JP2022058256A - Agents, pharmaceuticals, and pharmaceutical compositions for inhibiting growth of cd20-positive cancer cells resistant to type ii anti-cd20 antibody and for treating cd20-positive cancer resistant to type ii anti-cd20 antibody - Google Patents

Abstract

To provide methods for treating a CD20-positive cancer resistant to a type II anti-CD20 antibody or a CD20-positive cancer that has relapsed after a treatment with a type II anti-CD20 antibody.SOLUTION: Provided is a method in which a type II anti-CD20 antibody is used in combination with at least one compound selected from the group consisting of bendamustine and its salts and prodrugs, or alternatively natural killer cells affected by at least one compound selected from the group consisting of bendamustine and its salts and prodrugs.SELECTED DRAWING: None

Description

There is an application for exception of loss of novelty

The present invention relates to agents, pharmaceuticals, and pharmaceutical compositions for the growth inhibition of type II anti-CD20 antibody resistant CD20 positive cancer cells and the treatment of type II anti-CD20 antibody resistant CD20 positive cancers.

Patent Document 1 describes a combination of antineoplastic agents such as bendamustine and obinutuzumab. However, Patent Document 1 does not describe an effective treatment method for CD20-positive cancer resistant to type II anti-CD20 antibody.

Special table 2019-528282

In CD20-positive cancer, the cancer may be resistant to type II anti-CD20 antibody or may relapse after treatment with type II anti-CD20 antibody.
The present inventors used the type II anti-CD20 antibody for a CD20-positive cancer resistant to the type II anti-CD20 antibody or a CD20-positive cancer that recurred after treatment containing the type II anti-CD20 antibody. Means to enhance the effect of treatment were investigated.

By using bendamustine in combination with CD20-positive cancer resistant to type II anti-CD20 antibody or CD20-positive cancer that has relapsed after treatment containing type II anti-CD20 antibody, the present inventors II. It was found that the effect of treatment with type anti-CD20 antibody was enhanced.
Based on these findings, the following inventions are provided.

[1] Type II anti-CD20 antibody resistant CD20 positive, which contains type II anti-CD20 antibody and is used in combination with chemotherapy including administration of at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug. An agent for suppressing the growth of cells.
[2] Proliferation of obinutuzumab-resistant CD20-positive cancer cells by combination with chemotherapy including administration of at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug containing type II anti-CD20 antibody. Agent to suppress.
[3] Cells of type II anti-CD20 antibody-resistant CD20-positive cancer containing at least one compound selected from the group consisting of bendamustine and its salts and prodrugs and used in combination with treatment with type II anti-CD20 antibody. An agent for suppressing growth.
[4] Proliferation of cells of type II anti-CD20 antibody-resistant CD20-positive cancer in combination with treatment with type II anti-CD20 antibody containing at least one compound selected from the group consisting of bendamustine and its salts and prodrugs. Agent to suppress.
[5] The agent according to any one of [1] to [4], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[6] The agent according to any one of [1] to [5], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment.
[7] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody [1] to Any agent of [6].
[8] The agent according to any one of [1] to [7], wherein the type II anti-CD20 antibody is obinutuzumab.
[9] Type II anti-CD20 antibody, which is administered in combination with at least one compound selected from the group consisting of bendamustine and its salts and prodrugs, simultaneously, separately or sequentially. A drug for suppressing the growth of cells in antibody-resistant CD20-positive cancer.
[10] The drug according to [9], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[11] The drug according to [9] or [10], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment.
[12] The type II anti-CD20 antibody CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody monotherapy after induction therapy using the type II anti-CD20 antibody, [9] to The drug according to any one of [11].
[13] The drug according to any one of [9] to [12], wherein the type II anti-CD20 antibody is obinutuzumab.
[14] Introduction with type II anti-CD20 antibody, which contains type II anti-CD20 antibody and is used in combination with chemotherapy comprising administration of at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs. A drug for suppressing the growth of cancer cells that have recurred after the start of maintenance therapy with type II anti-CD20 antibody alone after therapy.

[2-1] Type II anti-CD20 antibody resistant CD20 containing type II anti-CD20 antibody and used in combination with chemotherapy comprising administration of at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs. A pharmaceutical composition for treating positive cancer.
[2-2] Type II anti-CD20 antibody resistant CD20 positive by combination with chemotherapy including administration of at least one compound selected from the group consisting of bendamustine and its salts and prodrugs containing type II anti-CD20 antibody. A pharmaceutical composition for treating antibodies.
[2-3] Type II anti-CD20 antibody-resistant CD20-positive cancer containing at least one compound selected from the group consisting of bendamustine and its salts and prodrugs and used in combination with treatment with type II anti-CD20 antibody. A pharmaceutical composition for treatment.
[2-4] Treatment of type II anti-CD20 antibody-resistant CD20-positive cancer in combination with treatment with type II anti-CD20 antibody containing at least one compound selected from the group consisting of bendamustine and its salts and prodrugs. Pharmaceutical composition for.
[2-5] Type II anti-CD20 antibody, combined with at least one compound selected from the group consisting of bendamustine and its salts and prodrugs, administered simultaneously, separately or sequentially. A pharmaceutical composition for treating anti-CD20 antibody-resistant CD20-positive cancer.
[2-6] The pharmaceutical composition according to any one of [2-1] to [2-5], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[2-7] The pharmaceutical composition according to any one of [2-1] to [2-6], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment. thing.
[2-8] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [ The pharmaceutical composition according to any one of 2-1] to [2-7].
[2-9] The pharmaceutical composition according to any one of [2-1] to [2-8], wherein the type II anti-CD20 antibody is obinutuzumab.

[3-1] To suppress the growth of cells of type II anti-CD20 antibody-resistant CD20-positive cancer in combination with chemotherapy including administration of at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug. Use of type II anti-CD20 antibody in the manufacture of the drug.
[3-2] A group consisting of bendamustine and its salts and prodrugs in the production of a drug for suppressing the growth of cells of type II anti-CD20 antibody-resistant CD20-positive cancer in combination with treatment with type II anti-CD20 antibody. Use of at least one compound selected from.
[3-3] A pharmaceutical composition for treating type II anti-CD20 antibody-resistant CD20-positive cancer in combination with chemotherapy including administration of at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug. Use of type II anti-CD20 antibody in the manufacture of.
[3-4] Selected from the group consisting of bendamustine and its salts and prodrugs in the manufacture of pharmaceutical compositions for treating type II anti-CD20 antibody resistant CD20-positive cancer in combination with treatment with type II anti-CD20 antibody. Use of at least one compound.
[3-5] At least one selected from the group consisting of type II anti-CD20 antibody and bendamustine and salts and prodrugs thereof in the manufacture of a pharmaceutical composition for treating type II anti-CD20 antibody resistant CD20-positive cancer. Use of compounds.
[3-6] Use of any of [3-1] to [3-5], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[3-7] Use of any of [3-1] to [3-6], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment.
[3-8] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [3] Use of any of -1] to [3-7].
[3-9] Use of any of [3-1] to [3-8], wherein the type II anti-CD20 antibody is obinutuzumab.

[4-1] In suppressing the growth of cells of type II anti-CD20 antibody-resistant CD20-positive cancer by combination with chemotherapy including administration of at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug. Type II anti-CD20 antibody for use.
[4-2] From the group consisting of bendamustine and its salts and prodrugs for use in suppressing the growth of cells of type II anti-CD20 antibody resistant CD20-positive cancer in combination with treatment with type II anti-CD20 antibody. At least one compound selected.
[4-3] At least one selected from the group consisting of type II anti-CD20 antibody and bendamustine and salts and prodrugs thereof for use in suppressing the growth of cells of type II anti-CD20 antibody resistant CD20-positive cancer. Combination of compounds.
[4-4] For use in the treatment of type II anti-CD20 antibody resistant CD20-positive cancer in combination with chemotherapy including administration of at least one compound selected from the group consisting of bendamustine and its salts and prodrugs. , Type II anti-CD20 antibody.
[4-5] At least one selected from the group consisting of bendamustine and its salts and prodrugs for use in the treatment of type II anti-CD20 antibody resistant CD20-positive cancer in combination with treatment with type II anti-CD20 antibody. Compound.
[4-6] A combination of a type II anti-CD20 antibody and at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs for use in the treatment of type II anti-CD20 antibody resistant CD20-positive cancer.
[4-7] A type II anti-CD20 antibody of [4-1] or [4-4], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[4-8] A compound of [4-2] or [4-5], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[4-9] The combination of [4-3] or [4-6], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[4-10] The type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer that has been treated with type II anti-CD20 antibody, [4-1], [4-4], and [4-7]. ] Any type II anti-CD20 antibody.
[4-11] The type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer that has been treated with type II anti-CD20 antibody, [4-2], [4-5], and [4-8]. ] Any one compound.
[4-12] The type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer that has been treated with type II anti-CD20 antibody, [4-3], [4-6], and [4-9]. ] Any combination.
[4-13] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody [4]. -1], [4-4], [4-7] and [4-10] type II anti-CD20 antibody.
[4-14] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody [4]. -2], [4-5], [4-8], and one compound of [4-11].
[4-15] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody [4]. -3], [4-6], [4-9], and any combination of [4-12].
[4-16] A type II anti-CD20 antibody according to any one of [4-1], [4-4], [4-7], [4-10], and [4-13], which is obinutuzumab.
[4-17] One of [4-2], [4-5], [4-8], [4-11], and [4-14], wherein the type II anti-CD20 antibody is obinutuzumab. One compound.
[4-18] One of [4-3], [4-6], [4-9], [4-12], and [4-15], wherein the type II anti-CD20 antibody is obinutuzumab. combination.

[5-1] For cells of type II anti-CD20 antibody-resistant CD20-positive cancer
i) Administering type II anti-CD20 antibody,
ii) A method for suppressing the growth of cells of type II anti-CD20 antibody resistant CD20 positive cancer, which comprises administering at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug.
[5-2] The method of [5-1], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[5-3] The method of [5-1] or [5-2], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience of type II anti-CD20 antibody treatment.
[5-4] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [ Any method from 5-1] to [5-3].
[5-5] The method according to any one of [5-1] to [5-4], which is an in vitro and non-human in vivo method.
[5-6] The method according to any one of [5-1] to [5-5], wherein the type II anti-CD20 antibody is obinutuzumab.

[6-1] For subjects with type II anti-CD20 antibody-resistant CD20-positive cancer
i) Administering type II anti-CD20 antibody,
ii) A method for treating type II anti-CD20 antibody resistant CD20-positive cancer, which comprises administering at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug.
[6-2] The method for treating [6-1], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[6-3] The treatment method of [6-1] or [6-2], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment.
[6-4] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [ The treatment method according to any one of 6-1] to [6-3].
[6-5] The therapeutic method according to any one of [6-1] to [6-4], wherein the type II anti-CD20 antibody is obinutuzumab.

[7-1] To increase the ADCC activity of type II anti-CD20 antibody against type II anti-CD20 antibody-resistant CD20-positive cancer cells containing at least one compound selected from the group consisting of bendamustine and its salts and prodrugs. Agent.
[7-2] The agent of [7-1], which is administered in vitro to natural killer cells separately or continuously at the same time as the type II anti-CD20 antibody.
[7-3] The agent of [7-2], wherein natural killer cells are derived from the therapeutic target.
[7-4] The agent of [7-1] administered to a subject separately or continuously in vivo at the same time as the type II anti-CD20 antibody.
[7-5] The agent according to any one of [7-1] to [7-4], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[7-6] The agent according to any one of [7-1] to [7-5], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment.
[7-7] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [ 7-1] to [7-6].
[7-8] The agent according to any one of [7-1] to [7-7], wherein the type II anti-CD20 antibody is obinutuzumab.
[7-9] Type II, which is a combination of at least one compound selected from the group consisting of bendamustine and its salts and prodrugs, and a type II anti-CD20 antibody, administered simultaneously, separately or sequentially. Anti-CD20 antibody-resistant drug for increasing ADCC activity of type II anti-CD20 antibody against CD20-positive cancer cells.
[7-10] The drug according to [7-9], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[7-11] The drug of [7-9] or [7-10], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment.
[7-12] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [ The drug according to any one of 7-9] to [7-11].
[7-13] The drug according to any one of [7-9] to [7-12], wherein the type II anti-CD20 antibody is obinutuzumab.

[8-1] At least selected from the group consisting of bendamustine and its salts and prodrugs in the manufacture of agents for increasing ADCC activity of type II anti-CD20 antibody against type II anti-CD20 antibody resistant CD20-positive cancer cells. Use of one compound.
Use of [8-1], wherein the agent is administered in vitro to natural killer cells separately or sequentially at the same time as the type II anti-CD20 antibody.
[8-3] Use of [8-2], in which natural killer cells are derived from the therapeutic target.
Use of [8-1], wherein the agent [8-4] is administered to the subject separately or consecutively in vivo at the same time as the type II anti-CD20 antibody.
[8-5] Use of any of [8-1] to [8-4], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[8-6] Use of any of [8-1] to [8-5], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment.
[8-7] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [ Use of any of 8-1] to [8-6].
[8-8] Use of any of [8-1] to [8-7], wherein the type II anti-CD20 antibody is obinutuzumab.

[9-1] At least selected from the group consisting of bendamustine and its salts and prodrugs for use to increase ADCC activity of type II anti-CD20 antibody against type II anti-CD20 antibody resistant CD20-positive cancer cells. One compound.
[9-2] A compound of [9-1] administered in vitro to natural killer cells separately or sequentially at the same time as type II anti-CD20 antibody.
[9-3] A compound of [9-2] from which natural killer cells are derived from the therapeutic target.
[9-4] A compound of [9-1] administered to a subject separately or continuously in vivo at the same time as a type II anti-CD20 antibody.
[9-5] A compound according to any one of [9-1] to [9-4], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[9-6] The compound of any one of [9-1] to [9-5], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience of type II anti-CD20 antibody treatment. ..
[9-7] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [ One compound of any one of 9-1] to [9-6].
[9-8] A compound according to any one of [9-1] to [9-7], wherein the type II anti-CD20 antibody is obinutuzumab.

[10-1] Type II anti-CD20 for type II anti-CD20 antibody-resistant CD20-positive cancer, which comprises administering to natural killer cells at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug. A method for increasing ADCC activity of an antibody.
[10-2] The method of [10-1], wherein at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs is administered in vitro.
[10-3] The method of [10-2], wherein the natural killer cells are derived from the therapeutic target.
[10-4] At least one compound selected from the group consisting of bendamustine and its salts and prodrugs is administered to the subject separately or sequentially in vivo at the same time as the type II anti-CD20 antibody [10-4]. -1] method.
[10-5] The method according to any one of [10-1] to [10-4], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[10-6] The method according to any one of [10-1] to [10-5], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment.
[10-7] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [ Any method from 10-1] to [10-6].
[10-8] The method according to any one of [10-1] to [10-7], wherein the type II anti-CD20 antibody is obinutuzumab.

[11-1] Type II anti-CD20 antibody against type II anti-CD20 antibody-resistant CD20-positive cancer cells containing natural killer cells reacted with at least one compound selected from the group consisting of bendamustine and its salts and prodrugs. Agent for increasing ADCC activity of.
[11-2] The agent of [11-1], which comprises natural killer cells exposed in vitro to at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs.
[11-3] [11-1] or [11-2] administered to type II anti-CD20 antibody-resistant CD20-positive cancer cells separately or continuously in vitro at the same time as type II anti-CD20 antibody. Agent.
[11-4] The agent according to any one of [11-1] to [11-3], wherein natural killer cells are derived from the therapeutic target.
[11-5] Either [11-1], [11-2] or [11-4] administered to a subject separately or continuously in vivo at the same time as the type II anti-CD20 antibody. Agent.
[11-6] The agent according to any one of [11-1] to [11-5], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[11-7] The agent according to any one of [11-1] to [11-6], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment.
[11-8] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [ Any agent from 11-1] to [11-7].
[11-9] The agent according to any one of [11-1] to [11-8], wherein the type II anti-CD20 antibody is obinutuzumab.

[12-1] Type II anti-CD20 containing natural killer cells treated with at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug, and used in combination with treatment with type II anti-CD20 antibody. A pharmaceutical composition for treating antibody-resistant CD20-positive cancer.
[12-2] Type II anti-CD20 antibody resistance in combination with treatment with type II anti-CD20 antibody containing natural killer cells treated with at least one compound selected from the group consisting of bendamustine and its salts and prodrugs. A pharmaceutical composition for treating CD20-positive cancer.
[12-3] Type II, which contains a type II anti-CD20 antibody and is used in combination with a composition comprising a natural killer cell which contains at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug. A pharmaceutical composition for treating anti-CD20 antibody-resistant CD20-positive cancer.
[12-4] Type II anti-CD20 antibody by combination with a composition containing natural killer cells containing bentamustine and at least one compound selected from the group consisting of salts thereof and prodrugs. A pharmaceutical composition for treating CD20 antibody-resistant CD20-positive cancer.
[12-5] A composition comprising a type II anti-CD20 antibody and a natural killer cell reacted with at least one compound selected from the group consisting of bendamustine and its salts and prodrugs is combined, simultaneously and separately. , Or a pharmaceutical composition for treating type II anti-CD20 antibody-resistant CD20-positive cancer, which is administered continuously.
[12-6] The pharmaceutical composition according to any one of [12-1] to [12-5], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[12-7] The pharmaceutical composition according to any one of [12-1] to [12-6], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment. thing.
[12-8] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [12-8]. The pharmaceutical composition according to any one of 12-1] to [12-7].
[12-9] The pharmaceutical composition according to any one of [12-1] to [12-8], wherein the type II anti-CD20 antibody is obinutuzumab.

[13-1] At least selected from the group consisting of bendamustine and its salts and prodrugs in the manufacture of agents for increasing ADCC activity of type II anti-CD20 antibody against type II anti-CD20 antibody resistant CD20-positive cancer cells. Use of natural killer cells treated with one compound.
[13-2] Use of [13-1], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[13-3] Use of [13-2] or [13-3], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer that has been treated with type II anti-CD20 antibody.
[13-4] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [ Use of any of 13-1] to [13-3].
[13-5] Use of any of [13-1] to [13-4], wherein the type II anti-CD20 antibody is obinutuzumab.

[14-1] Type II anti-CD20 antibody-resistant CD20-positive cancer cells in combination with a composition containing natural killer cells reacted with at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug. Use of type II anti-CD20 antibody in the manufacture of agents to suppress proliferation.
[14-2] A group consisting of bendamustine and its salts and prodrugs in the production of agents for suppressing the growth of cells of type II anti-CD20 antibody-resistant CD20-positive cancer in combination with treatment with type II anti-CD20 antibody. Use of natural killer cells treated with at least one compound selected from.
[14-3] Treatment of type II anti-CD20 antibody-resistant CD20-positive cancer in combination with a composition comprising natural killer cells treated with at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug. Use of type II anti-CD20 antibody in the manufacture of pharmaceutical compositions for.
[14-4] Selected from the group consisting of bendamustine and its salts and prodrugs in the manufacture of pharmaceutical compositions for treating type II anti-CD20 antibody resistant CD20-positive cancer in combination with treatment with type II anti-CD20 antibody. Use of natural killer cells treated with at least one compound.
[14-5] At least one selected from the group consisting of type II anti-CD20 antibody and bendamustine and salts and prodrugs thereof in the manufacture of a pharmaceutical composition for treating type II anti-CD20 antibody resistant CD20-positive cancer. Use of natural killer cells reacted with the compound.
[14-6] Use of any of [14-1] to [14-5], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[14-7] Use of any of [14-1] to [14-6], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment.
[14-8] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [14]. Use any of -1] to [14-7].
[14-9] Use of any of [14-1] to [14-8], wherein the type II anti-CD20 antibody is obinutuzumab.

[15-1] Type II anti-CD20 antibody-resistant CD20-positive cancer cells when used in combination with a composition containing natural killer cells reacted with at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug. Type II anti-CD20 antibody for use in suppressing the growth of.
[15-2] From the group consisting of bendamustine and its salts and prodrugs for use in suppressing the growth of cells of type II anti-CD20 antibody resistant CD20 positive cancer in combination with treatment with type II anti-CD20 antibody. Natural killer cells treated with at least one compound of choice.
[15-3] At least one selected from the group consisting of type II anti-CD20 antibody and bendamustine and salts and prodrugs thereof for use in suppressing the growth of cells of type II anti-CD20 antibody resistant CD20 positive cancer. A combination of compositions containing natural killer cells inflicted with the compound.
[15-4] Treatment of type II anti-CD20 antibody resistant CD20-positive cancer by concomitant use with a composition containing natural killer cells reacted with at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug. Type II anti-CD20 antibody for use in.
[15-5] At least one selected from the group consisting of bendamustine and its salts and prodrugs for use in the treatment of type II anti-CD20 antibody resistant CD20-positive cancer in combination with treatment with type II anti-CD20 antibody. Natural killer cells on which a compound is allowed to act.
[15-6] A type II anti-CD20 antibody for use in the treatment of type II anti-CD20 antibody resistant CD20-positive cancer, and at least one compound selected from the group consisting of bendamustine and its salts and prodrugs are allowed to act. A combination of natural killer cells.
[15-7] A type II anti-CD20 antibody of [15-1] or [15-4], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[15-8] Natural killer cells of [15-2] or [15-5], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[15-9] A combination of [15-3] or [15-6], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[15-10] The type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer that has been treated with type II anti-CD20 antibody, [15-1], [15-4], and [15-7]. ] Any type II anti-CD20 antibody.
[15-11] The type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer that has been treated with type II anti-CD20 antibody, [15-2], [15-5], and [15-8]. ] Any natural killer cell.
[15-12] The type II anti-CD20 antibody resistant CD20-positive cancer is a CD20-positive cancer that has been treated with type II anti-CD20 antibody, [15-3], [15-6], and [15-9]. ] Any combination.
[15-13] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody [15]. -1], [15-4], [15-7] and [15-10] type II anti-CD20 antibody.
[15-14] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody [15]. -2], [15-5], [15-8], and [15-11] natural killer cells.
[15-15] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody [15]. -3], [15-6], [15-9], and any combination of [15-12].
[15-16] A type II anti-CD20 antibody of any of [15-1], [15-4], [15-7], [15-10], and [15-13], which is obinutuzumab.
[5-17] One of [15-2], [15-5], [15-8], [15-11], and [15-14], wherein the type II anti-CD20 antibody is obinutuzumab. Natural killer cells.
[15-18] One of [15-3], [15-6], [15-9], [15-12], and [15-15], wherein the type II anti-CD20 antibody is obinutuzumab. combination.

[16-1] For cells of type II anti-CD20 antibody-resistant CD20-positive cancer,
i) Administering a composition comprising natural killer cells reacted with at least one compound selected from the group consisting of bendamustine and its salts and prodrugs,
ii) Methods of suppressing the growth of cells of type II anti-CD20 antibody resistant CD20-positive cancer, including administration of type II anti-CD20 antibody.
[16-2] The method of [16-1], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[16-3] The method of [16-1] or [16-2], wherein the type II anti-CD20 antibody resistant CD20-positive cancer is a CD20-positive cancer having experience of type II anti-CD20 antibody treatment.
[16-4] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [ 16-1] to [16-3].
[16-5] The method according to any one of [16-1] to [16-4], which is an in vitro and non-human in vivo method.
[16-6] The method according to any one of [16-1] to [16-5], wherein the type II anti-CD20 antibody is obinutuzumab.

[17-1] For cells of type II anti-CD20 antibody-resistant CD20-positive cancer,
i) Administering a composition comprising natural killer cells reacted with at least one compound selected from the group consisting of bendamustine and its salts and prodrugs,
ii) A method for increasing ADCC activity of type II anti-CD20 antibody against type II anti-CD20 antibody resistant CD20-positive cancer, including administration of type II anti-CD20 antibody.
[17-2] The method of [17-1], wherein natural killer cells and type II anti-CD20 antibody are administered to a subject simultaneously, separately or sequentially.
[17-3] The method of [17-1] or [17-2], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[17-4] The method according to any one of [17-1] to [17-3], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment.
[17-5] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [ 17-1] to [17-4].
[17-6] The method according to any one of [17-1] to [17-5], which is an in vitro and non-human in vivo method.
[17-7] The method of [17-6], which is an in vivo method in which natural killer cells are derived from a therapeutic target.
[17-8] The method according to any one of [17-1] to [17-7], wherein the type II anti-CD20 antibody is obinutuzumab.

[18-1] For subjects with type II anti-CD20 antibody-resistant CD20-positive cancer,
i) Administering a composition comprising natural killer cells reacted with at least one compound selected from the group consisting of bendamustine and its salts and prodrugs,
ii) A method of treating type II anti-CD20 antibody resistant CD20-positive cancer, including administration of type II anti-CD20 antibody.
[18-2] The method for treating [18-1], wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma.
[18-3] The method for treating [18-1] or [18-2], wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in treating type II anti-CD20 antibody.
[18-4] The type II anti-CD20 antibody-resistant CD20-positive cancer relapsed after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy using type II anti-CD20 antibody, [18-4]. The treatment method according to any one of 18-1] to [18-3].
[18-5] The therapeutic method according to any one of [18-1] to [18-4], wherein the type II anti-CD20 antibody is obinutuzumab.

According to the present invention, for CD20-positive cancer resistant to type II anti-CD20 antibody or CD20-positive cancer relapsed after treatment containing type II anti-CD20 antibody, from bendamustine and its salts and prodrugs. By using at least one compound selected from the above group in combination, the effect of suppressing the growth of CD20-positive cancer cells using the type II anti-CD20 antibody can be enhanced.
According to the present invention, for CD20-positive cancer resistant to type II anti-CD20 antibody or CD20-positive cancer relapsed after treatment containing type II anti-CD20 antibody, from bendamustine and its salts and prodrugs. By using at least one compound selected from the above group in combination, the effect of treatment with type II anti-CD20 antibody can be enhanced.
According to the present invention, for CD20-positive cancer resistant to type II anti-CD20 antibody or CD20-positive cancer relapsed after treatment containing type II anti-CD20 antibody, from bendamustine and its salts and prodrugs. The ADCC activity of type II anti-CD20 antibody against CD20-positive cancer can be increased by concomitant use of at least one compound selected from the group.
According to the present invention, for CD20-positive cancer resistant to type II anti-CD20 antibody or CD20-positive cancer relapsed after treatment containing type II anti-CD20 antibody, from bendamustine and its salts and prodrugs. The ADCC activity of type II anti-CD20 antibody against CD20-positive cancer can be increased by concomitant use of natural killer cells treated with at least one compound selected from the group.
According to the present invention, for CD20-positive cancer resistant to type II anti-CD20 antibody or CD20-positive cancer relapsed after treatment containing type II anti-CD20 antibody, from bendamustine and its salts and prodrugs. By using a natural killer cell on which at least one compound selected from the above group is allowed to act, the effect of suppressing the growth of CD20-positive cancer cells using the type II anti-CD20 antibody can be enhanced.
According to the present invention, for CD20-positive cancer resistant to type II anti-CD20 antibody or CD20-positive cancer relapsed after treatment containing type II anti-CD20 antibody, from bendamustine and its salts and prodrugs. By using a natural killer cell on which at least one compound selected from the above group is allowed to act, the effect of treatment with the type II anti-CD20 antibody can be enhanced.

FIG. 1 shows the change in tumor volume due to obinutuzumab in the SU-DHL-4 strain (parent strain) (A) and the change in tumor volume due to obinutuzumab in the 18-8 strain (B). FIG. 2 is an optical micrograph of immunohistochemical staining of CD20 protein in SU-DHL-4 strain (parent strain) (left) and 18-8 strain (right). FIG. 3 shows changes in tumor volume with control, obinutuzumab monotherapy (OBI), bendamustine monotherapy (Benda), and obinutuzumab plus bendamustine combination (OBI + Benda) in an 18-8 strain xenograft model. FIG. 4 shows ADCC sensitivity (% ADCC) in the parent strain (RL) and the ADCC resistant strains (RL-E300-1, RL-E300-2, RL-E300-8, RL-E300-22). FIG. 5 shows the effector cells (NK-92 cells) with or without bendamustine pretreatment (black bar: Benda) or no (white bar) in the parent strain (RL) and ADCC resistant strains (RL-E300-8, RL-E300-22). :-) Indicates ADCC activity (% ADCC) mediated by type II anti-CD20 antibody. FIG. 6 shows the change in the ratio of CD107a-positive NK-92 cells depending on the presence or absence of type II anti-CD20 antibody addition (+ OBI) and the presence or absence of pretreatment with bendamustine (black bar: Benda) and no (white bar:-). Figure 7 shows IgG administration (IgG (30 mg / kg)), single agent administration (OBI or Benda), obinutuzumab + bendamustine combination (OBI (30 mg / kg) + Benda (13.3)) in ADCC-resistant strain RL-E300-22. The change in tumor volume due to mg / kg)) is shown.

A. Agents, Pharmaceuticals, and Pharmaceutical Compositions (a1) In one embodiment, the invention comprises at least one compound selected from the group consisting of type II anti-CD20 antibody and / or bendamustine and salts and prodrugs thereof. Provided are an agent, a pharmaceutical, or a pharmaceutical composition to be contained. The agent, drug, or pharmaceutical composition is used to suppress the growth of cells of type II anti-CD20 antibody resistant CD20 positive cancer and / or to treat type II anti-CD20 antibody resistant CD20 positive cancer. be able to.

When a type II anti-CD20 antibody is used in combination with at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug for type II anti-CD20 antibody-resistant CD20-positive cancer, only the type II anti-CD20 antibody or the above-mentioned Compared with the case where only the compound is used, the growth of cancer cells is significantly suppressed, that is, a high antitumor effect can be obtained (for example, Example 3).
Therefore, the combination of type II anti-CD20 antibody with at least one compound selected from the group consisting of bendamustine and its salts and prodrugs is to suppress the growth of cells of type II anti-CD20 antibody resistant CD20 positive cancer, and It is effective for treating type II anti-CD20 antibody-resistant CD20-positive cancer.

In the first example of the first aspect, the agent comprises a type II anti-CD20 antibody. The agent is used simultaneously, separately or in combination with chemotherapy comprising administration of at least one compound selected from the group consisting of bendamustine and its salts and prodrugs.
In the second example of the first aspect, the agent comprises at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs. The agent is used in combination with treatment with type II anti-CD20 antibody simultaneously, separately or sequentially.
In a third example of the embodiment, the drug is a combination of a type II anti-CD20 antibody and at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs, simultaneously, separately or sequentially. It is a drug that is administered.
In the fourth example of the first aspect, the pharmaceutical composition contains a type II anti-CD20 antibody. The pharmaceutical composition is used simultaneously, separately or in combination with chemotherapy comprising administration of at least one compound selected from the group consisting of bendamustine and its salts and prodrugs.
In the fifth example of the first aspect, the pharmaceutical composition contains at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs. The pharmaceutical composition is used in combination with treatment with type II anti-CD20 antibody simultaneously, separately or sequentially.
In the sixth example of the first aspect, the pharmaceutical composition is a combination of a type II anti-CD20 antibody and at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs, simultaneously and separately. A pharmaceutical composition that is administered to or continuously.

== Type II anti-CD20 antibody resistant CD20 positive cancer ==
In a first aspect, B-cell lymphoma is exemplified as the CD20 positive cancer. In that embodiment, B-cell non-Hodgkin's lymphoma is exemplified as the B-cell lymphoma. In that embodiment, the CD20-positive cancer is preferably B-cell non-Hodgkin's lymphoma.
B-cell non-hodgkin lymphomas, according to the WHO classification, are B-cell chronic, originating from precursor B-cell tumors, precursor B lymphoblastic leukemia / lymphoma, and mature B-cell tumors. Lymphocytic leukemia / small lymphocytic lymphoma, pre-B cell lymphocytic leukemia, lymphoplasmacytic lymphoma, splenic B cell marginal zone lymphoma (± hairy lymphocytes), hairy cell leukemia, plasmacell myeloma / trait Celloma, MALT-type extranodal marginal B-cell lymphoma, nodal marginal B-cell lymphoma (± monocytic B-cell), follicular lymphoma, mantle-cell lymphoma, diffuse large-cell B-cell lymphoma (diffuse large-cell B-cell lymphoma) Medial large cell type B cell lymphoma, and primary exudate lymphoma), and Berkit lymphoma are exemplified. In this aspect, among these, B-cell non-Hodgkin's lymphoma is preferably follicular lymphoma.
Non-Hodgkin's lymphoma is classified into low-grade lymphoma, medium-grade lymphoma, and high-grade lymphoma based on its malignancy. When B-cell non-Hodgkin's lymphoma is classified in more detail, Grade 1 and 2 follicular lymphoma and MALT lymphoma are classified as low-grade lymphoma, and Grade 3 follicular lymphoma, mantle cell lymphoma, and diffuse large cell type. B-cell lymphoma is exemplified as moderate-grade lymphoma, and Berkit lymphoma is exemplified as high-grade lymphoma. Based on malignancy, B-cell non-Hodgkin's lymphoma is preferably low-grade and moderate-grade lymphoma because it includes follicular lymphoma.

In the first aspect, a CD20-positive cancer having experience of type II anti-CD20 antibody treatment is exemplified as a type II anti-CD20 antibody-resistant CD20-positive cancer. The "type II anti-CD20 antibody resistance" can be replaced with "type II anti-CD20 antibody resistance". CD20-positive cancers that have recurred after treatment with type II anti-CD20 antibody are included in CD20-positive cancers that have been treated with the type II anti-CD20 antibody. CD20-positive cancer that recurred after treatment with type II anti-CD20 antibody, for example, relapsed after initiation of maintenance therapy with type II anti-CD20 antibody alone after induction therapy with type II anti-CD20 antibody. It may be. In that embodiment, the type II anti-CD20 antibody resistant CD20-positive cancer is preferably a CD20-positive cancer that has been treated with type II anti-CD20 antibody. The type II anti-CD20 antibody resistant CD20-positive cancer is more preferably a B-cell non-Hodgkin's lymphoma who has been treated with a type II anti-CD20 antibody, and even more preferably a follicular lymphoma.

Here, "resistance" as used herein means that a cell or individual is not responsive (also referred to as susceptibility) to the treatment or treatment of a disease and / or has a significant response (eg, partial response and / or completeness). It is not limited as long as the ability to produce success) is reduced. For example, cancers that are resistant to type II anti-CD20 antibody are cancers that are completely unresponsive to treatment with type II anti-CD20 antibody or do not show a significant response, such as partial or complete response. .. Administering a drug to a cancer that is "resistant" to the drug does not have the desired effect, but may even progress or even transform into a highly malignant cancer. be. The "tolerance" may be "natural resistance" or "acquisition resistance". In particular, the acquired resistance in the agent, drug, pharmaceutical composition, etc. of the present invention may be the resistance developed after treatment with the conventional type II anti-CD20 antibody. For example, cancer may eventually develop resistance to the treatment, even if it is effective in the early stages of treatment, but will no longer regress or progress in the presence of type II anti-CD20 antibody. May even do.

Another example of type II anti-CD20 antibody resistant CD20-positive cancer is cancer that has recurred after initiation of maintenance therapy with type II anti-CD20 antibody alone after induction therapy with type II anti-CD20 antibody. The induction therapy is a therapy aimed at obtaining a response greater than a partial response or preventing disease progression by intensive treatment with a combination of type II anti-CD20 antibody and other chemotherapy. The antitumor effect including the partial response or the disease progression is evaluated based on the "Malignant Lymphoma Effect Criteria (Revised Edition)" of the International Working Group (IWG). The induction therapy is usually continued for 24 weeks. For example, in induction therapy with obinutuzumab, which is an example of type II anti-CD20 antibody, in combination with CHOP therapy or CVP therapy, obinutuzumab is administered in 8 cycles with 3 weeks as 1 cycle. In induction therapy in combination with bendamustine, obinutuzumab is administered in 6 cycles with 4 weeks as 1 cycle. In the first cycle of the induction therapy, obinutuzumab is administered on days 1, 8 and 15, and after the second cycle, it is administered on day 1. If other chemotherapy is discontinued during induction therapy due to causes such as toxicity, obinutuzumab monotherapy may be continued. On the other hand, the maintenance therapy is a treatment that is continued for up to 2 years with obinutuzumab alone after the induction therapy in patients who have achieved a partial response or more in the induction therapy. In the maintenance therapy, for example, obinutuzumab, which is an example of a type II anti-CD20 antibody, is administered once every two months. In the induction therapy and the maintenance therapy, 1000 mg of obinutuzumab is administered once a day. The administration method is not particularly limited, but is preferably intravenous injection.
In addition to the cancer that recurred after the start of maintenance therapy with type II anti-CD20 antibody alone after induction therapy with the type II anti-CD20 antibody described above, the other chemotherapy was discontinued during the above-mentioned induction therapy. CD20-positive cancer that became resistant to type II anti-CD20 antibody when monotherapy of type II anti-CD20 antibody was continued is cited as another example of type II anti-CD20 antibody-resistant CD20-positive cancer. Be done.
In all of the first embodiments, the cancer type of type II anti-CD20 antibody resistant CD20 positive cancer is preferably B-cell non-Hodgkin's lymphoma, more preferably follicular lymphoma.

== Type II anti-CD20 antibody ==
In the first aspect, the type II anti-CD20 antibody is appropriately selected from those known at the time of manufacture of the agent. Type II anti-CD20 antibodies are classified differently from type I based on their binding to the CD20 antigen and their biological activity, the details of which are as described in WO 2009/118142.
In the first aspect, the "type II anti-CD20 antibody" in the "type II anti-CD20 antibody-resistant CD20-positive cancer" and the "type II anti-CD20 antibody" contained in the agent, drug, or pharmaceutical composition are different from each other II. Aspects of type anti-CD20 antibody are included, but the same type II anti-CD20 antibody is preferred. Obinutuzumab is exemplified as a type II anti-CD20 antibody. In that embodiment, preferably the type II anti-CD20 antibody is obinutuzumab.
In the present specification, obinutuzumab is specified by "Obinutuzumab (Genetical Recombination)" (trade name: Gazaiba) in the Japanese Accepted Name (JAN), and a range in which the same effect can be obtained. And can include the biosimilars and biobetters.

Obinutuzumab is generally a glycoprotein-modified recombinant humanized anti-CD20 monoclonal antibody that exhibits the characteristics of a type II anti-CD20 antibody, with two heavy chains (H chains) consisting of 449 amino acid residues and 219. It is a glycoprotein composed of two light chains (L chains) consisting of individual amino acid residues, and has a molecular weight of about 148,000 to 150,000. In the H chain, CDR (abbreviation of complementarity-determining region. The same shall apply hereinafter) 1 is an amino acid sequence consisting of SEQ ID NO: 1, CDR2 is an amino acid sequence consisting of SEQ ID NO: 2, and CDR3 is. SEQ ID NO:: Represented by an amino acid sequence consisting of 3. In the L chain, CDR1 is represented by an amino acid sequence consisting of SEQ ID NO: 4, CDR2 is represented by an amino acid sequence consisting of SEQ ID NO: 5, and CDR3 is represented by an amino acid sequence consisting of SEQ ID NO: 6. The variable region (HV region) of the H chain is represented by SEQ ID NO: 7. The variable region (LV region) of the L chain is represented by SEQ ID NO: 8. The H chain is a polypeptide having an amino acid sequence consisting of SEQ ID NO: 9, and the L chain is a polypeptide having an amino acid sequence consisting of SEQ ID NO: 10.

In the first embodiment, the type II anti-CD20 antibody is an antibody having an amino acid sequence having at least 80%, 85%, 90%, 98%, 99% sequence identity with respect to the amino acid sequence of obinutuzumab, or an antigen thereof. It may be a bound fragment. In one aspect, the type II anti-CD20 antibody may be an antibody comprising each CDR in the heavy and light chains of obinutuzumab or an antigen binding fragment thereof. In one aspect, the type II anti-CD20 antibody has at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to the amino acid sequence of each CDR in the heavy and light chains of obinutuzumab. It may be an antibody containing each CDR containing an amino acid sequence having or an antigen-binding fragment thereof. In one aspect, the type II anti-CD20 antibody comprises each CDR in the heavy and light chains of obinutuzumab, at least 80%, 85%, 90%, 95% for the amino acid sequences of the HV and LV regions of obinutuzumab, respectively. , 98%, 99%, an antibody containing an HV region and an LV region containing an amino acid sequence having sequence identity, or an antigen-binding fragment thereof. In one aspect, the type II anti-CD20 antibody is an amino acid having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to the amino acid sequences of the HV and LV regions of obinutuzumab, respectively. It may be an antibody containing an HV region containing a sequence and an LV region or an antigen-binding fragment thereof.

As used herein, the biosimyla of obinutuzumab has the same amino acid sequences as the above H and L chains, and may have a sugar chain different from that of "Obinutuzumab (Genetical Recombination)". It means an antibody having and having a biological activity equal to or higher than that of "Obinutuzumab (Genetical Recombination)".
As used herein, the biobetter of obinutuzumab has 90% or more and less than 100% amino acid sequence homology with the above H and L chains, and in some cases, "Obinutuzumab (Genetical Recombination)". )) "means an antibody having a different sugar chain and having a biological activity equal to or higher than that of" Obinutuzumab (Genetical Recombination) ".

Here, the term "anti-CD20 antibody" used herein is an antibody that specifically binds to CD20. Depending on the binding and biological activity of the anti-CD20 antibody to the CD20 antigen, the two types of anti-CD20 antibody (type I and type II anti-CD20 antibodies) are Cragg, M.S. et al., Blood 103 (2004) 2738. -2743 and Cragg, M. S. et al., Blood 101 (2003) 1045-1052. See Table 1.

One essential property of type I and type II anti-CD20 antibodies is their binding form. Therefore, type I and type II anti-CD20 antibodies are classified by the ratio of the binding capacity of the antibody to rituximab to anti-CD20 Raji cells (ATCC No. CCL-86).

Type II anti-CD20 antibody is 0.3-0.6, preferably 0.35-0.55, more preferably 0.4-0.5, the ratio of the ability of the anti-CD20 antibody to rituximab to bind to Raji cells (ATCC No. CCL-86). Has. Examples of such type II anti-CD20 antibodies include, for example, tositumomab (B1IgG2a), humanized B-Ly1 antibody IgG1 (chimeric humanized IgG1 antibody disclosed in International Publication No. 2005/044859), 11B8IgG1 (International Publication No. 2004). / 035607), and AT80I g G1. Preferably, the type II anti-CD20 antibody is a monoclonal antibody that binds to the same epitope as the humanized B-Ly1 antibody (disclosed in WO 2005/044859).

The ratio of the binding ability of the anti-CD20 antibody to rituximab to Raji cells (ATCC No. CCL-86) is 0.8 to 1.2, preferably 0.9 to 1.1 for the type I anti-CD20 antibody to the type II anti-CD20 antibody. Has. Examples of such type I anti-CD20 antibodies include, for example, rituximab, 1F5IgG2a (ECACC, hybridoma; Press, O.W. et al., Blood 69/2 (1987) 584-591), HI47IgG3 (ECACC, hybridoma), 2C6IgG1 (International Publication No. 2005). / 103081), 2F2IgG1 (disclosed in International Publication No. 2004/0356567 and International Publication No. 2005/103081) and 2H7IgG1 (disclosed in International Publication No. 2004/056312). include.

"Ratio of CD20 binding ability of anti-CD20 antibody to rituximab to Raji cells (ATCC No. CCL-86)" was obtained in FACS Array (Becton Dickinson) by Raji cells (ATCC No. CCL-86) described in Example 2. Then, using the anti-CD20 antibody combined with Cy5 and rituximab combined with Cy5, it was measured by direct immunofluorescence measurement (mean fluorescence intensity (MFI) is measured) and calculated as follows. Rituximab.

MFI is the average fluorescence intensity. As used herein, "Cy5 labeling rate" means the number of Cy5 labeled molecules per antibody molecule. Typically, the type II anti-CD20 antibody is 0.3-0.6, preferably 0.35-0.55, more preferably 0.4-0.5, Raji cells of the type II anti-CD20 antibody against rituximab (ATCC No. CCL-86). ) Has the ratio of the binding ability of CD20 to. Type II anti-CD20 antibodies herein have increased antibody-dependent cellular cytotoxicity (ADCC). "Antibody with increased antibody-dependent cellular cytotoxicity (ADCC)" or "antibody with increased antibody-dependent cellular cytotoxicity (ADCC)" is arbitrary known to those of skill in the art as defined herein. Has increased ADCC as determined by the preferred method of. One recognized in vitro ADCC assay is:
1) The assay uses target cells known to express the target antigen recognized by the antigen binding region of the antibody;
2) The assay uses human peripheral blood mononuclear cells (PBMCs) isolated from the blood of randomly selected healthy donors as effector cells;
3) The above assay is performed according to the following protocol:
i) PBMCs are isolated using standard concentration centrifugation and suspended at 5 x 10 ⁶ cells / mL in RPMI cell culture medium;
ii) Target cells proliferate by standard tissue culture methods, harvest from exponential growth phase with greater than 90% viability, washed with RPMI cell culture medium, labeled with 100 μCi ⁵¹ Cr, and 2 in cell culture medium. Wash once and resuspend in cell culture medium at a concentration of ¹⁰⁵ cells / mL;
iii) Transfer 100 μL of the above final target cell suspension to each well of a 96-well microtiter plate;
iv) Antibodies were serially diluted to 4000 ng / mL to 0.04 ng / mL in cell culture medium and 50 μL of the resulting antibody solution was added to the target cells of the 96-well microtiter plate over the entire concentration range above. Test various antibody concentrations at 3 points to cover;
v) For maximal release (MR) controls, instead of antibody solution (iv above), 50 μL of 2% (VN) nonionic surfactant in 3 additional wells in a plate containing labeled target cells. Add aqueous solution (Nonidet, Sigma, St. Louis);
vi) For spontaneous release (SR) controls, replace the antibody solution (iv above) with 50 μL RPMI cell culture medium in 3 additional wells in a plate containing labeled target cells;
vii) 96-well microtiter plates are then centrifuged at 50xg for 1 minute and incubated at 4 ° C for 1 hour;
viii) Add 50 μL of PBMC suspension (I above) to each well to obtain effector: target cells in a 25: 1 ratio and plate in an incubator at 37 ° C. for 4 hours in a 5% CO ₂ atmosphere. put;
ix) Cell-free supernatants from each well are collected and experimentally released radioactivity (ER) is quantified using a gamma counter;
x) The percentage of the particular lysate is the formula (ER-MR) / (MR-SR) x 100 (ER is the average radioactivity quantified for antibody concentration (see ix above), MR is Quantified mean radioactivity (see ix above) for MR controls (see v above) and SR is quantified mean radioactivity (see ix above) for SR controls (see vi above). ) Is calculated for each antibody concentration;
4) "Increased ADCC" is an increase in the maximum percentage of the particular lysate observed within the above tested antibody concentration range and / or the specific observed within the above tested antibody concentration. Defined as a decrease in antibody concentration required to achieve half of the maximum percentage of lysate. Increased ADCC was measured using the above assay mediated by the same antibody produced by the same host cell using the same standard production, purification, formation and storage methods known to those of skill in the art. For ADCC, which was not produced by a host cell designed to overexpress GnTIII.

The "increased ADCC" is obtained by the sugar chain engineering technique of the antibody. It is a natural, cell-mediated effector of monoclonal antibodies by making its oligosaccharide components described in Umana P. et al., Nature Biotechnol. 17 (1999) 176-180, and US Pat. No. 6,602,684. Means function.

The term "complement-dependent cytotoxicity (CDC)" refers to the lysis of human tumor target cells by an antibody according to the invention in the presence of complement. CDC is preferably measured by treatment of a preparation of CD20 expressing cells with an anti-CD20 antibody according to the invention in the presence of complement. CDC is seen when the antibody induces lysis (cell death) of 20% or more of tumor cells after 4 hours at a concentration of 100 nM. The assay preferably uses measurements of ⁵¹ Cr or Eu labeled tumor cells and released ⁵¹ Cr or Eu. Controls include incubation of tumor target cells with complement rather than the antibody.

Typically, IgG1 isotype type II anti-CD20 antibody exhibits characteristic CDC. Type II anti-CD20 antibody has reduced CDC (if it is IgG1 isotype) compared to type I anti-CD20 antibody of IgG1 isotype. Preferably, the type II anti-CD20 antibody is an IgG1 isotype antibody.

The "rituximab" antibody (reference antibody; an example of a type I anti-CD20 antibody) is a chimeric mouse monoclonal antibody against a human CD20 antigen that is recombinantly produced and contains a human gamma 1 constant domain. This chimeric antibody is identified by the name "C2B8" in US Pat. No. 5,736,137 (Anderson, K.C. et al.) Issued April 17, 1998 by IDEC Pharmaceuticals Corporation. Rituximab is approved for the treatment of patients with relapsed, refractory, follicular, CD20-positive non-Hodgkin's B-cell lymphoma. In vitro mechanisms of action studies have revealed that rituximab exhibits human complement-dependent cytotoxicity (CDC) (Reff, M. E. et al., Blood 83 (2) (1994) 435-445). In addition, it exhibits significant activity in assays that measure antibody-dependent cellular cytotoxicity (ADCC).

The oligosaccharide components significantly affect properties related to the efficacy of therapeutic glycoproteins, including physical stability, resistance to protease attacks, immune system interactions, drug kinetics, and specific biological activity. do. Such properties may depend not only on the presence or absence of oligosaccharides, but also on their particular structure. Some generalization can be made between oligosaccharide structure and glycoprotein function. For example, one oligosaccharide structure mediates a rapid clearance of the glycoprotein from the bloodstream by interacting with a particular carbohydrate-binding protein, while another oligosaccharide structure is bound by an antibody and causes an undesired immune response. Induce (Jenkins, N. et al., Nature Biotechnol. 14 (1996) 975 -81).

Mammalian cells are the preferred host for the production of therapeutic glycoproteins due to their ability to glycosylate proteins in the most compatible form for human application (Cumming, D.A. et al., Glycobiology 1 (Glycobiology 1). 1991) 115-30; Jenkins, N. et al., Nature Biotechnol. 14 (1996) 975-81). Bacteria rarely glycosylate proteins and, like other types of common hosts such as yeast, filamentous fungi, insects and plant cells, have rapid clearance from the bloodstream, unwanted immune interactions, and in some cases. Gives a glycosylation pattern associated with reduced biological activity. Among mammalian cells, Chinese hamster ovary (CHO) cells have been the most commonly used for the last 20 years. In addition to certain suitable glycosylation patterns, these cells allow for the consistent production of genetically stable and highly productive crawl cell lines. They are cultured in high concentrations in a single bioreactor using serum-free media, enabling the development of safe and reproducible bioprocesses. Other commonly used animal cells include baby hamster kidney (BHK) cells, NSO- and SP2 / 0-mouse myeloma cells. Most recently, production from transgenic animals has also been tested (Jenkins, N. et al., Nature Biotechnol. 14 (1996) 975-981).

All antibodies contain glycan structures at conservative positions in the heavy chain constant region, and each isotope has a different array of N-linked glycan structures that variably affect protein assembly, secretion or functional activity. (Wright, A. and Monison, S.L., Trends Biotech. 15 (1997) 26-32). The structure of bound N-linked sugar chains varies considerably depending on the degree of processing and can include complex oligosaccharides of high-mannose, multi-branched and bi-branched (Wright, A. and Morrison, S. L.). , Trends Biotech. 15 (1997) 26-32). Typically, there is heterogeneous processing of the oligosaccharide structure of the core bound to a particular glycosylation site, such that even a monoclonal antibody exists as multiple glycoforms. Similarly, it has been found that major differences in antibody glycosylation occur between cell lines, with slight differences in the number of cells grown under different culture conditions (Lifely, M.R. et al., Glycobiology 5). (8) (1995) 813-22).

Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180 is one way to gain a significant increase in capacity while at the same time maintaining a single production process and potentially avoiding significant and unwanted side effects. And by making the oligosaccharide component described in US Pat. No. 6,602,684, it is to enhance the cell-mediated effector function of the natural fuel of the monoclonal antibody. IgG1 type antibody is the most commonly used antibody in cancer immunotherapy and is a glycoprotein having a conservative N-linked glycosylation site in Asn297 of each CH2 domain. The two-branched oligosaccharides of the two complexes bound to Asn297 are buried between the CH2 domains and make extensive contact with the polypeptide backbone, the presence of which the antibody is antibody-dependent cellular cytotoxicity ( Essential for intervening effector functions such as ADCC) (Lifely, M. R. et al., Glycobiology 5 (1995) 813-822; Jefferis, R. et al., Immunol. Rev. 163 (1998) 59-76; Wright, A. and Morrison, S. L., Trends Biotechnol. 15 (1997) 26-32).

Overexpression of β (1, 4) -N-acetylglucosaminyl transferase III ("GnTIII7y", a glucosyl transferase that catalyzes the formation of dichotomous oligosaccharides, in Chinese hamster ovary (CHO) cells is produced. It has been previously shown that the in vitro ADCC activity of the anti-neuropathy chimeric monoclonal antibody (chCE7) produced by the CHO cells produced was significantly increased (Umana, P. et al., Nature Biotechnol. 17 (1999)). 176-180 and WO 99/154342, the entire contents of which are incorporated herein by reference). The antibody chCE7 is a number of non-complexes with high tumor affinity and specificity. Belonging to monoclonal antibodies, but unlikely to be clinically advantageous when produced in standard industrial cell lines lacking GnTIII enzyme (Umana, P., et al., Nature Biotechnol. 17 (1999) 176- 180). The study was obtained by creating antibody-producing cells for expressing GnTIII with a large increase in ADCC activity, which contained a bifurcated, non-fucosylated oligosaccharide (Fc). It also resulted in an increase in the proportion of related bifurcated oligosaccharides, which was the first to show the levels found in natural antibodies.

The type II anti-CD20 antibody is mixed with other acceptable raw materials exemplified in (a5) using known techniques at the time of manufacture, as described in "B. Production method and use in production". Can be manufactured.

== Bendamustine and its salts and prodrugs ==
For example, bendamustine hydrochloride is exemplified as at least one compound selected from the group consisting of bendamustine and its salts and prodrugs. In that embodiment, preferably at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs is bendamustine hydrochloride.

== Administration method ==
The description of "combined" use or administration of multiple active ingredients, or "combination" means administration of separate or single pharmaceutical formulations at the same time, and preferably both (or all). It comprises administering the active agent separately or consecutively in any order within the period when the active agent exerts biological activity at the same time. Those skilled in the art can arbitrarily determine the administration order, administration frequency, administration time, etc. of each component to be used in combination. Further, for the purpose of treatment, another drug other than those described, another drug, radiation therapy, surgery, etc. may be further used in combination.
The specific method for administering the formulated active ingredient to a living body is the same as that described in "E. Therapeutic method" described later.

(A2) Second Aspect In the second aspect, the present invention provides an agent or a pharmaceutical substance containing at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug.
The agent can be used to increase ADCC activity of type II anti-CD20 antibody against type II anti-CD20 antibody resistant CD20-positive cancer cells in vivo or in vitro.

Natural killer cells are exposed to at least one compound selected from the group consisting of bendamstin and salts thereof and prodrugs, and after the compound is allowed to act on the natural killer cells, the natural killer cells are resistant to type II anti-CD20 antibody together with type II anti-CD20 antibody. When administered to CD20-positive cancer cells, ACDD to type II anti-CD20 antibody-resistant CD20-positive cancer cells of type II anti-CD20 antibody compared to the case of using natural killer cells without pretreatment with the compound. The activity is significantly increased (eg, Example 5).
That is, at least one compound selected from the group consisting of bendamstin and its salts and prodrugs is administered to a therapeutic subject in vivo and acts on natural killer cells in vivo, or acts on natural killer cells in vitro. By doing so, ADCC activity of the type II anti-CD20 antibody against type II anti-CD20 antibody-resistant CD20-positive cancer cells can be increased.

In a second embodiment, the agent may be exposed in vitro to the natural killer cells and the compound may be allowed to act, or the agent may be administered in vivo to the subject to be treated and the natural killer cells in vivo. The compound may be allowed to act.

When the agent is exposed in vitro to the natural killer cells and the compound is allowed to act on the natural killer cells, the natural killer cells are preferably collected from the treatment target, that is, derived from the treatment target. Here, when natural killer cells are exposed in vitro to at least one compound selected from the group consisting of bendamustine and its salts and prodrugs, for example, selected from the group consisting of bendamustine and its salts and prodrugs in its culture medium. At least one compound to be added may be added.

The agent may be administered to the therapeutic subject separately or sequentially in vivo at the same time as the type II anti-CD20 antibody. The administration method is the same as that described in "E. Treatment method" described later.

The specific configuration other than the above in the second aspect is the same as the aspect described in (a1).

(A3) Third aspect In another aspect, the present invention provides an agent containing natural killer cells.
The agent can be used to increase ADCC activity of type II anti-CD20 antibody against type II anti-CD20 antibody resistant CD20-positive cancer cells in vivo or in vitro.

Natural killer cells are exposed to at least one compound selected from the group consisting of bendamstin and salts thereof and prodrugs, and after the compound is allowed to act on the natural killer cells, the natural killer cells are resistant to type II anti-CD20 antibody together with type II anti-CD20 antibody. When administered to CD20-positive cancer cells, ADCC for type II anti-CD20 antibody-resistant CD20-positive cancer cells of type II anti-CD20 antibody compared to the case of using natural killer cells without pretreatment with the compound. The activity is significantly increased (eg, Example 5).
That is, natural killer cells exposed to and acted on at least one compound selected from the group consisting of bendamustine and its salts and prodrugs are treated with type II anti-CD20 antibody-resistant CD20-positive cancer in vivo or in vitro. It is effective for increasing ADCC activity of type II anti-CD20 antibody against cells.

In a third aspect, the natural killer cell is preferably a natural killer cell that has been exposed in vitro to at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug and allowed to act on the compound. The natural killer cells are preferably collected from the treatment target, that is, derived from the treatment target.
Here, when natural killer cells are exposed in vitro to at least one compound selected from the group consisting of bendamustine and its salts and prodrugs, for example, selected from the group consisting of bendamustine and its salts and prodrugs in its culture medium. At least one compound to be added may be added.
As used herein, a "compound-acted" natural killer cell is exposed to at least one compound selected from the group consisting of bendamustine and its salts and prodrugs as described above and is exposed to type II anti-CD20 antibody II. This is a group of cells that has acquired the effect of increasing ADCC activity against CD20-positive cancer cells resistant to type anti-CD20 antibody. It also includes the natural killer cell group when a part of the natural killer cell group exposed to the compound acquires the effect.
Natural killer cells can be formulated as described in "B. Production method and use in production".

The agent is administered to the subject separately or sequentially at the same time as the type II anti-CD20 antibody. The administration method is the same as that described in "E. Treatment method" described later.

The specific configuration other than the above in the third aspect is the same as the aspect described in (a1).

(A4) Fourth Aspect In another embodiment, the present invention provides a pharmaceutical composition containing natural killer cells or type II anti-CD20 antibody.
The pharmaceutical composition can be used to treat type II anti-CD20 antibody resistant CD20 positive cancer.

Natural killer cells are exposed to at least one compound selected from the group consisting of bendamstin and salts thereof and prodrugs, and after the compound is allowed to act on the natural killer cells, the natural killer cells are resistant to type II anti-CD20 antibody together with type II anti-CD20 antibody. When administered to CD20-positive cancer cells, ACDD to type II anti-CD20 antibody-resistant CD20-positive cancer cells of type II anti-CD20 antibody compared to the case of using natural killer cells without pretreatment with the compound. The activity is significantly increased (eg, Example 5).
That is, a combination of natural killer cells exposed to and acted on by at least one compound selected from the group consisting of bendamustine and its salts and prodrugs and type II anti-CD20 antibody is a type II anti-CD20 antibody resistant CD20. Since it increases the ADCC activity of type II anti-CD20 antibody against positive cancer, it is effective for treating anti-CD20 antibody-resistant CD20-positive cancer.

In the first example of the fourth aspect, the agent contains natural killer cells. The agent is used in combination with treatment with type II anti-CD20 antibody.
In the second example of the fourth aspect, the agent comprises a type II anti-CD20 antibody. The agent is used in combination with a composition comprising natural killer cells.

In the fourth aspect, the preferred embodiment of the natural killer cell is the same as that described in (a3).

The agent is administered to the subject separately or sequentially at the same time as the type II anti-CD20 antibody. The administration method is the same as that described in "E. Treatment method" described later.

The specific configuration other than the above in the fourth aspect is the same as the aspect described in (a1).

(A5) Other Configurations in 1st to 4th Aspects In the 1st to 4th embodiments described above, when the agent is formulated, an antibody having a desired purity may be used as one or more of any of the antibodies. Prepared in the form of a lyophilized or aqueous solution by mixing with a pharmaceutically acceptable carrier (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at doses and concentrations when used and include, but are not limited to, the following: phosphates, citrus. Buffers such as acid salts and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, Or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); small molecules (less than about 10 residues) polypeptides; serum albumin, gelatin, or Proteins such as immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; Chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, sorbitol; salt-forming counterions such as sodium; metal complexes (eg, Zn-protein complexes); and / or non-polyethylene glycol (PEG). Ionic surface activator. Exemplary pharmaceutically acceptable carriers herein are further human soluble such as soluble neutral active hyaluronidase glycoprotein (sHASEGP) (eg, rHuPH20 (HYLENEX®, Baxter International, Inc.)). Contains interstitial drug dispersants such as PH-20 hyaluronidase glycoprotein). Specific exemplary sHASEGPs and their uses (including rHuPH20) are described in US Patent Application Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

An exemplary lyophilized antibody formulation is described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006/044908, the latter formulation containing histidine-acetate buffer.

The active ingredient is incorporated into microcapsules prepared, for example, by a droplet formation (core selvation) technique or by interfacial polymerization (eg, hydroxymethyl cellulose or gelatin microcapsules, and poly (methyl methacrylate) microcapsules, respectively). It may be incorporated into a colloidal drug delivery system (eg, liposomes, albumin globules, microemulsions, nanoparticles, and nanocapsules) or it may be incorporated into macroemulsions. Such a technique is disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained release formulations may be prepared. A suitable example of a sustained release formulation comprises a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, which matrix is in the form of a shaped article such as a film or microcapsules.

Formulations used for in vivo administration are usually sterile. Aseptic conditions are easily achieved, for example by filtering through a sterile filtration membrane.

When these active ingredients are formulated separately to form two or more active ingredients, the individual formulations may be administered simultaneously, separately at regular intervals, or in succession. It is possible. The two or more kinds of preparations can be administered at different times a day. The agent, drug, or pharmaceutical composition according to the present invention can be orally or parenterally administered systemically or topically. When these active ingredients are formulated separately to form two or more kinds of preparations, the individual preparations can be administered by different routes.

B. Production Method and Use in Production (b1) In one embodiment, the invention comprises a type II anti-CD20 antibody and / or at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs. Provided are an agent, a pharmaceutical, and a pharmaceutical composition (the agent, pharmaceutical, and pharmaceutical composition according to (a1)).
The agent, drug, or pharmaceutical composition is used to suppress the growth of cells of type II anti-CD20 antibody resistant CD20 positive cancer and / or to treat type II anti-CD20 antibody resistant CD20 positive cancer. be able to.

In the first example of the first aspect, a type II anti-CD20 antibody is used in the production of an agent, drug, or pharmaceutical composition.

In the second example of the first aspect, at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs is used in the manufacture of agents, pharmaceuticals or pharmaceutical compositions.

In the third example of the first embodiment, a type II anti-CD20 antibody and at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs are used in the production of the drug or pharmaceutical composition.

(B2) Second Aspect In the second aspect, the present invention comprises the production of an agent (the agent according to (a2)) containing at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug. offer. The agent can be used to increase ADCC activity of type II anti-CD20 antibody against type II anti-CD20 antibody resistant CD20-positive cancer cells in vivo or in vitro.

In a second embodiment, at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs is used in the production of the agent.

(B3) Third Embodiment In the third embodiment, the present invention contains natural killer cells exposed to and allowed to act on at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs. The manufacture of an agent, a pharmaceutical, and a pharmaceutical composition (the agent, the pharmaceutical, and the pharmaceutical composition according to (a3)) is provided. The agents, pharmaceuticals, and pharmaceutical compositions can be used in vivo or in vitro to increase the ADCC activity of type II anti-CD20 antibody against type II anti-CD20 antibody resistant CD20-positive cancer cells.

In a third embodiment, natural killer cells exposed to and acted on by at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs are used in the production of agents, pharmaceuticals, and pharmaceutical compositions. Will be done.

In a third aspect, the natural killer cells are preferably harvested from the subject of treatment by known means. The collected natural killer cells consist of bendamustine and its salts and prodrugs in vitro, for example by adding at least one compound selected from the group consisting of bendamustine and its salts and prodrugs to its culture medium. After exposure to at least one compound selected from the group and allowing the compound to act, it is mixed with other acceptable raw materials exemplified in (a5) by conventional techniques to prepare agents, pharmaceuticals, and pharmaceutical compositions. Can be used for.
A "compound-acted" natural killer cell is exposed to at least one compound selected from the group consisting of bendamustine and its salts and prodrugs as described above and is resistant to type II anti-CD20 antibody type II anti-CD20 antibody. It is a group of cells that has acquired the effect of increasing ADCC activity against CD20-positive cancer cells. It also includes the natural killer cell group when a part of the natural killer cell group exposed to the compound acquires the effect.

(B4) Fourth Aspect In a fourth aspect, the present invention relates to an agent, a pharmaceutical, and a pharmaceutical composition containing natural killer cells and / or a type II anti-CD20 antibody (the agent, pharmaceutical, according to (a4)). And the manufacture of pharmaceutical compositions).
The agent, pharmaceutical, and pharmaceutical composition are effective for treating type II anti-CD20 antibody resistant CD20-positive cancer.

In the first example of the embodiment, the agent contains natural killer cells. The agent is used in combination with treatment with type II anti-CD20 antibody.

In the first example of this embodiment, natural killer cells are used in the production of agents, pharmaceuticals, or pharmaceutical compositions. The natural killer cells can be prepared as described in (b3).

In a second example of that embodiment, a type II anti-CD20 antibody is used in the production of an agent, drug, or pharmaceutical composition.

In these first to fourth embodiments ((b1) to (b4)), the type II anti-CD20 antibody can be obtained as a polyclonal antibody or a monoclonal antibody by using known means, and the preferred embodiment thereof is (). It is the same as the aspect described in a1). Obinutuzumab, which is a preferred embodiment of the type II anti-CD20 antibody, can be produced by one of ordinary skill in the art according to a known method shown in WO 2005/044859. The agent, the pharmaceutical, and the pharmaceutical composition usually contain the active ingredient described in the first to fourth aspects ((b1) to (b4)) and other acceptable raw materials exemplified in (a5). It can be manufactured by mixing according to the above techniques.

In each of the embodiments described in this section "B. Production Method and Use in Production Method", "type II anti-CD20 antibody", "at least one compound selected from the group consisting of bendamustine and its salts and prodrugs", and. General and preferred examples of "type II anti-CD20 antibody resistant CD20-positive cancer" are the same as those described in "A. Agents, Pharmaceuticals, and Pharmaceutical Compositions" above.
In addition, other specific configurations of these examples are the same as those described in "A. Agents, Pharmaceuticals, and Pharmaceutical Compositions" above.

C. Method of suppressing cell proliferation In one aspect, the present invention provides a method of suppressing cell proliferation. In that embodiment, the cell is a type II anti-CD20 antibody resistant CD20 positive cancer cell.
(C1) First aspect In the first aspect, the method comprises administering i) type II anti-CD20 antibody to cells of type II anti-CD20 antibody resistant CD20 positive cancer, ii) bendamustine and a salt thereof and Includes administration of at least one compound selected from the group consisting of prodrugs.
The method in this embodiment is an in vitro or in vivo method. The method in this embodiment is preferably an in vitro or non-human in vivo method.

Here, the type II anti-CD20 antibody and bendamustine and their salts and prodrugs may be formulated separately or individually to the extent pharmaceutically acceptable. In addition, at least one compound selected from the group consisting of type II anti-CD20 antibody and bendamustine and its salts and prodrugs is simultaneously, separately, sequentially, in vitro or in vivo resistant to type II anti-CD20 antibody CD20 positive. It can be administered to cancer cells.
The method is selected from, for example, the group consisting of agents, pharmaceuticals, or pharmaceutical compositions containing the type II anti-CD20 antibody described in "A. Agents, Pharmaceuticals, and Pharmaceutical Compositions," and bendamustine and salts and prodrugs thereof. A method comprising administering an agent, a pharmaceutical, or a pharmaceutical composition comprising at least one compound thereof.

(C2) Second Embodiment In the second embodiment, the method administers a composition comprising i) bentamustine and a natural killer cell on which at least one compound selected from the group consisting of a salt thereof and a prodrug has been acted on. That, ii) includes administration of type II anti-CD20 antibody.
The method in the second aspect is an in vitro or in vivo method. The method in this embodiment is preferably an in vitro or non-human in vivo method.

Here, the natural killer cells in the second aspect are the same as those described in (a1) above, and are prepared as described in (b3).

Natural killer cells and type II anti-CD20 antibody may be formulated separately or integrally as long as they are pharmaceutically acceptable. In addition, natural killer cells and type II anti-CD20 antibody can be administered simultaneously, separately, and sequentially to cells of type II anti-CD20 antibody-resistant CD20-positive cancer in vitro or in vivo.
The method is, for example, an agent, a drug, or a pharmaceutical composition containing a natural killer cell according to "A. Agent, a drug, and a pharmaceutical composition", and an agent, a drug, or a pharmaceutical composition containing a type II anti-CD20 antibody. A method that involves administering a substance.

In each of the embodiments described in this section "C. Method for Inhibiting Cell Proliferation", "Type II anti-CD20 antibody", "At least one compound selected from the group consisting of bendamustine and its salts and prodrugs", and "Type II". General and preferred examples of "anti-CD20 antibody resistant CD20-positive cancer" are the same as those described in "A. Agents, Pharmaceuticals, and Pharmaceutical Compositions" above.

In addition, other specific configurations of these examples are the same as those described in "A. Agents, Pharmaceuticals, and Pharmaceutical Compositions" above.

D. Methods for Increasing ADCC Activity of Type II Anti-CD20 Antibodies In one aspect, the invention provides methods for increasing ADCC activity of Type II anti-CD20 antibodies.
(D1) First Embodiment In the first embodiment, the method comprises administering to natural killer cells at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug.
The method in this embodiment is an in vitro or in vivo method. The method in this embodiment is preferably an in vitro or non-human in vivo method.

Here, at least one compound selected from the group consisting of the type II anti-CD20 antibody and bendamustine and a salt thereof and a prodrug is formulated into one, even if they are separately formulated, to the extent pharmaceutically acceptable. It may be made into. The administration method is the same as that described in "E. Treatment method" described later.

Type II anti-CD20 antibody resistant CD20-positive cancer cells exposed to and acted on by at least one compound selected from the group consisting of bendamstin and its salts and prodrugs. Administration of anti-CD20 antibody can significantly increase the ACDD activity of the type II anti-CD20 antibody against antibody-resistant CD20-positive cancer cells.

In a first aspect, the method may be a method of administering at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug to natural killer cells in vitro.

The method comprises administering at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs to natural killer cells in vitro, and further administering a type II anti-CD20 antibody to the compound. At the same time, the method may include administration to type II anti-CD20 antibody-resistant CD20-positive cancer cells in vitro, either separately or continuously.

In a first aspect, the method exposes natural killer cells exposed to at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs to which the compound has been applied to type II anti-CD20 antibody resistant CD20 positive. Treatment subjects with cancer cells may further include administration of type II anti-CD20 antibody simultaneously, separately or sequentially, in vivo.

Alternatively, in the first embodiment, the method comprises at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug at the same time as a type II anti-CD20 antibody against natural killer cells in vivo to be treated. , Separately or continuously, may be administered in vivo. In the body to be treated, natural killer cells are exposed to and acted upon by at least one compound selected from the group consisting of bendamustine and its salts and prodrugs. When administered in vivo in this manner, the agent is administered to the subject separately or sequentially at the same time as the type II anti-CD20 antibody. The administration method is the same as that described in "E. Treatment method" described later.

(D2) Second aspect In the second aspect, the method comprises cells of type II anti-CD20 antibody resistant CD20 positive cancer, i) at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs. Includes administration of a composition comprising a natural killer cell that has been acted upon, ii) administration of type II anti-CD20 antibody.
By this method, ADCC activity of type II anti-CD20 antibody against type II anti-CD20 antibody-resistant CD20-positive cancer can be increased.
The method in the second aspect is an in vitro or in vivo method. The method in this embodiment is preferably an in vitro or non-human in vivo method.
Type II anti-CD20 antibody against type II anti-CD20 antibody-resistant CD20-positive cancer by in vivo administration in combination with type II anti-CD20 antibody to treatment subjects with type II anti-CD20 antibody-resistant CD20-positive cancer ADCC activity can be increased.

Natural killer cells and type II anti-CD20 antibody may be formulated separately or integrally as long as they are pharmaceutically acceptable. In addition, natural killer cells and type II anti-CD20 antibody can be administered simultaneously, separately, and sequentially to cells of type II anti-CD20 antibody-resistant CD20-positive cancer in vitro or in vivo. The administration method is the same as that described in "E. Treatment method" described later.

The method for increasing ADCC activity of a type II anti-CD20 antibody according to the present invention is, for example, an agent, pharmaceutical, or pharmaceutical composition containing natural killer cells according to "A. Agent, Pharmaceutical, and Pharmaceutical Composition". , And a method comprising administering a drug, drug, or pharmaceutical composition comprising a type II CD20 antibody.
The natural killer cells in each aspect described in this section "Method for increasing ADCC activity of D. type II anti-CD20 antibody" are the same as those described in (a1) above, and are described in (b3). It is prepared as it is. When the natural killer cells are administered to the treatment target, the natural killer cells are preferably collected from the treatment target, that is, derived from the treatment target.

In each embodiment described in this section "Methods for increasing ADCC activity of D. type II anti-CD20 antibody", it is selected from the group consisting of "type II anti-CD20 antibody", "bendamustine and its salts and prodrugs". The general and preferred examples of "at least one compound" and "type II anti-CD20 antibody resistant CD20 positive cancer" are the same as those described in "A. Agents, Pharmaceuticals, and Pharmaceutical Compositions" above. ..

In addition, other specific configurations of these examples are the same as those described in "A. Agents, Pharmaceuticals, and Pharmaceutical Compositions" above.

E. Therapeutic Methods In one aspect, the invention provides a method of treating CD20-positive cancer. In that embodiment, the CD20-positive cancer is a type II anti-CD20 antibody resistant CD20-positive cancer.
(E1) First aspect The therapeutic method comprises administering i) a type II anti-CD20 antibody, ii) administering at least one compound selected from the group consisting of bendamustine and its salts and prodrugs.

In this embodiment, the therapeutically effective amount of the type II anti-CD20 antibody can be administered to a therapeutic subject by an administration method known to those skilled in the art. Intravenous administration and subcutaneous administration are exemplified as the administration method.
In this embodiment, at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug can be administered in a therapeutically effective amount thereof by an administration method known to those skilled in the art. Intravenous administration, subcutaneous administration and oral administration are exemplified as the administration method.

In that embodiment, the type II anti-CD20 antibody and at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs are the agents described in "A. Agents, Pharmaceuticals, and Pharmaceutical Compositions" herein. It is preferable to administer in combination as a drug, a pharmaceutical composition, or the like, and they may be administered simultaneously or at regular intervals, separately or continuously. Here, at least one compound selected from the group consisting of the type II anti-CD20 antibody and bendamustine and a salt thereof and a prodrug thereof is combined into one, even if they are separately formulated, to the extent pharmaceutically acceptable. It may be formulated. At least one compound selected from the group consisting of type II anti-CD20 antibody and bendamustine and salts thereof and prodrugs can be administered at different doses and dosing regimens for each. For example, at least one compound selected from the group consisting of type II anti-CD20 antibody, bendamustine and its salt and prodrug is formulated and administered to one treatment subject at the dosage and administration schedule as exemplified below. May be.

The administration plan (frequency, duration, administration rate, etc.) of type II anti-CD20 antibody is not limited to the extent that the desired effect can be obtained, and the patient's condition, treatment history, treatment process (induction therapy or maintenance therapy), combination therapy. Those skilled in the art can make appropriate decisions based on other drugs to be treated and their administration plans. Administration of type II anti-CD20 antibody may differ in the length of one cycle for treatment and the number of cycles in induction therapy and maintenance therapy.

The dosing regimen (frequency, duration, rate of administration, etc.) of at least one compound selected from the group consisting of bendamustine and its salts and prodrugs is not limited to the extent that the desired effect is obtained, and the patient's condition, treatment history, etc. It can be appropriately determined by those skilled in the art based on the treatment process (induction therapy or maintenance therapy), other drugs to be used in combination, and its administration plan.

(E2) Second Embodiment In the second embodiment, the therapeutic method administers a composition comprising i) a natural killer cell on which at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug is acted on. Including ii) administration of type II anti-CD20 antibody.

In the second aspect, the therapeutically effective amount of the type II anti-CD20 antibody can be administered by an administration method known to those skilled in the art. Intravenous administration and subcutaneous administration are exemplified as the administration method.
In this embodiment, a therapeutically effective amount of the natural killer cell can be administered by an administration method known to those skilled in the art. Intravenous administration, subcutaneous administration and oral administration are exemplified as the administration method.

In the second aspect, the type II anti-CD20 antibody and the natural killer cell are administered in combination as the agent, drug, pharmaceutical composition, etc. described in "A. Agent, Medicine, and Pharmaceutical Composition" of the present specification. It is preferable to administer the cells separately or continuously at the same time or at regular intervals. Here, the type II anti-CD20 antibody and the natural killer cells may be formulated separately or individually as long as they are pharmaceutically acceptable. Type II anti-CD20 antibody and natural killer cells can be administered at different doses and dosing regimens, respectively. For example, type II anti-CD20 antibody and natural killer cells may be formulated and administered to one therapeutic subject at the dosage and administration schedule as exemplified below.

The type II anti-CD20 antibody can be administered to a subject according to the dose, administration rate, administration method, and administration plan described in (e1) above, but is not limited to the extent that the desired effect can be obtained, and the patient. Can be appropriately modified by those skilled in the art based on the condition of the patient, treatment history, treatment process (induction therapy or maintenance therapy), other drugs to be used in combination, and its administration plan.

The dose, administration rate, administration method, and administration plan of natural killer cells are not limited to the extent that the desired effect can be obtained, and the patient's condition, treatment history, treatment process (induction therapy or maintenance therapy), concomitant use, etc. Can be appropriately changed by those skilled in the art based on the above-mentioned medicine and its administration plan.

In each of the embodiments described in "E. Therapeutic Methods" of this section, the "therapeutically effective amount" is at least the minimum concentration required to have an effect on measurable amelioration or prevention of a particular disorder. Therapeutically effective amounts herein may vary depending on factors such as the patient's disease state, age, gender, and body weight, as well as the ability of the antibody to elicit the desired response in an individual. A therapeutically effective amount also outweighs any toxic or detrimental effects of the antibody for therapeutically beneficial effects.

In each of the embodiments described in "E. Therapeutic Method" of this section, and in the present specification, the "treatment target" or "subject" is a living body having a type II anti-CD20 antibody-resistant CD20-positive cancer. The subject is not particularly limited as long as it is a living body having a CD20-positive cancer. In this embodiment, a mammal including a human is exemplified as the living body. In that embodiment, the organism is preferably a mammal, including a human. In that embodiment, primates, including rodents and humans, are exemplified as said mammals. In that embodiment, mice and rats are exemplified as rodents. In that embodiment, humans and cynomolgus monkeys are exemplified as primates that include humans. In that embodiment, the mammal is preferably a primate, including humans. In that embodiment, the primates including humans are preferably humans and cynomolgus monkeys, more preferably humans.

In each of the embodiments described in this section "E. Therapeutic Method", "type II anti-CD20 antibody", "at least one compound selected from the group consisting of bendamustine and its salts and prodrugs", "natural killer cells", and. The general and preferred examples of "type II anti-CD20 antibody resistant CD20-positive cancer" are the same as those of the above-mentioned "A. Agent, drug, and pharmaceutical composition", and the acquisition method thereof is described in "B. As described in "Manufacturing method and use in manufacturing".

In addition, other specific configurations of these examples are the same as those described in "A. Agents, Pharmaceuticals, and Pharmaceutical Compositions" above.

F. Type II anti-CD20 antibody, compound, natural killer cell, and combination In one embodiment, the present invention inhibits the growth of cells of type II anti-CD20 antibody resistant CD20 positive cancer, enhances ADCC activity of type II anti-CD20 antibody, And / or provide type II anti-CD20 antibody, compounds, natural killer cells, and / or combinations thereof for use in the treatment of type II anti-CD20 antibody resistant CD20 positive cancer.

In a first example of this embodiment, the type II anti-CD20 antibody of the invention is a type II anti-CD20 antibody for use in suppressing the growth of cells of type II anti-CD20 antibody resistant CD20 positive cancer. The type II anti-CD20 antibody is used in combination with at least one compound selected from the group consisting of bendamustine and salts thereof and prodrugs.
In a second example of that embodiment, the compound of the invention is selected from the group consisting of bendamustine and salts thereof and prodrugs for use in suppressing the growth of cells of type II anti-CD20 antibody resistant CD20 positive cancer. At least one compound. The compound is used in combination with a type II anti-CD20 antibody.
In a third example of that embodiment, the combination of the invention is a type II anti-CD20 antibody for use in suppressing the growth of cells of type II anti-CD20 antibody resistant CD20 positive cancer, and bendamustine and salts and prodrugs thereof. It is a combination of at least one compound selected from the group consisting of.
In a fourth example of the embodiment, the type II anti-CD20 antibody of the invention is bendamustine and a salt thereof for use in enhancing ADCC activity of the type II anti-CD20 antibody against type II anti-CD20 antibody resistant CD20 positive cancer cells. And at least one compound selected from the group consisting of prodrugs. The compound is used in combination with a type II anti-CD20 antibody.
In a fifth example of the embodiment, the combination of the invention is a type II anti-CD20 antibody for use in enhancing ADCC activity of the type II anti-CD20 antibody against CD20-positive cancer cells resistant to type II anti-CD20 antibody, and bendamustine. And a combination of at least one compound selected from the group consisting of salts thereof and prodrugs.
In a sixth example of this embodiment, the type II anti-CD20 antibody of the invention is a type II anti-CD20 antibody for use in suppressing the growth of cells of type II anti-CD20 antibody resistant CD20 positive cancer. The type II anti-CD20 antibody is exposed to at least one compound selected from the group consisting of bendamustine and a salt thereof and a prodrug, and is used in combination with a composition containing natural killer cells on which the compound is allowed to act.
In a seventh example of the embodiment, the natural killer cells of the invention consist of a group consisting of bendamustine and salts thereof and prodrugs for use in suppressing the growth of cells of type II anti-CD20 antibody resistant CD20 positive cancer. Natural killer cells treated with at least one compound of choice. The natural killer cells are used in combination with treatment with type II anti-CD20 antibody.
In an eighth example of the embodiment, the combinations of the invention are type II anti-CD20 antibody, and bendamustine and salts thereof and prodrugs for use in suppressing the growth of cells of type II anti-CD20 antibody resistant CD20 positive cancer. A combination of natural killer cells treated with at least one compound selected from the group consisting of drugs.

In each embodiment described in this section "F. Type II anti-CD20 antibody, compound, natural killer cell, and combination", it is selected from the group consisting of "type II anti-CD20 antibody", "bendamustine and its salts and prodrugs". General and preferred examples of "at least one compound", "natural killer cells", and "type II anti-CD20 antibody resistant CD20 positive cancer" are described in "A. Agents, Pharmaceuticals, and Pharmaceutical Compositions" above. The acquisition method is the same as that described in "B. Manufacturing method and use in manufacturing".

In addition, other specific configurations of these examples are the same as those described in "A. Agents, Pharmaceuticals, and Pharmaceutical Compositions" above.

Incorporation by explicit source All publications, patents, and patent applications mentioned herein are as if each individual publication, patent, or patent application should be specifically and individually incorporated by source. The whole is incorporated here by specifying the source. In the event of a contradiction, the present application containing the definition herein shall prevail.

(Example 1)
== Acquisition of type II anti-CD20 antibody-resistant cancer cell xenograft model ==
Human germinal center B cell-like diffuse large B-cell lymphoma cell line SU-DHL-4 was treated with 100 μg / mL N-ethyl-N-nitrosourea for 24 hours, and gene mutations were randomly introduced into the cell line. Was subcutaneously transplanted into mouse CB-17 / Icr-scid / scidJcl (Claire Japan) 5 x 10 ⁶ cells / animal each.
After tumor engraftment, 18 animals were intraperitoneally administered with obinutuzumab (Obinutuzumab (Genetical Recombination)) 10 mg / kg once a week, three times a week, and then once every three weeks. Intraperitoneal administration was performed twice. This series of administrations showed complete disappearance of the tumor in 16 of 18 animals, but tumor regrowth was observed in 2 animals. Tumor regrowth was observed in 2 animals. The tumor was removed from the individual and re-implanted in another mouse CB-17 / Icr-scid / scidJcl. After tumor engraftment, obinutuzumab 10 mg / kg was intraperitoneally administered once a week three times to grow the tumor. Later, re-implantation was performed again. By this operation, a type II anti-CD20 antibody in vivo resistant strain SU-DHL-4-OR-18-8 (hereinafter referred to as 18-8 strain) was established.

SU-DHL-4 strain (parent strain) and 18-8 strain were subcutaneously transplanted into mouse CB-17 / Icr-scid / scidJcl, and after tumor engraftment, each was randomly assigned to the following groups in n = 6 / group. bottom.

SU-DHL-4 strain (parent strain) Xenograft model:
1. 1. Control (human IgG (HuIgG) 10 mg / kg) group 2. Obinutuzumab (OBI) 3 mg / kg group 3. Obinutuzumab (OBI) 6 mg / kg group 4. Obinutuzumab (OBI) 10 mg / kg group

18-8 strain (type II anti-CD20 antibody resistant strain) Xenograft model:
1. 1. Control (human IgG (HuIgG) 10 mg / kg) group 2. Obinutuzumab (OBI) 10 mg / kg group

Obinutuzumab or human IgG (MP Biomedicals, LLC, Cat # 55908) as a control was administered to each of the above groups at the above dose (mg / kg body weight) once a week (qw) three times in the tail vein ( iv) The first administration start date is Day 1, and SU-DHL-4 strain (parent strain) is on Day 1, 5, 8, 12, 15, 19, 22 and 18-8 strain (type II anti-CD20 antibody resistant strain). Measured tumor volume on Days 1, 4, 8, 11, 15, 18, and 22 to evaluate the antitumor effect of obinutuzumab (Fig. 1).

As a result, the antitumor effect of obinutuzumab was reduced in the 18-8 strain compared to the antitumor effect of obinutuzumab observed in the SU-DHL-4 strain, and the 18-8 strain was against the type II anti-CD20 antibody. It has been shown to be resistant.

(Example 2)
== Maintaining CD20 protein expression in SU-DHL-4-OR-18-8 (18-8 strain) ==
Engrafted tumor tissue was isolated from the SU-DHL-4 strain (parent strain) Xenograft model and the 18-8 strain Xenograft model obtained in Example 1, and promptly 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Industries, Ltd.). Yaku Co., Ltd .; # 060-01667) was soaked and fixed for 24 hours, embedded in paraffin, and then sections were prepared. After deparaffinizing the tumor tissue section, it was incubated overnight with anti-CD20 antibody (primary antibody: Abcam, # ab78237, rabbit monoclonal anti-CD20 antibody [EP459Y], dilution concentration: 0.25 μg / mL) at 4 ^o C. After washing the anti-CD20 antibody, the localization of the anti-CD20 antibody bound on the tumor tissue section was visualized using the Anti-Rabbit polymer-HRP method (Dako EnVision + System-HRP Labelled Polymer, Agilent, # K400311-2). Nuclei were counterstained with hematoxylin (Sakura Finetech Japan KK, Tissue Tech Hematoxylin 3G, # 8656).
Tumor tissue sections were observed under a light microscope to assess CD20 protein expression.

As a result, it was shown that the CD20 protein expression of the 18-8 strain was similar to that of the parent strain, and that the acquisition of type II anti-CD20 antibody resistance did not attenuate the CD20 protein expression (Fig. 2).

(Example 3)
== Effect of combined use of type II anti-CD20 antibody and bendamustine on SU-DHL-4-OR-18-8 (18-8 strain) ==
The 18-8 strain Xenograft model obtained in Example 1 was randomly assigned to the following groups with n = 6 / group. For each group, the administration start date is Day 1, obinutuzumab or human IgG is Day 1, 8, 15, and bendamustine (100 mg for intravenous infusion of Treakisym, SymBio Pharmaceuticals Limited) or physiological saline (Bendamustine non-administration group). Was administered to the tail vein on Days 1 and 2, respectively.

1. 1. Control (human IgG (HuIgG) 6 mg / kg) group 2. Obinutuzumab (OBI) 6 mg / kg group 3. Bendamustine (Benda) 25 mg / kg group 4. Obinutuzumab 6 mg / kg + bendamustine 25 mg / kg combination group (OBI + Benda)

The administration start date was set to Day 1, and the tumor volume was measured after Days 1, 4, 8, 11, 15, 18, 22, 25, and 29 days, and the antitumor effect of each administration was evaluated (Fig. 3).

As a result, in the 18-8 strain Xenograft model, a significant improvement in the antitumor effect was observed in the obinutuzumab + bendamustine combination group as compared with the respective antitumor effects obtained with obinutuzumab or bendamustine alone (Fig.). 3).

(Example 4)
== Establishment of obinutuzumab-inducible ADCC-resistant strain ==
Human non-Hodgkin's lymphoma cell line RL was treated with 300 μg / mL N-ethyl-N-nitrosourea for 24 hours and gene mutations were randomly introduced. CD16 (158V) / NK-92 cells were added as effector cells at an ET ratio of 20: 1, and ADCC reaction was performed overnight at 37 ° C with obinutuzumab 0.1 μg / mL. For the proliferated cells, negative selection using anti-CD56 antibody (Biolegend) and positive selection using anti-CD20 antibody (BD Pharmingen) were performed by MACS.

After repeating the above ADCC reaction and selection by MACS a total of 3 times, the proliferated cells were cloned and used as obinutuzumab-induced ADCC resistant strains RL-E300-1, RL-E300-2, RL-E300-8, RL-E300-22 was established.

For the RL parent strain and each resistant strain, live cells were stained with calcein-AM (FUJIFILM Wako Pure Chemical Corporation) and then seeded at 1x10 ⁴ cells / well on a 96-well plate, and obinutuzumab was added at a concentration of 0.0001 to 100 ng / mL. CD16 (158V) / NK-92 cells as effector cells were added at an ET ratio of 1: 1 and incubated at 37 ° C. for 4 hours. In addition, 1% Triton X-100 was added as Maximum Lysis. After centrifuging the plate and collecting the supernatant, the fluorescence of calcein was measured with a plate reader. ADCC susceptibility was assessed by (measured-background) / (Maximum Lysis-background) x100 (%).

As a result, it was confirmed that all resistant strains had a significant decrease in ADCC sensitivity as compared with the parent strain (RL) (Fig. 4).

(Example 5)
== Enhancement of ADCC activity by pretreatment of effector cells with bendamustine ==
Bendamustine (Selleck, Cat # S1212) (2 μM) was added to the CD16 (158V) / NK-92 cell line, and the cells were precultured at 37 ° C. for 72 hours. After staining the RL parent strain and each resistant strain cell with calcein-AM (FUJIFILM Wako Pure Chemical Corporation), 1x10 ⁴ cells / well were seeded on a 96-well plate, and as Effector cells, bendamustine pretreated NK-92 cells or NK-92 cells without bendamustine pretreatment were added at an ET ratio of 1: 1. Obinutuzumab was added to these at a concentration of 1 ng / mL and incubated at 37 ° C. for 4 hours. In addition, 1% Triton X-100 was added as Maximum Lysis. After centrifuging the plate and collecting the supernatant, the fluorescence of calcein was measured with a plate reader. ADCC activity was assessed by (measured-background) / (Maximum Lysis-background) x 100 (%).

As a result, it was clarified that ADCC activity by the type II anti-CD20 antibody was enhanced by the bendamustine pretreatment of Effector cells (NK-92 cells) in the parent strain and the resistant strain (Fig. 5).

(Example 6)
== Activation of natural killer (NK) cells by type II anti-CD20 antibody treatment and pretreatment with bendamustine ==
Bendamustine (Selleck, Cat # S1212) (2 μM) was added to the CD16 (158V) / NK-92 cell line, and the cells were precultured at 37 ° C. for 72 hours. After staining live cells with calcein-AM (FUJIFILM Wako Pure Chemical Corporation), the RL parent strain and resistant strains RL-E300-8 and RL-E300-22 were seeded on a 96-well plate at 5x10 ⁵ cells / well as effector cells. , Bendamustine pretreated NK-92 cells (Benda), or undamustine untreated NK-92 cells (-) were added at an ET ratio of 1: 1. Obinutuzumab was added to these at a concentration of 0 to 0.1 μg / mL, anti-CD107a antibody (BD Biosciences) and GolgiStop (BD Biosciences) were added, and the mixture was incubated at 37 ° C. for 3 hours.
Cells were harvested, stained with anti-CD56 antibody (Biolegend), and the CD107a positive rate in CD56-positive NK92 cells was evaluated by FACS Fortessa (Becton Dickinson).

As a result, the ratio of CD107a-positive NK-92 cells increased with the addition of type II anti-CD20 antibody, and tended to increase with the pretreatment with bendamustine (Fig. 6). CD107a expression is an indicator of natural killer cell activation, indicating that type II anti-CD20 antibody treatment and bendamustine pretreatment activate natural killer cells.

(Example 7)
== Antitumor effect of type II anti-CD20 antibody treatment and combined use of bendamustine ==
RL-E300-8 resistant strains were subcutaneously transplanted into mouse CB-17 / Icr-scid / scidJcl (Claire Japan) 5x10 ⁶ cells / animal each, and after tumor engraftment, they were grouped as follows (n = 6 each). ..

1. 1. IgG (30 mg / kg) + vehicle administration group 2. Obinutuzumab (OBI) (30 mg / kg) + vehicle administration group 3. IgG (30 mg / kg) + bendamustine (13.3 mg / kg) administration group 4. Obinutuzumab (OBI) (30 mg / kg) + bendamustine (13.3 mg / kg) administration group

For each of the above groups, IgG (human IgG, MP Biomedicals, LLC, Cat # 55908), obinutuzumab, bendamustine (Selleck, Cat # S1212), or vehicle (physiological saline (Otsuka Pharmaceutical Factory)) was administered at the above doses. Each was administered to the tail vein (iv). Obinutuzumab or IgG was administered on Days 1, 8, and 15, and bendamustine or vehicle was administered on Days 1, and 2, with the administration start date as Day 1. Tumor volume was measured by tumor diameter measurement on Days 1, 4, 8, 11, 15, 18, 22, 25, and 29, and the antitumor effect in each administration group was evaluated (Fig. 7).

As a result, when compared on Day 18, a significant antitumor effect was observed in the obinutuzumab + bendamustine combination group as compared with the IgG (30 mg / kg) administration group or each single agent administration group (Fig. 7).

Claims (13)

Cells of type II anti-CD20 antibody resistant CD20-positive cancer containing type II anti-CD20 antibody and used in combination with chemotherapy comprising administration of at least one compound selected from the group consisting of bendamustine and its salts and prodrugs. An agent for suppressing the growth of. Inhibits the growth of cells of type II anti-CD20 antibody-resistant CD20-positive cancer that contains at least one compound selected from the group consisting of bendamustine and its salts and prodrugs and is used in combination with treatment with type II anti-CD20 antibody. Agent to do. The agent according to claim 1 or 2, wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma. The agent according to any one of claims 1 to 3, wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment. 2. The agent according to any one item. The agent according to any one of claims 1 to 5, wherein the type II anti-CD20 antibody is obinutuzumab. Treatment of type II anti-CD20 antibody-resistant CD20-positive cancer that contains type II anti-CD20 antibody and is used in combination with chemotherapy including administration of at least one compound selected from the group consisting of bendamustine and its salts and prodrugs. Pharmaceutical composition for use. A drug for treating type II anti-CD20 antibody-resistant CD20-positive cancer that contains at least one compound selected from the group consisting of bendamustine and its salts and prodrugs and is used in combination with treatment with type II anti-CD20 antibody. Composition. The pharmaceutical composition according to claim 7 or 8, wherein the CD20-positive cancer is B-cell non-Hodgkin's lymphoma. The pharmaceutical composition according to any one of claims 7 to 9, wherein the type II anti-CD20 antibody-resistant CD20-positive cancer is a CD20-positive cancer having experience in type II anti-CD20 antibody treatment. Claims 7 to 10, wherein the type II anti-CD20 antibody-resistant CD20-positive cancer has recurred after the start of maintenance therapy by single administration of the type II anti-CD20 antibody after induction therapy using the type II anti-CD20 antibody. The pharmaceutical composition according to any one of the above. The pharmaceutical composition according to any one of claims 7 to 11, wherein the type II anti-CD20 antibody is obinutuzumab. After induction therapy with type II anti-CD20 antibody, which contains type II anti-CD20 antibody and is used in combination with chemotherapy comprising administration of at least one compound selected from the group consisting of bendamustine and its salts and prodrugs. A drug for suppressing the growth of cancer cells that have recurred after the start of maintenance therapy with type II anti-CD20 antibody alone.

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