant individuals and the amount of label that does not specifically bind to cells or accumulate in tissues. Therefore, when the amount of tracer measured in a subject diagnosed with cancer exceeds the standard amount, overexpression of the 5AC subtype mucin is assumed. In this case, the subject may be diagnosed with cancer. In this method, contact between a cell or tissue of a subject and a labeled antibody of the present invention can be accomplished using a cell or tissue obtained from the subject, and in an in vitro embodiment. It is also possible to contact a cell or tissue of a subject and a labeled antibody of the present invention in an in vivo embodiment without removing the cell or tissue from the subject.

In another embodiment, a humanized antibody or antigen binding fragment thereof of the present invention is labeled so that it can be detected, cells or tissues are collected from a subject, the collected cells or tissues are contacted with the labeled humanized antibody of the present invention, the amount of label in the collected cells or tissues is measured and compared the amount of labeled antibody bound to cells or tissues taken from a subject with the amount of labeled antibody bound to normal cells or tissues, which can be used to diagnose cancer. When the amount of label found in a cell or tissue taken from a subject is greater than the amount of label found in a normal cell or tissue, the cell or tissue can be diagnosed as being affected by a cancer overexpressing subtype 5AC mucin.

As used herein, the term normal cell or tissue refers to a cell or tissue taken from a subject to be diagnosed with cancer in accordance with the present invention, or from a healthy subject, and previously diagnosed as a normal cell or tissue. Such normal cells or tissues do not overexpress mucin of the 5AC subtype and can be used as a control.

In another embodiment, a humanized antibody or antigen-binding fragment thereof of the present invention is labeled so that it can be detected, the labeled humanized antibody or antigen-binding fragment thereof is administered to a subject, the label is detected in the subject's tissue, and accumulation of the labeled antibody is detected, whereby cancer can be diagnosed. When accumulation of a labeled humanized antibody or antigen-binding fragment thereof is detected in a subject's tissue, the subject is diagnosed with a cancer that overexpresses mucin subtype 5AC. In this method, tissue label detection can be performed using tissue obtained from a subject and in an in vitro embodiment. It is also possible to perform label detection in tissue of a subject in an in vivo embodiment without removing tissue from the subject.

In another embodiment of the invention, a detectable label is attached to a humanized antibody or antigen binding fragment thereof of the present invention, and the label is detected in the tissue of a subject to whom the labeled humanized antibody or antigen binding fragment thereof is administered. When accumulation of a labeled humanized antibody or antigen-binding fragment thereof is detected in a subject's tissue, the subject may be diagnosed with cancer.

When a subject is diagnosed with a cancer overexpressing mucin subtype 5AC using the method described above, the cancer can be treated by administering to the subject a pharmaceutical composition containing a humanized antibody or antigen binding fragment thereof described in (1) and a pharmaceutically acceptable carrier.

Specific examples of the label described herein include, but are not specifically limited to, labeling with a fluorescent labeling agent, as long as they can detect the humanized antibody or antigen binding fragment thereof of the present invention. Linking between the antibody and the label can be accomplished through a linker described in (3) antibody-drug conjugate.

(8) Method for measuring cancer volume.

The present invention relates to a method for measuring the volume of cancer overexpressing mucin subtype 5AC in a subject using a humanized antibody or antigen binding fragment thereof described in (1). As used herein, the term cancer volume refers to the volume of the portion of a subject's tissue consisting of cancer cells, and excludes the volume of the portion consisting of normal, non-cancerous cells.

- 14 044968

In one embodiment, a humanized antibody or antigen binding fragment thereof of the present invention is labeled so that it can be detected, tissue from the subject and tissue from a healthy subject are contacted with the labeled humanized antibody or antigen binding fragment thereof, and the amount of label in the subject tissue and tissue from the healthy subject is measured. . The amount of tracer measured in the subject's tissue is compared with a standard amount, which represents the amount of tracer measured in the tissue of a healthy subject, and the part in which the amount measured in the subject exceeds the standard amount represents cancerous tissue, its volume is measured, whereby the volume of cancer can be measured.

As used herein, the term standard amount refers to the amount of label measured in a healthy subject and indicates the overall level of expression of the 5AC subtype mucin in non-malignant individuals and the amount of label that does not specifically bind to cells or accumulate in tissues. Thus, a portion in which the amount of tracer exceeds the standard amount in the subject's tissue can be considered as cancerous tissue, and the volume of cancer can be determined by measuring the volume of that portion.

In yet another embodiment of the invention, a detectable label is attached to a humanized antibody or antigen-binding fragment thereof of the present invention, the label is detected in the tissue of a subject to whom the labeled antibody or antigen-binding fragment thereof is administered, and the volume of the portion where accumulation of the labeled humanized antibody or antigen-binding fragment thereof is detected is measured. antigen-binding fragment, whereby the volume of cancer can be measured.

To measure the volume of cancer, a label such as a fluorescent labeling agent and the like is detected in an image from a subject using a device that can detect the label, and the volume can be calculated from the size of the cancer tissue in which the label is detected in the image, using as necessary the corresponding calculation formula. Volume can also be determined by removing tissue from a sample cut from the subject and directly measuring the volume of the part where the mark is found.

Measuring the volume of cancer in a subject is useful for diagnosing the severity of cancer and the like. at the subject. In addition, when a subject is undergoing treatment, measuring the volume of cancer in the subject is useful in determining the effect of treatment.

Although the present invention is described in more detail below with reference to examples, the present invention is not limited to these examples.

Examples

Although the present invention is described in more detail below with reference to examples and comparative examples, the present invention is not limited to them. The experimental methods and test methods in the examples and comparative examples are as follows.

Experimental example 1. Preparation of various antibodies.

The amino acid sequences of the various variable regions with the signal sequence appended thereto and the amino acid sequences of the various constant regions were converted into base sequences using codons suitable for expression in CHO cells. A Kozak sequence was added to the start codon site of the signal sequence, and a stop codon was added to the C-terminal side of the constant region. In addition, restriction enzyme sites were added before the Kozak sequence and after the stop codon so that they could be introduced into the expression gene transfer site of the mammalian cell expression plasmid (pcDNA3.4). Each DNA fragment constructed in this way was obtained through chemical synthesis. The DNA fragment containing the variable region and the DNA fragment containing the constant region were ligated by fusion PCR to form the desired H chain and the desired L chain.

The produced genes for various antibodies were subjected to restriction digestion and then purified. Similarly, a plasmid for transient expression in mammalian cells (pcDNA3.4) was also treated with the same restriction enzyme and then purified. Both fragments were mixed in the appropriate ratio and ligated. The ligation reaction solution was mixed with competent Escherichia coli DH5α cells and transformed. Colony PCR, isolation of one colony, extraction of the plasmid from a small volume of culture medium and determination of the base sequence of the insert were carried out from the resulting transformant and plasmid (Escherichia coli clone), in which the constructed full-length antibody gene was correctly inserted in the intended direction with the selected sequence. Large scale cultivation was carried out on the selected Escherichia coli clone and plasmid extraction and purification was carried out, including the endotoxin removal step. The absorbance of the purified plasmid was measured at a wavelength of 260 nm and the concentration was calculated.

Using the ExpiCHO system (Thermo Fisher Scientific), CHO cells were transiently expressed. From each H chain expression plasmid and each expression

- 15 044968 L chain plasmids were selected for one H chain and one L chain to obtain the desired combination, transfected by lipofection, cultured and fed. The culture medium was collected 7–13 days after transfection. The centrifuged and filtered culture supernatant was added to a protein A column, and the antibody was purified by conventional affinity chromatography on the column (post-adsorption wash, acidic buffer elution, neutralization of eluate). The absorbance of the purified antibody was measured at a wavelength of 280 nm and the concentration was calculated.

Antibodies 1 to 20 were prepared using the method described above. The antibody numbers assigned to the combination of the heavy chain variable region and the light chain variable region are shown below:

antibody 1: H01L01, antibody 2: H01L02, antibody 3: H01L03, antibody 4: H01L04, antibody 5: H02L01, antibody 6: H02L02, antibody 7: H02L03, antibody 8: H02L04, antibody 9: H03L01, antibody 10: H03L02, antibody 11: H03L03, antibody 12: H03L04, antibody 13: H04L01, antibody 14: H04L02, antibody 15: H04L03, antibody 16: H04L04, antibody 17: H05L05, antibody 18: H06L06, antibody 19: H05L07, antibody 20 : H07L08.

As used herein, the designations H01, H02, H03 and H04 are the heavy chain variable regions respectively shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and the designations L01, L02, L03 and L04 are the light chain variable regions shown respectively in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. Antibodies of the present invention used in Examples 1-1 to 1 -16, consist of combinations of heavy chain constant region 1 and light chain constant region 1, as well as heavy chain variable regions and light chain variable regions of the above-mentioned antibody 1 to 16.

On the other hand, H05 and H06 are the heavy chain variable regions of the humanized antibody disclosed in Patent Document 3, and L05, L06 and L07 are the light chain variable regions of the humanized antibody disclosed in Patent Document 3. The humanized antibodies of Patent Document 3 used Comparative Examples 1-1 to 1-3 are humanized antibodies of the prior art, which consist of combinations of heavy chain constant region 1 and light chain constant region 1, as well as heavy chain variable regions and light chain variable regions, of the above-mentioned antibodies 17-19. The amino acid sequences of H05 and H06 are shown in SEQ ID NO: 9 and SEQ ID NO: 10, and the amino acid sequences of L05, L06 and L07 are shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. Amino acid sequences of H05 and H06 and the amino acid sequences of L05-L07 are shown in FIG. 3.

In addition, H07 is the heavy chain variable region of the chimeric antibody disclosed in Patent Document 1, L08 is the light chain variable region of the chimeric antibody disclosed in Patent Document 1. The chimeric antibodies used in Comparative Examples 1 to 4 are humanized antibodies , known in the art, which consist of combinations of heavy chain constant region 1 and light chain constant region 1, as well as heavy chain variable regions and light chain variable regions of the above-mentioned antibody 20. The amino acid sequence of H07 is shown in SEQ ID NO: 14, and the amino acid sequence of L08 is shown in SEQ ID NO: 15. In addition, the amino acid sequences of H07 and L08 are shown in FIG. 4.

Test case 1. Evaluation of the physical properties of the antibody.

The resulting various antibodies were diluted with 150 mM acetate buffer (pH 4.7) to an antibody concentration of approximately 1 mg/ml. Since the antibody concentration in Comparative Example 1-2 was approximately 0.5 mg/ml, it was used without dilution. In these different antibody solutions, the temperature of the midpoint of denaturation, the temperature of the onset of aggregation and the particle distribution were determined.

- 16 044968 sizes using the UNcle device (manufactured by UNchained Labs Co., Ltd.), which is a device for assessing the physical properties of proteins. Specifically, to measure the average denaturation temperature and onset of aggregation temperature, various antibodies were heated from approximately room temperature to 75 °C at a rate of HS/min and held for 30 s, and the fluorescence spectrum measurement and static light scattering measurement were performed simultaneously in time. Based on the results of measuring the fluorescence spectrum, the temperature of the midpoint of the displacement of the center of gravity of the peak was determined, i.e. the temperature of the midpoint of denaturation, using the phenomenon that the center of gravity of the fluorescence spectrum peak of an amino acid inherent in an antibody shifts when the antibody is denatured. In addition, based on the results of measuring static light scattering, the temperature at which scattered light with a wavelength of 266 nm begins to intensify was obtained, i.e. the temperature at which aggregation begins, taking advantage of the fact that the scattering intensity becomes stronger during aggregation. Particle size distribution was determined by measuring dynamic light scattering of various antibodies at near room temperature.

For antibodies 1-20, the assessment of the physical properties of various antibodies was carried out in accordance with Test Example 1. The results are presented in table. 1. As mentioned above, Examples 1-1 to 1-16 are humanized antibodies of the present invention, Comparative Examples 1-1 to 1-3 are humanized antibodies from Patent Document 3, and Comparative Examples 1-4 are chimeric antibodies from patent document 1.

Table 1

Midpoint Denaturation Temperature (°C) Aggregation onset temperature (°C) Most common particle size (nm) Humanized other antibody (present invention) example 1-1 antibody 1 59.7 66.4 10.6 example 1-2 antibody 2 60.2 70.0 9.9 example 1-3 antibody 3 59.4 67.8 9.9 example 1-4 antibody 4 58.8 64.8 9.9 example 1-5 antibody 5 59.6 68.1 10.7 example 1-6 antibody 6 59.6 66.6 9.8 example 1-7 antibody 7 59.5 69.6 9.8 example 1-8 antibody 8 59.3 68.5 9.9 example 1-9 antibody 9 59.1 65.8 9.9 example 1-10 antibody 10 59.6 68.7 10.6 example 1-11 antibody 11 59.1 68.2 10.7 example 1-12 antibody 12 59.3 65.5 11.5 example 1-13 antibody 13 58.5 69.8 9.9 example 1-14 antibody 14 63.5 64.3 10.6 example 1-15 antibody 15 59.8 67.3 10.6 example 1-16 antibody 16 59.5 70.4 9.9 Humanized Other Antibody (Patent Document 3) Comparative Example 1-1 antibody 17 59.1 61.0 12.22 Comparative example 1-2 antibody 18 55.5 55.9 11.76 Comparative Example 1-3 antibody 19 60.5 61.1 10.86 Chimeric Antibody (Patent Document 1) Comparative Example 1-4 antibody 20 49.5 45.4 203.15

The average denaturation temperature was low and was 49.5°C for the chimeric antibody and high for the humanized antibody of the present invention and was 58.5-63.5°C. It has been found that the humanized antibody of the present invention has stable physical properties compared to the chimeric antibody.

The aggregation onset temperature was lowest for the chimeric antibody at 45.4°C, moderate for the humanized antibody of Patent Document 3 at approximately 55,961.1°C, and highest for the humanized antibody of the present invention at 64.3°C.

70.4°C. It was found that the humanized antibody of the present invention has stable physical properties compared with the chimeric antibody and the humanized antibody of Patent Document 3.

The particle size of the chimeric antibody was approximately 203 nm and clear aggregation was detected. In the humanized antibody of the present invention, the particle size was about 10 nm, and the humanized antibody was found to be stable and present as monodisperse particles.

Based on the above results, it has been found that the humanized antibody of the present invention has more stable physical properties than the chimeric antibody of Patent Document 1 and the humanized antibody of Patent Document 3. That is, the antibody of the present invention is expected to stably produce a compound of interest without aggregation etc., even if it undergoes an additional production step (for example, the step of binding a conjugate to a fluorescent labeling agent).

Test Example 2. In vitro binding activity evaluation.

Various antibodies were added to a 96-well plate coated with various antigens, and the plate was incubated for 1 h at room temperature. The plate was washed, the blocking solution was added, and the mixture was left for 30 min at room temperature. The plate was washed, HRP-conjugated anti-human IgG was added, and the mixture was incubated for 1 hour or longer at room temperature. The plate was washed, color development reagent (Bethyl Laboratories, Inc.) was added, and color development was measured using a microplate reader (VersaMax, Molecular Devices, LLC).

The results of assessing the binding activity between antibody and antigen are shown in table. 2-4. In table 2 and 3, a tumor mass obtained from a cancer mouse model transplanted with the human pancreatic cancer cell line SW1990 was homogenized, and the supernatant (crude antigen extract solution) after centrifugation was used as the antigen. In table 4, the mucin fraction obtained by isopycnic centrifugation using cesium chloride from a solution of the crude antigen extract was used as the antigen.

For antibodies 1-20, the in vitro binding activity of various antibodies was assessed in accordance with Test Example 2. The results are presented in table. 2-4.

In table 2 shows the ratio of in vitro binding activity of antibodies 2-8 to antibody 1 (example 2-1), in table. 3 shows the ratio of in vitro binding activity of antibodies 10-16 to antibody 9 (example 2-9), and table 4 shows the ratio of in vitro binding activity of antibodies 3, 4, 8, 16-19 to antibody 20 (comparative example 2- 4).

As can be seen from these results, it was found that the humanized antibody of the present invention has an in vitro binding activity comparable to that of the antibody of Comparative Examples 2 to 4, which is a chimeric antibody in Patent Document 1. In general, humanizing a chimeric antibody often reduces the activity binding to antigen. Surprisingly, the humanized antibody of the present invention retained the same level of binding activity as the chimeric antibody. In addition, it was found that the humanized antibody of the present invention has in vitro binding activity comparable to or higher than that of the humanized antibody of Patent Document 3.

table 2

Humanized Antibody (Present Invention) example 2-1 antibody 1 1.00 example 2-2 antibody 2 1.03 example 2-3 antibody 3 0.99 example 2-4 antibody 4 0.99 example 2-5 antibody 5 0.60 example 2-6 antibody 6 0.71 example 2-7 antibody 7 0.65 example 2-8 antibody 8 0.72

Table 3

Humanized Antibody (Present Invention) example 2-9 antibody 9 1.00 example 2-10 antibody 10 0.96 example 2-11 antibody 11 1.25

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example 2-12 antibody 12 0.91 example 2-13 antibody 13 0.90 example 2-14 antibody 14 1.04 example 2-15 antibody 15 1.36 example 2-16 antibody 16 0.88

Table 4

Humanized Antibody (Present Invention) example 2-3 antibody 3 1.56 example 2-4 antibody 4 0.97 example 2-8 antibody 8 0.70 example 2-16 antibody 16 1.09 Humanized Antibody (Patent Document 3) Comparative Example 2-1 antibody 17 0.75 Comparative Example 2-2 antibody 18 0.13 Comparative Example 2-3 antibody 19 0.49 Chimeric antibody (patent document 1) Comparative Example 2-4 antibody 20 1.00

Test Case 3: In Vivo Tumor Accumulation Assessment.

The human pancreatic cancer cell line SW1990 (1*10 ⁷ cells) was injected subcutaneously into Balb/c nude mice from side to back. When tumor size reached approximately 100-200 mm ² 13-16 days after SW1990 transplantation, mice were injected with 2 mg/kg of various fluorescently labeled antibodies via the tail vein (n=2). The antibody was fluorescently labeled using Dye SE CF (trade name) protein labeling kits (manufactured by Biotium). Tumor volume was calculated using the following calculation formula:

tumor volume=(minor axis of tumor ² *major axis of tumor)/2.

Assessment of tumor accumulation over time.

As an assessment of tumor accumulation over time in vivo, fluorescence emitted by antibody-treated mice was photographed using IVIS LuminaIII (Perkin Elmer Inc.) before, one day after, 2 days after, 3 days after, and 7 days after administration of various antibodies with a fluorescent label.

Assessment of tumor accumulation at end point.

In addition, 7 days after administration of various fluorescently labeled antibodies, the tumor, liver, and kidney were removed and photographed using IVIS Lumina III (Perkin Elmer Inc.). The photographed image data was analyzed using an analysis computer (Living Image software) and numerical data were obtained. The numerical value was calculated as the average brightness ([f/s/cm ² /sr]/[μW/cm ² ]) in the area indicated in the image.

Tumor accumulation was calculated as follows. The tumor accumulation amount here represents the average brightness of the tumor area calculated by the above formula, and the liver or kidney accumulation represents the average brightness of the liver or kidney area calculated by the above formula.

Tumor-liver ratio = tumor accumulation 7 days after fluorescently labeled antibody/liver accumulation 7 days after fluorescently labeled antibody.

Tumor-kidney ratio = tumor accumulation 7 days after fluorescently labeled antibody/kidney accumulation 7 days after fluorescently labeled antibody.

For antibodies 3, 4, 8, 9, 10, 16-20, the accumulation of various antibodies in the tumor in vivo was assessed in accordance with Test Example 3. The results are shown in table. 5 and fig. 5. In table. Figure 5 shows the accumulation of various antibodies in the tumor in vivo (average n=2).

Table 5

Tumor-liver relationship Tumor-kidney relationship Humanized example 3-1 antibody 3 23.5 16.8

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Claims (7)

antibody (present invention) example 3-2 antibody 4 11.7 12.7 example 3-3 antibody 8 14.8 17.7 example 3-4 antibody 9 20.5 15.4 example 3-5 antibody 10 17.5 14.9 example 3-6 antibody 16 18.1 14.0 Humanized Antibody (Patent Document 3) Comparative Example 3-1 antibody 17 14.1 9.4 Comparative Example 3-2 antibody 18 17.7 18.2 Comparative Example 3-3 antibody 19 15.3 14.3 Chimeric Antibody (Patent Document 1) Comparative Example 3-4 antibody 20 6.0 7.0 Based on the results of table. 5, it was found that the humanized antibody of the present invention (Antibodies 3, 4, 8, 9, 10, 16) has a higher tumor-to-liver ratio and a higher tumor-to-kidney ratio than the chimeric antibody (Antibody 20) of Patent Document 1; those. a humanized antibody with high tumor accumulation was obtained. In fig. 5 shows the accumulation over time of the antibody of example 3-1 (antibody 3), which has the highest tumor-liver ratio in table. 5. The antibody was found to accumulate in a tumor on the lower right side of the mouse's back. As described above, in general, since humanization of a chimeric antibody often reduces antigen binding activity, tumor accumulation in vivo is also often reduced. Surprisingly, the humanized antibody of the present invention was found to exhibit much higher tumor accumulation than the chimeric antibody. When it is intended to produce an antibody-drug conjugate conjugated with a drug having a very strong cell killing effect, high tumor accumulation of the antibody as its delivery vehicle (i.e., the humanized antibody of the present invention) is very useful because it not only enhances the antitumor effect, but can also reduce side effects on normal tissues. Industrial applicability The present invention relates to a humanized antibody or antigen-binding fragment thereof capable of binding to subtype 5AC mucin. The humanized antibody of the present invention has stable physical properties, as well as the ability to bind to mucin of the 5AC subtype and accumulates better in the tumor. Thus, the humanized antibody or antigen-binding fragment thereof of the present invention is extremely useful for the treatment and/or diagnosis of cancer overexpressing 5AC mucin subtype and, further, for preparing an antibody-drug conjugate for the treatment and/or diagnosis of cancer overexpressing 5AC mucin subtype . This application is based on Patent Application No. 2019-191560, filed in Japan (filing date: October 18, 2019), the entire contents of which are incorporated into this application. CLAIM 1. A humanized antibody capable of binding to mucin of the 5AC subtype, containing a heavy chain variable region consisting of: (1) an amino acid sequence having at least 98% sequence identity with the amino acid sequence SEQ ID NO: 1, (2) an amino acid sequence having at least 98% sequence identity with the amino acid sequence SEQ ID NO: 2, (3) an amino acid sequence a sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 3, or (4) an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 4; and a light chain variable region consisting of: (5) an amino acid sequence having at least 98% sequence identity - 20 044968 amino acid sequence SEQ ID NO: 5, (6) an amino acid sequence having at least 98% sequence identity with the amino acid sequence SEQ ID NO: 6, (7) an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 7, or (8) an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 8. 2. The humanized antibody according to claim 1, containing a heavy chain variable region consisting of: (1) an amino acid sequence having at least 98% sequence identity to the amino acid sequence SEQ ID NO: 1, (3) an amino acid sequence having at least 98% sequence identity to the amino acid sequence SEQ ID NO: 3, or (4) an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 4; and a light chain variable region consisting of: (5) an amino acid sequence having at least 98% sequence identity with the amino acid sequence SEQ ID NO: 5, (6) an amino acid sequence having at least 98% sequence identity with the amino acid sequence SEQ ID NO: 6, (7) amino acid a sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 7, or (8) an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 8. 3. The humanized antibody according to claim 2, containing a heavy chain variable region consisting of: (1) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1, (3) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 3, or (4) an amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO: 4; and a light chain variable region consisting of: (5) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 5, (6) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 6, (7) an amino acid sequence, that is at least 99% identical to the amino acid sequence of SEQ ID NO: 7, or (8) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 8. 4. The humanized antibody of claim 2, comprising a heavy chain variable region consisting of: (1) amino acid sequence SEQ ID NO: 1, (3) amino acid sequence SEQ ID NO: 3, or (4) amino acid sequence SEQ ID NO: 4 ; and a light chain variable region consisting of: (5) amino acid sequence SEQ ID NO: 5, (6) amino acid sequence SEQ ID NO: 6, (7) amino acid sequence SEQ ID NO: 7, or (8) amino acid sequence SEQ ID NO: 8. 5. A humanized antibody capable of binding to mucin of the 5AC subtype, containing: (1) a heavy chain variable region consisting of an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, and (7) a light chain variable region consisting of an amino acid sequence having at least 95% identity in sequence with the amino acid sequence SEQ ID NO: 7. 6. The humanized antibody according to claim 5, comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 7. 7. Composition for the treatment of cancer overexpressing mucin subtype 5AC, containing humane -