A pharmaceutical composition comprising a bispecific antibody as an active ingredient

Abstract

To provide novel drugs for prevention, suppression of symptom progression, suppression of recurrence, or treatment of autoimmune diseases.SOLUTION: As a result of diligent studies, the inventors of the present invention focused on PD-1 / CD19 bispecific antibodies and the like as substances that can solve this problem, and confirmed that the bispecific antibody can be a novel drug for the prevention, the suppression of symptom progression, the suppression of recurrence, or the treatment of autoimmune diseases. We also confirmed that the bispecific antibody has a characteristic that allows interaction with PD-1 and its ligand, PD-L1.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition containing, as an active ingredient, a bispecific antibody capable of specifically binding to PD-1 and CD19 respectively (hereinafter sometimes abbreviated as "PD-1 / CD19 bispecific antibody") or an antibody fragment thereof (hereinafter sometimes abbreviated as "PD-1 / CD19 bispecific antibody etc."), and their pharmaceutical therapeutic uses etc.

Background Art

[0002] PD-1 is an immunosuppressive receptor belonging to the immunoglobulin family, and is a molecule having a function of suppressing the immune activation signal of B cells activated by stimulation from an antigen receptor. From the analysis of PD-1 knockout mice etc., it is known that the PD-1 signal plays an important role in the suppression of autoimmune diseases such as autoimmune dilated cardiomyopathy, lupus-like syndrome, autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease, type I diabetes and rheumatoid arthritis. Therefore, it has been pointed out that a substance that enhances the PD-1 signal can be a preventive or therapeutic agent for autoimmune diseases etc.

[0003] On the other hand, CD19 is a membrane protein expressed on B cells, and is a molecule that transmits stimulation to B cells together with the B cell receptor complex.

[0004] Regarding PD-1 bispecific antibodies for the purpose of preventing or treating autoimmune diseases, there have been several reports so far (Patent Documents 1 to 3), but one of the targets is CD3 which is a member of the T cell receptor complex.

[0005] There are also reports on bispecific antibodies targeting PD-1 and CD19 for the purpose of cancer treatment (Patent Document 4), but there are no reports on PD-1 / CD19 bispecific antibodies for the purpose of preventing, suppressing symptom progression, suppressing recurrence or treating autoimmune diseases.

Prior Art Documents

Patent Documents

[0006] Patent Document 1 International Publication No. 2003 / 011911 Patent Document 2 International Publication No. 2004 / 072286 Patent Document 3 International Publication No. 2013 / 022091 Patent Document 4 Specification of Chinese Patent Application Publication No. 106939050 Summary of the Invention Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a new drug for the prevention, suppression of symptom progression, suppression of recurrence, or treatment of autoimmune diseases and the like. Means for Solving the Problems

[0008] As a result of intensive studies, the inventors of the present invention focused on the PD-1 / CD19 bispecific antibody of the present invention as a substance capable of solving such problems, and completed the present invention.

[0009] Furthermore, the inventors of the present invention confirmed that the PD-1 / CD19 bispecific antibody has a characteristic of allowing interaction with PD-1 and its ligand PD-L1. In addition, the inventors of the present invention have found a PD-1 / CD19 bispecific antibody that is easily separable from by-products in its purification process by a predetermined one or more amino acid substitutions or additions to the heavy chain of the second arm that specifically binds to CD19 constituting the PD-1 / CD19 bispecific antibody.

[0010] That is, the present invention is as follows.

[0011] [1] A bispecific antibody or an antibody fragment thereof having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19, wherein the first arm that specifically binds to PD-1 is (A) (a) Complementary determining region 1 of the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 6 (hereinafter, "complementary determining region 1 of the heavy chain variable region" may be abbreviated as VH-CDR1). (b) Complementary determining region 2 of the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 (hereinafter, "complementary determining region 2 of the heavy chain variable region" may be abbreviated as VH-CDR2) and (c) A heavy chain variable region having complementary determining region 3 of the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 8 (hereinafter, "complementary determining region 3 of the heavy chain variable region" may be abbreviated as VH-CDR3) (hereinafter, the "heavy chain variable region" may be abbreviated as "VH"). (B) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 9, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 10 and (c) A VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 11, (C) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and (c) A VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 14, (D) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 15, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) A VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17, and (E) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 18, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) A VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 20, and having any one VH selected therefrom, The second arm that specifically binds to CD19 is (A) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 36 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 37, (B) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 40, (C) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, (D) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 45 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 47, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, and the PD-1 / CD19 bispecific antibody or an antibody fragment thereof, wherein in any one or more VH-CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3 in the first arm that specifically binds to PD-1, any 1 to 5 amino acid residues thereof may each be substituted with other amino acids, and / or in any one or more VH-CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3 in the second arm that specifically binds to CD19, any 1 to 5 amino acid residues thereof may each be substituted with other amino acids. [2] The first arm that specifically binds to PD-1 is (A) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 6, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8, (B) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 9, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 10 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 11, (C) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 14, (D) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 15, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17, and (E) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 18, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 20, and having any one VH selected from among them, The second arm that specifically binds to CD19 is (A) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 36 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 37, (B) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 and (c) A VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 40, (C) (a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42 and (c) A VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, (D) (a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 45 and (c) A VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) (a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 47, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and (c) The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding [1], having any one VH selected from VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49. [3] (i) The VH of the first arm that specifically binds to PD-1 is (a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 6, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) Having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8, (ii) The VH of the second arm that specifically binds to CD19 is (A) (a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 36 and (c) A VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 37, (B) (a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 and (c) A VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 40, (C) (a) VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 42 and (c) VH having VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, (D) (a) VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, (b) VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 45 and (c) VH having VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) (a) VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 47, (b) VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and (c) VH having VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, which is any one selected from the group consisting of the PD - 1 / CD19 bispecific antibody or an antibody fragment thereof according to item [1] or [2] above. [4] (i) The VH of the first arm that specifically binds to PD - 1 is (a) VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 9, (b) VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 10 and (c) having VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 11, (ii) The VH of the second arm that specifically binds to CD19 is (A) (a) VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, (b) VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 36 and (c) VH having VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 37, (B) (a) VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, (b) VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 and (c) VH having VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 40, (C) (a) VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) VH composed of the amino acid sequence of SEQ ID NO: 42 and VH-CDR2 and (c) VH having VH-CDR3 composed of the amino acid sequence of SEQ ID NO: 43, (D) (a) VH-CDR1 composed of the amino acid sequence of SEQ ID NO: 44, (b) VH-CDR2 composed of the amino acid sequence of SEQ ID NO: 45 and (c) VH having VH-CDR3 composed of the amino acid sequence of SEQ ID NO: 46, and (E) (a) VH-CDR1 composed of the amino acid sequence of SEQ ID NO: 47, (b) VH-CDR2 composed of the amino acid sequence of SEQ ID NO: 48 and (c) VH having VH-CDR3 composed of the amino acid sequence of SEQ ID NO: 49, and any one selected from the group consisting of VH, the PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to the preceding item [1] or [2]. [5] (i) The VH of the first arm that specifically binds to PD-1 is (a) VH-CDR1 composed of the amino acid sequence of SEQ ID NO: 12, (b) VH-CDR2 composed of the amino acid sequence of SEQ ID NO: 13 and (c) having VH-CDR3 composed of the amino acid sequence of SEQ ID NO: 14, (ii) The VH of the second arm that specifically binds to CD19 is (A) (a) VH-CDR1 composed of the amino acid sequence of SEQ ID NO: 35, (b) VH-CDR2 composed of the amino acid sequence of SEQ ID NO: 36 and (c) VH having VH-CDR3 composed of the amino acid sequence of SEQ ID NO: 37, (B) (a) VH-CDR1 composed of the amino acid sequence of SEQ ID NO: 38, (b) VH-CDR2 composed of the amino acid sequence of SEQ ID NO: 39 and (c) VH having VH-CDR3 composed of the amino acid sequence of SEQ ID NO: 40, (C) (a) VH-CDR1 composed of the amino acid sequence of SEQ ID NO: 41, (b) VH-CDR2 composed of the amino acid sequence of SEQ ID NO: 42 and (c) A VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, (D) (a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 45 and (c) A VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) (a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 47, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and (c) A VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, which is any one selected from the VH described in the preceding item [1] or [2], the PD-1 / CD19 bispecific antibody or an antibody fragment thereof. [6] (i) The VH of the first arm that specifically binds to PD-1 is (a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 15, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) Having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17, (ii) The VH of the second arm that specifically binds to CD19 is (A) (a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 36 and (c) A VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 37, (B) (a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 and (c) A VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 40, (C) (a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42 and (c) A VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, (D)(a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 45 and (c) A VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E)(a) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 47, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and (c) A VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, being any one selected from the VH's described in the preceding item [1] or [2], the PD-1 / CD19 bispecific antibody or its antibody fragment. [7] (i) The VH of the first arm that specifically binds to PD-1 (a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 18, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 20, (ii) The VH of the second arm that specifically binds to CD19 (A)(a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 36 and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 37, (B)(a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 40, (C)(a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42 and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, (D)(a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 45 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 47, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, and being any one selected from the VH, the PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to the preceding [1] or [2]. [8] (i) The VH of the first arm that specifically binds to PD-1 (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 6, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8, (ii) The VH of the second arm that specifically binds to CD19 (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 36 and (c) having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 37, the PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to the preceding [1] or [2]. [9] (i) The VH of the first arm that specifically binds to PD-1 (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 6, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8, (ii) The VH of the second arm that specifically binds to CD19 (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 and (c) The PD-1 / CD19 bispecific antibody or its antibody fragment according to item [1] or [2] above, having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 40.

[10] (i) The VH of the first arm that specifically binds to PD-1 (a) having a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 6, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8, (ii) The VH of the second arm that specifically binds to CD19 (a) having a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42 and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, the PD-1 / CD19 bispecific antibody or its antibody fragment according to item [1] or [2] above.

[11] (i) The VH of the first arm that specifically binds to PD-1 (a) having a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 6, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8, (ii) The VH of the second arm that specifically binds to CD19 (a) having a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 45 and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, the PD-1 / CD19 bispecific antibody or its antibody fragment according to item [1] or [2] above.

[12] (i) The VH of the first arm that specifically binds to PD-1 (a) having a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 6, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8, (ii) The VH of the second arm that specifically binds to CD19 is (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 47, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48, and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, and the PD-1 / CD19 bispecific antibody or antibody fragment thereof described in the preceding item [1] or [2].

[13] (i) The VH of the first arm that specifically binds to PD-1 is (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 9, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 11, and (ii) The VH of the second arm that specifically binds to CD19 is (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 36, and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 37, and the PD-1 / CD19 bispecific antibody or antibody fragment thereof described in the preceding item [1] or [2].

[14] (i) The VH of the first arm that specifically binds to PD-1 is (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 9, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 11, and (ii) The VH of the second arm that specifically binds to CD19 is (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39, and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 40, and the PD-1 / CD19 bispecific antibody or antibody fragment thereof described in the preceding item [1] or [2].

[15] (i) The VH of the first arm that specifically binds to PD-1 has (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 9, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 10 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 11, and (ii) The VH of the second arm that specifically binds to CD19 has (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, the PD-1 / CD19 bispecific antibody or its antibody fragment according to item [1] or [2] above.

[16] (i) The VH of the first arm that specifically binds to PD-1 has (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 9, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 10 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 11, and (ii) The VH of the second arm that specifically binds to CD19 has (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 45 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, the PD-1 / CD19 bispecific antibody or its antibody fragment according to item [1] or [2] above.

[17] (i) The VH of the first arm that specifically binds to PD-1 has (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 9, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 10 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 11, and (ii) The VH of the second arm that specifically binds to CD19 has (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 47, (b) VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and the PD - 1 / CD19 bispecific antibody or its antibody fragment according to item [1] or [2] above, having (c) VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 49.

[18] (i) The VH of the first arm that specifically binds to PD - 1 is (a) VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, (b) VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and (c) having VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 14, (ii) The VH of the second arm that specifically binds to CD19 is (a) VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, (b) VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 36 and (c) having VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 37, the PD - 1 / CD19 bispecific antibody or its antibody fragment according to item [1] or [2] above.

[19] (i) The VH of the first arm that specifically binds to PD - 1 is (a) VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, (b) VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and (c) having VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 14, (ii) The VH of the second arm that specifically binds to CD19 is (a) VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, (b) VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 and (c) having VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 40, the PD - 1 / CD19 bispecific antibody or its antibody fragment according to item [1] or [2] above.

[20] (i) The VH of the first arm that specifically binds to PD - 1 is (a) VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and (c) having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 14, (ii) the VH of the second arm that specifically binds to CD19 is (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42 and (c) having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, the PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1] or [2].

[21] (i) The VH of the first arm that specifically binds to PD-1 is (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and (c) having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 14, (ii) the VH of the second arm that specifically binds to CD19 is (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 45 and (c) having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, the PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1] or [2].

[22] (i) The VH of the first arm that specifically binds to PD-1 is (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and (c) having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 14, (ii) the VH of the second arm that specifically binds to CD19 is (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 47, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and (c) The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1] or [2], which has a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49.

[23] (i) The VH of the first arm that specifically binds to PD-1 (a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 15, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16, and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17, (ii) The VH of the second arm that specifically binds to CD19 (a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 36, and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 37. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1] or [2].

[24] (i) The VH of the first arm that specifically binds to PD-1 (a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 15, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16, and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17, (ii) The VH of the second arm that specifically binds to CD19 (a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39, and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 40. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1] or [2].

[25] (i) The VH of the first arm that specifically binds to PD-1 (a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 15, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16, and (c) having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17, (ii) the VH of the second arm that specifically binds to CD19, (a) a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42 and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, the PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to item [1] or [2] above.

[26] (i) the VH of the first arm that specifically binds to PD-1, (a) a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 15, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17, (ii) the VH of the second arm that specifically binds to CD19, (a) a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 45 and (c) a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, the PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to item [1] or [2] above.

[27] (i) the VH of the first arm that specifically binds to PD-1, (a) a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 15, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17, (ii) the VH of the second arm that specifically binds to CD19, (a) a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 47, (b) a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and (c) The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [2], which has a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49.

[28] (i) The VH of the first arm that specifically binds to PD-1 (a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 18, (b) has a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19, and (c) has a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 20, (ii) The VH of the second arm that specifically binds to CD19 (a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, (b) has a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 36, and (c) has a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 37. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [2].

[29] (i) The VH of the first arm that specifically binds to PD-1 (a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 18, (b) has a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19, and (c) has a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 20, (ii) The VH of the second arm that specifically binds to CD19 (a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, (b) has a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39, and (c) has a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 40. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [2].

[30] (i) The VH of the first arm that specifically binds to PD-1 (a) has a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 18, (b) has a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19, and (c) having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 20, (ii) the VH of the second arm that specifically binds to CD19, (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42 and (c) having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to item [1] or [2] above.

[31] (i) the VH of the first arm that specifically binds to PD-1, (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 18, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 20, (ii) the VH of the second arm that specifically binds to CD19, (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 45 and (c) having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to item [1] or [2] above.

[32] (i) the VH of the first arm that specifically binds to PD-1, (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 18, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 20, (ii) the VH of the second arm that specifically binds to CD19, (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 47, (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and (c) The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [2], which has a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49.

[33] Among the framework regions of VH of the first arm that specifically binds to PD-1 (hereinafter, the "framework region" may be abbreviated as FR), framework region 1 (hereinafter, may be abbreviated as FR1), framework region 2 (hereinafter, may be abbreviated as FR2), and framework region 3 (hereinafter, may be abbreviated as FR3) respectively correspond to the amino acid sequences encoded by the germline V gene IGHV7-4-1 or the gene thereof that has undergone somatic mutation thereof. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[32] .

[34] The framework region 4 (hereinafter, the "framework region 4" may be abbreviated as FR4) of VH of the first arm that specifically binds to PD-1 consists of the amino acid sequence encoded by the germline J gene JH6c or the gene thereof that has undergone somatic mutation thereof (however, excluding the amino acid sequence contained in the VH-CDR3 region). The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding item

[33] .

[35] The FR of VH of the first arm that specifically binds to PD-1 is encoded by the gene IGHV7-4-1 of the germline V gene, which may have undergone somatic mutation, and due to the somatic mutation, lysine at position 13 in the amino acid sequence of SEQ ID NO: 21 is substituted with glutamine, alanine at position 16 is substituted with valine, or lysine at position 19 is substituted with methionine, or any combination of them is substituted or may be substituted. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding item

[33] or

[34] .

[36] The FR of the VH of the first arm that specifically binds to PD-1 may be encoded by the gene IGHV7-4-1 of the germline V gene, which may have somatic mutations, and due to the somatic mutations, the valine at position 37 in the amino acid sequence of SEQ ID NO: 21 is replaced by leucine or may be replaced, and consists of FR2, which is the PD-1 / CD19 bispecific antibody or its antibody fragment according to any one of the preceding items

[33] to

[35] .

[37] The FR of the VH of the first arm that specifically binds to PD-1 may be encoded by the gene IGHV7-4-1 of the germline V gene, which may have somatic mutations, and due to the somatic mutations, the serine at position 77 in the amino acid sequence of SEQ ID NO: 21 is replaced by threonine, or the cysteine at position 84 is replaced by serine or asparagine, or any combination of them may be replaced or may be replaced, and consists of FR3, which is the PD-1 / CD19 bispecific antibody or its antibody fragment according to any one of the preceding items

[33] to

[36] .

[38] The FR4 of the VH of the first arm that specifically binds to PD-1 may be encoded by the gene JH6c of the germline J gene (excluding the gene region encoding the VH-CDR3 region), and lysine (Lys) in its FR4 amino acid sequence (Trp-Gly-Lys-Gly-Thr-Thr*-Val-Thr-Val-Ser-Ser) (SEQ ID NO: 61) is replaced by glutamine or asparagine, and / or the threonine (* marked) is replaced by leucine or may be replaced, which is the PD-1 / CD19 bispecific antibody or its antibody fragment according to any one of the preceding items

[33] to

[37] .

[39] Among the FRs of the VH of the second arm that specifically binds to CD19, FR1, FR2, and FR3 each correspond to the amino acid sequence encoded by the germline V gene IGHV5-51 or the gene that has undergone somatic mutations thereof, which is the PD-1 / CD19 bispecific antibody or its antibody fragment according to any one of the preceding items [1] to

[38] .

[40] The FR of the VH of the second arm that specifically binds to CD19 may be encoded by the gene that has undergone somatic mutation of the germline V gene IGHV5-51, and due to the somatic mutation, glutamic acid at position 1 in the amino acid sequence of SEQ ID NO: 22 is replaced by glutamine, proline at position 14 is replaced by serine, tyrosine at position 27 is replaced by phenylalanine, or threonine at position 30 is replaced by isoleucine, or any combination of these is replaced or may be replaced, and consists of FR1, the PD-1 / CD19 bispecific antibody or antibody fragment thereof described in the preceding item

[39] .

[41] The FR of the VH of the second arm that specifically binds to CD19 consists of FR2 encoded by the gene that may have undergone somatic mutation of the germline V gene IGHV5-51, the PD-1 / CD19 bispecific antibody or antibody fragment thereof described in the preceding item

[39] or

[40] .

[42] The FR of the VH of the second arm that specifically binds to CD19 may be encoded by the gene that has undergone somatic mutation of the germline V gene IGHV5-51, and due to the somatic mutation, isoleucine at position 76 in the amino acid sequence of SEQ ID NO: 22 is replaced by phenylalanine, serine at position 77 is replaced by threonine or asparagine, threonine at position 78 is replaced by valine, serine at position 84 is replaced by asparagine, methionine at position 93 is replaced by isoleucine or leucine, or alanine at position 97 is replaced by valine, or any combination of these is replaced or may be replaced, and consists of FR3, the PD-1 / CD19 bispecific antibody or antibody fragment thereof described in any one of the preceding items

[39] to

[41] .

[43] The VH of the first arm that specifically binds to PD-1 consists of any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, or an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the VH, the PD-1 / CD19 bispecific antibody or antibody fragment thereof described in any one of the preceding items [1] to

[42] .

[44] The VH of the first arm that specifically binds to PD-1 consists of any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of items [1] to

[43] above.

[45] The VH of the second arm that specifically binds to CD19 consists of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, or an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the VH, and the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of items [1] to

[44] above.

[46] The VH of the second arm that specifically binds to CD19 consists of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, wherein glutamine at position 114 in the sequence is substituted with arginine, or an amino acid sequence that may or may not be substituted, and the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of items [1] to

[45] above.

[47] The VH of the second arm that specifically binds to CD19 consists of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, and the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of items [1] to

[45] above.

[48] The VH of the first arm that specifically binds to PD-1 consists of any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and the VH of the second arm that specifically binds to CD19 consists of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, and the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to item [1] or [2] above.

[49] A bispecific antibody or an antibody fragment thereof having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19, wherein the VH of the first arm that specifically binds to PD-1 consists of any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, or an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the VH; and the VH of the second arm that specifically binds to CD19 consists of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, or an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the VH; a PD-1 / CD19 bispecific antibody or an antibody fragment thereof.

[50] The PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to the preceding [1] or [2], wherein the VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 1, and the VH in the second arm that specifically binds to CD19 consists of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66.

[51] The PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to the preceding [1] or [2], wherein the VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 2, and the VH in the second arm that specifically binds to CD19 consists of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66.

[52] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 3, and the VH in the second arm that specifically binds to CD19 consists of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1] or [2].

[53] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 4, and the VH in the second arm that specifically binds to CD19 consists of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1] or [2].

[54] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 5, and the VH in the second arm that specifically binds to CD19 consists of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1] or [2].

[55] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 1, and the VH in the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 62. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1], [2], or [8].

[56] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 1, and the VH in the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 63. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1], [2], or [9].

[57] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 1, and the VH in the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 64. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1], [2] or

[10] .

[58] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 1, and the VH in the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 65. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1], [2] or

[11] .

[59] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 1, and the VH in the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 66. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1], [2] or

[12] .

[60] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 2, and the VH in the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 62. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1], [2] or

[13] .

[61] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 2, and the VH in the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 63. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1], [2] or

[14] .

[62] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 2, and the VH in the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 64. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1], [2] or

[15] .

[63] A PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [1], [2] or

[16] , wherein the VH of the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 2, and the VH of the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 65.

[64] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [1], [2] or

[17] , wherein the VH of the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 2, and the VH of the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 66.

[65] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [1], [2] or

[18] , wherein the VH of the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 3, and the VH of the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 62.

[66] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [1], [2] or

[19] , wherein the VH of the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 3, and the VH of the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 63.

[67] A PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [1], [2] or

[20] , wherein the VH of the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 3, and the VH of the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 64.

[68] A PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [1], [2] or

[21] , wherein the VH of the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 3, and the VH of the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 65.

[69] A PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [1], [2] or

[22] , wherein the VH of the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 3, and the VH of the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 66.

[70] A PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [1], [2] or

[23] , wherein the VH of the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 4, and the VH of the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 62.

[71] A PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [1], [2] or

[24] , wherein the VH of the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 4, and the VH of the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 63.

[72] A PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [1], [2] or

[25] , wherein the VH of the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 4, and the VH of the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 64.

[73] A PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [1], [2] or

[26] , wherein the VH of the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 4, and the VH of the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 65.

[74] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [1], [2] or

[27] , wherein the VH of the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 4, and the VH of the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 66.

[75] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 5, and the VH in the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 62. The PD-1 / CD19 bispecific antibody or antibody fragment thereof described in the preceding item [1], [2] or

[28] .

[76] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 5, and the VH in the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 63. The PD-1 / CD19 bispecific antibody or antibody fragment thereof described in the preceding item [1], [2] or

[29] .

[77] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 5, and the VH in the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 64. The PD-1 / CD19 bispecific antibody or antibody fragment thereof described in the preceding item [1], [2] or

[30] .

[78] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 5, and the VH in the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 65. The PD-1 / CD19 bispecific antibody or antibody fragment thereof described in the preceding item [1], [2] or

[31] .

[79] The VH in the first arm that specifically binds to PD-1 consists of the amino acid sequence of SEQ ID NO: 5, and the VH in the second arm that specifically binds to CD19 consists of the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 66. The PD-1 / CD19 bispecific antibody or antibody fragment thereof described in the preceding item [1], [2] or

[32] .

[80] The first arm that specifically binds to PD-1 and / or the second arm that specifically binds to CD19 each (a) Complementary determining region 1 of the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 26 (hereinafter, "complementary determining region 1 of the light chain variable region" may be abbreviated as VL-CDR1). (b) Complementary determining region 2 of the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 27 (hereinafter, "complementary determining region 2 of the light chain variable region" may be abbreviated as VL-CDR2), and (c) A light chain variable region having a complementary determining region 3 of the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 28 (hereinafter, "complementary determining region 3 of the light chain variable region" may be abbreviated as VL-CDR3) (hereinafter, the "light chain variable region" may be abbreviated as "VL"), the PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to any one of the preceding items [1] to

[79] .

[81] The PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to any one of the preceding items [1] to

[80] , wherein the first arm specifically binding to PD-1 and / or the second arm specifically binding to CD19 each has a VL consisting of the amino acid sequence of SEQ ID NO: 25.

[82] A bispecific antibody or an antibody fragment thereof having a first arm specifically binding to PD-1 and a second arm specifically binding to CD19, and specifically binding to PD-1 and CD19 respectively, (A) The first arm specifically binding to PD-1 has a VH consisting of any one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a VL consisting of the amino acid sequence of SEQ ID NO: 25, and (B) The second arm specifically binding to CD19 has a VH consisting of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66 and a VL consisting of the amino acid sequence of SEQ ID NO: 25, the PD-1 / CD19 bispecific antibody or an antibody fragment thereof.

[83] A PD-1 / CD19 bispecific antibody or antibody fragment thereof, comprising a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19, and which specifically binds to both PD-1 and CD19, respectively, wherein the first arm that specifically binds to PD-1 cross-competes with (1) the binding to PD-1 of the first arm that specifically binds to PD-1, which has a VH consisting of any one of the amino acid sequences selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and a VL consisting of the amino acid sequence of SEQ ID NO:25, or (2) the binding to PD-1 of the variable region of a monoclonal antibody that specifically binds to PD-1, which comprises the VH and VL.

[84] A PD-1 / CD19 bispecific antibody or antibody fragment thereof, which has a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19, and which specifically binds to PD-1 and CD19, respectively, wherein the binding of the first arm that specifically binds to PD-1 is cross-competed by either (1) the first arm that specifically binds to PD-1, which has a VH consisting of any one of the amino acid sequences selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and a VL consisting of the amino acid sequence of SEQ ID NO:25, or (2) the variable region of a monoclonal antibody that specifically binds to PD-1, which comprises the VH and VL.

[85] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph

[83] or

[84] , wherein the second arm that specifically binds to CD19 cross-competes with the binding to CD19 of (1) the second arm that specifically binds to CD19, which has a VH consisting of any one of the amino acid sequences selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, and a VL consisting of the amino acid sequence of SEQ ID NO: 25, or (2) the binding to CD19 of the variable region of a monoclonal antibody that specifically binds to CD19, which comprises the VH and VL.

[86] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph

[83] or

[84] , wherein the binding to CD19 of the second arm that specifically binds to CD19 is cross-competed by (1) a second arm that specifically binds to CD19, the second arm having a VH consisting of any one of the amino acid sequences selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, and a VL consisting of the amino acid sequence of SEQ ID NO: 25; or (2) a PD-1 / CD19 bispecific antibody or antibody fragment thereof that specifically binds to CD19, the VH and VL comprising the variable region of a monoclonal antibody that specifically binds to CD19.

[87] The second arm specifically binds to CD19. (A) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35; (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 36; and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 37; (B) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38; (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39; and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 40; (C) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 41; (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42; and (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 43; (D) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 44; (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 45; and (c) a VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46; and (E) (a) VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 47; (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48; and (c) Any one VH selected from VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, the PD-1 / CD19 bispecific antibody or an antibody fragment thereof described in the preceding item

[83] or

[84] .

[88] The VH of the second arm that specifically binds to CD19 is any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, or consists of an amino acid sequence that is at least 80%, 90%, 95%, 98% or 99% identical to the amino acid sequence of the VH, the PD-1 / CD19 bispecific antibody or an antibody fragment thereof described in the preceding item

[83] or

[84] .

[89] The VH of the second arm that specifically binds to CD19 consists of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, the PD-1 / CD19 bispecific antibody or an antibody fragment thereof described in the preceding item

[83] or

[84] .

[90] The first arm that specifically binds to PD-1 and / or the second arm that specifically binds to CD19 is (a) VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, (b) VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27 and (c) Any one VL having a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28, the PD-1 / CD19 bispecific antibody or an antibody fragment thereof described in any one of the preceding items

[83] to

[89] .

[91] The first arm that specifically binds to PD-1 and / or the second arm that specifically binds to CD19 has a VL consisting of the amino acid sequence of SEQ ID NO: 25, the PD-1 / CD19 bispecific antibody or an antibody fragment thereof described in any one of the preceding items

[83] to

[89] .

[92] The PD-1 / CD19 bispecific antibody is an IgG antibody, the PD-1 / CD19 bispecific antibody or an antibody fragment thereof described in any one of the preceding items [1] to

[91] .

[93] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph

[92] , wherein the IgG antibody described in the preceding paragraph

[92] is an IgG1 antibody or an IgG4 antibody.

[94] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph

[92] , wherein the IgG antibody described in the preceding paragraph

[92] is an IgG1 antibody.

[95] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph

[92] , wherein the IgG antibody described in the preceding paragraph

[92] is an IgG4 antibody.

[96] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph

[94] , wherein the 235th leucine in the EU numbering system in the two heavy chain constant regions of the IgG1 antibody described in the preceding paragraph

[94] is each replaced with glycine, and / or the 236th glycine is each replaced with arginine.

[97] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph

[94] or

[96] , wherein the 351st leucine and the 366th threonine in the constant region of the heavy chain having VH of the first arm specifically binding to PD-1 are both replaced with lysine, the 351st leucine in the constant region of the heavy chain having VH of the second arm specifically binding to CD19 is replaced with aspartic acid, and the 368th leucine is replaced with glutamic acid.

[98] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph

[94] or

[96] , wherein the 351st leucine in the constant region of the heavy chain having VH of the first arm specifically binding to PD-1 is replaced with aspartic acid, and the 368th leucine is replaced with glutamic acid, and the 351st leucine and the 366th threonine in the constant region of the heavy chain having VH of the second arm specifically binding to CD19 are both replaced with lysine.

[99] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items

[94] and

[96] to

[98] , wherein the 447th lysine according to the EU numbering system in the two heavy chain constant regions of the IgG1 antibody described in any one of the preceding items

[94] and

[96] to

[98] is absent.

[0100] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding item

[95] , wherein the 228th serine according to the EU numbering system in the two heavy chain constant regions of the IgG4 antibody described in the preceding item

[95] is each substituted with proline.

[0101] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items

[94] and

[96] to

[99] , wherein the heavy chain having the VH of the second arm that specifically binds to CD19 further has Gly (glycine), Gly-Lys-Lys-Ala (SEQ ID NO: 67), Gly-Lys-Ala-Lys-Ala (SEQ ID NO: 68), Gly-Arg-Arg-Ala (SEQ ID NO: 69) or Gly-Arg-Ala-Arg-Ala (SEQ ID NO: 70) at its C-terminus.

[0102] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[94] ,

[96] ,

[98] ,

[99] and

[0101] , wherein the heavy chain having the VH of the first arm that specifically binds to PD-1 comprises a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23.

[0103] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[94] ,

[96] ,

[98] ,

[99] ,

[0101] and

[0102] , wherein the heavy chain having the VH of the second arm that specifically binds to CD19 comprises a heavy chain constant region consisting of any one amino acid sequence selected from SEQ ID NO: 24, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75.

[0104] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0103] , wherein the light chain having a VL in a first arm that specifically binds to PD-1 and / or the light chain having a VL in a second arm that specifically binds to CD19 comprises a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29.

[0105] A bispecific antibody or an antibody fragment thereof, which has a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19, and which specifically binds to PD-1 and CD19, respectively; (A) a heavy chain having a VH of a first arm that specifically binds to PD-1, the heavy chain comprising a VH consisting of any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23; (B) a light chain having a VL of the first arm that specifically binds to PD-1 comprises a VL consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29; (C) a heavy chain having a VH of the second arm that specifically binds to CD19, comprising a VH consisting of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, and a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 24; and (D) The PD-1 / CD19 bispecific antibody or antibody fragment thereof, wherein the light chain having the VL of the second arm that specifically binds to CD19 comprises a VL consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29.

[0106] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0105] , wherein the first arm that specifically binds to PD-1 permits interaction between PD-1 and PD-L1.

[0107] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0106] , wherein the isoelectric point of a complex consisting of the heavy chain and light chain of the first arm that specifically binds to PD-1 is about 7.4 to about 7.7 (preferably about 7.5 to about 7.6).

[0108] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0107] , wherein the complex consisting of the heavy chain and light chain of the second arm that specifically binds to CD19 has an isoelectric point of about 8.3 to about 8.8 (preferably about 8.4 to about 8.6).

[0109] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0108] , which sufficiently reduces cytokine production in the blood or tissues during administration or within 24 hours after administration.

[0110] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0108] , wherein the first arm that specifically binds to PD-1 allows interaction between PD-1 and PD-L1, and cytokine production is sufficiently reduced.

[0111] A PD-1 / CD19 bispecific antibody or antibody fragment thereof described in the preceding paragraph

[0109] or

[0110] , wherein the cytokine is at least IL-2, IFN-γ, or TNF-α.

[0112] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0111] , which inhibits B cell activation by binding to PD-1 and CD19, each of which is expressed on the same B cell.

[0113] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0111] , which suppresses the activation of T cells (preferably memory T cells).

[0114] A PD-1 / CD19 bispecific antibody or antibody fragment thereof described in the preceding paragraph

[0113] , which inhibits T cell activation by binding to CD19 expressed on B cells and PD-1 expressed on T cells, respectively.

[0115] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0114] , wherein PD-1 and CD19 are human PD-1 and human CD19, respectively.

[0116] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0115] , wherein the PD-1 / CD19 bispecific antibody is a monoclonal antibody.

[0117] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0116] , wherein the PD-1 / CD19 bispecific antibody is an isolated antibody. [1-1] A pharmaceutical composition comprising, as an active ingredient, a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[0117] . [1-2] The pharmaceutical composition according to the preceding paragraph [1-1], further comprising at least one pharmaceutically acceptable carrier.

[0012] [2-1] An agent for preventing, suppressing the progression of symptoms, suppressing recurrence, and / or treating an autoimmune disease, comprising as an active ingredient a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[0117] . [2-2] Autoimmune diseases include Behçet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis (systemic sclerosis, progressive systemic sclerosis), scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa, microscopic polyangiitis), aortitis syndrome (Takayasu's arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, and rheumatoid vasculitis. , large vessel vasculitis, ANCA-associated vasculitis (e.g., granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis), Cogan's syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid syndrome, eosinophilic fasciitis, IgG4-related disorders (e.g., primary sclerosing cholangitis, autoimmune pancreatitis, etc.), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, nonalcoholic steatohepatitis, primary biliary cirrhosis, Goodpasture's syndrome, rapidly progressive glomerular purpura Nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease (Graves' disease (hyperthyroidism)), Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, Addison's disease (chronic hypoadrenocorticism), idiopathic Addison's disease, type 1 diabetes, slowly progressive type 1 diabetes (adult latent autoimmune diabetes), localized scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, acquired The agent according to the preceding item [2-1], which is epidermolysis bullosa, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, transplantation immunity, familial Mediterranean fever, eosinophilic sinusitis, dilated cardiomyopathy, systemic mastocytosis, or inclusion body myositis. [2-3] An agent for preventing, suppressing the progression of symptoms, suppressing recurrence, and / or treating graft-versus-host disease (GVHD), comprising as an active ingredient a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[0117] . [2-4] Insulin preparations (e.g., human insulin, insulin glargine, insulin lispro, insulin detemir, insulin aspart, etc.), sulfonylureas (e.g., glibenclamide, gliclazide, glimepiride, etc.), rapid-acting insulin secretagogues (e.g., nateglinide, etc.), biguanides (e.g., metformin, etc.), insulin sensitizers (e.g., pioglitazone, etc.), α-glucosidase inhibitors (e.g., acarbose, voglibose, etc.), diabetic neuropathy medications (e.g., epalresta, an agent for preventing, inhibiting the progression of symptoms, inhibiting recurrence, and / or treating type 1 diabetes, the agent comprising as an active ingredient a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[0117] , which is administered in combination with one or more drugs selected from the group consisting of benzodiazepines (e.g., benzodiazepines), ... [2-5] A prophylactic, symptom progression inhibitor, relapse inhibitor and / or therapeutic agent for multiple sclerosis, characterized by being administered together with any one or more agents selected from steroid drugs (e.g., cortisone acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, fludrocortisone acetate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisolone butylacetate, prednisolone sodium phosphate, halopredone acetate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, triamcinolone, triamcinolone acetate, triamcinolone acetonide, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone palmitate, paramethasone acetate and betamethasone, etc.), interferon β-1a, interferon β-1b, glatiramer acetate, mitoxantrone, azathioprine, cyclophosphamide, cyclosporine, methotrexate, cladribine, adrenocorticotropic hormone (ACTH), corticotropin, mizoribine, tacrolimus, fingolimod and alemtuzumab, and containing as an active ingredient a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[0117] . [2-6] A prophylactic, symptom progression inhibitor, relapse inhibitor and / or therapeutic agent for systemic lupus erythematosus, characterized by being administered together with any one or more agents selected from steroid drugs (e.g., the steroid drugs described in the preceding item [2-5]), immunosuppressive agents (e.g., cyclosporine, tacrolimus and fingolimod, etc.) and belimumab, and containing as an active ingredient a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[0117] . [2-7] An agent for preventing, inhibiting the progression of symptoms, inhibiting recurrence, and / or treating rheumatoid arthritis, which comprises as an active ingredient a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[0117] , and which is administered in combination with one or more drugs selected from a steroid drug (e.g., a steroid drug described in the preceding item [2-5]), an antirheumatic drug (e.g., methotrexate, sulfasalazine, bucillamine, leflunomide, mizoribine, tacrolimus, etc.), an anticytokine drug (e.g., infliximab, adalimumab, tocilizumab, etanercept, golimumab, certolizumab, etc.), and abatacept. [2-8] An agent for preventing, suppressing the progression of symptoms, suppressing recurrence, and / or treating an autoimmune disease, characterized by being administered in combination with one or more of the drugs listed in the preceding paragraphs [2-4] to [2-7], and comprising as an active ingredient a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding paragraphs [1] to

[0117] . [2-9] A preventive, symptom progression suppressive, recurrence suppressive and / or therapeutic agent for each of the diseases described in the preceding paragraphs [2-4] to [2-8], characterized by being administered to a patient receiving any one or more of the drugs listed in the preceding paragraphs [2-4] to [2-7]. [2-10] A preventive, symptom progression suppressive, recurrence suppressive and / or therapeutic agent for each of the diseases described in the preceding paragraphs [2-4] to [2-8], characterized in that it is administered after the administration of any one or more of the drugs listed in the preceding paragraphs [2-4] to [2-7]. [2-11] A preventive, symptom progression suppressive, recurrence suppressive and / or therapeutic agent for each of the diseases described in the preceding paragraphs [2-4] to [2-8], characterized in that it is administered before the administration of any one or more of the drugs listed in the preceding paragraphs [2-4] to [2-7].

[0013] [3-1] An intravenous formulation comprising a PD-1 / CD19 bispecific antibody or antibody fragment thereof selected from any one of the preceding items [1] to

[0117] and at least one pharmaceutically acceptable carrier. [3-2] The intravenous preparation according to the preceding item [3-1] for preventing, inhibiting the progression of symptoms, inhibiting recurrence and / or treating an autoimmune disease. [3-3] An intravenous injection preparation according to the preceding item [3-1] or [3-2], which is for intravenous drip.

[0014] [4-1] Use of a pharmaceutical composition according to the preceding item [1-1] or [1-2] for the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of autoimmune diseases. [4-2] Autoimmune diseases include Behçet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis (systemic sclerosis, progressive systemic sclerosis), scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa, microscopic polyangiitis), aortitis syndrome (Takayasu's arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, and rheumatoid vasculitis. , large vessel vasculitis, ANCA-associated vasculitis (e.g., granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis), Cogan's syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid syndrome, eosinophilic fasciitis, IgG4-related disorders (e.g., primary sclerosing cholangitis, autoimmune pancreatitis, etc.), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, nonalcoholic steatohepatitis, primary biliary cirrhosis, Goodpasture's syndrome, rapidly progressive glomerular nephropathy inflammation, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease (Graves' disease (hyperthyroidism)), Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, Addison's disease (chronic hypoadrenocorticism), idiopathic Addison's disease, type 1 diabetes, slowly progressive type 1 diabetes (adult latent autoimmune diabetes), localized scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, acquired epidermal ulcers The use according to the preceding item [4-1], wherein the condition is hydrocephalus, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, transplantation immunity, familial Mediterranean fever, eosinophilic sinusitis, dilated cardiomyopathy, systemic mastocytosis, or inclusion body myositis. [4-3] Use of the pharmaceutical composition described in the preceding paragraph [1-1] or [1-2] for the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of graft-versus-host disease (GVHD). Use of the pharmaceutical composition according to the preceding item [1-1] or [1-2], which is administered together with any one or more drugs selected from insulin preparations (e.g., human insulin, insulin glargine, insulin lispro, insulin detemir, insulin aspart, etc.), sulfonylurea agents (e.g., glibenclamide, gliclazide, glimepiride, etc.), rapid-acting insulin secretagogues (e.g., nateglinide, etc.), biguanide preparations (e.g., metformin, etc.), insulin resistance-improving agents (e.g., pioglitazone, etc.), α-glucosidase inhibitors (e.g., acarbose, voglibose, etc.), drugs for treating diabetic neuropathy (e.g., epalrestat, mexiletine, imidapril, etc.), GLP-1 analog preparations (e.g., liraglutide, exenatide, lixisenatide, semaglutide, etc.), and DPP-4 inhibitors (e.g., sitagliptin, vildagliptin, alogliptin, etc.) in the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of type 1 diabetes. Use of the pharmaceutical composition according to the preceding item [1-1] or [1-2], which is administered together with any one or more drugs selected from steroid drugs (e.g., cortisone acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, fludrocortisone acetate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisolone butylacetate, prednisolone sodium phosphate, halopredone acetate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, triamcinolone, triamcinolone acetate, triamcinolone acetonide, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone palmitate, paramethasone acetate, and betamethasone, etc.), interferon β-1a, interferon β-1b, glatiramer acetate, mitoxantrone, azathioprine, cyclophosphamide, cyclosporine, methotrexate, cladribine, adrenocorticotropic hormone (ACTH), corticotropin, mizoribine, tacrolimus, fingolimod, and alemtuzumab in the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of multiple sclerosis. [4-6] Use of the pharmaceutical composition described in the preceding paragraph [1-1] or [1-2], administered together with one or more drugs selected from steroid drugs (e.g., steroid drugs described in the preceding paragraph [2-5]), immunosuppressants (e.g., cyclosporine, tacrolimus, fingolimod, etc.), and belimumab, in the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of systemic lupus erythematosus. [4-7] Use of the pharmaceutical composition described in the preceding paragraph [1-1] or [1-2] in combination with one or more drugs selected from steroid drugs (e.g., the steroid drugs described in the preceding paragraph [2-5]), antirheumatic drugs (e.g., methotrexate, sulfasalazine, bucillamine, leflunomide, mizoribine, tacrolimus, etc.), anticytokine drugs (e.g., infliximab, adalimumab, tocilizumab, etanercept, golimumab, certolizumab, etc.), and abatacept, in the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of rheumatoid arthritis. [4-8] Use of the pharmaceutical composition according to the preceding paragraph [1-1] or [1-2], administered together with one or more of the drugs listed in the preceding paragraphs [4-4] to [4-7], in the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of an autoimmune disease.

[0015] [5-1] A method for preventing, suppressing the progression of symptoms, suppressing recurrence, and / or treating an autoimmune disease, comprising administering to a patient an effective amount of a PD-1 / CD19 bispecific antibody or antibody fragment thereof selected from any one of the preceding items [1] to

[0117] .

[0016] [6-1] A PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[0117] , for use in the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of an autoimmune disease.

[0017] [7-1] Use of a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[0117] in the manufacture of a pharmaceutical for preventing, suppressing the progression of symptoms, suppressing recurrence, and / or treating an autoimmune disease.

[0018] [8-1] A preventive, symptom progression inhibitor, recurrence inhibitor and / or therapeutic agent for autoreactive B cell-mediated diseases, comprising as an active ingredient a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of items [1] to

[0117] in the preceding paragraph. [8-2] The preventive, symptom progression inhibitor, recurrence inhibitor and / or therapeutic agent according to the preceding item [8-1], wherein the autoreactive B cell-mediated disease is systemic lupus erythematosus, Graves' disease, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary biliary cirrhosis or pernicious anemia.

[0019] [9-1] An autoreactive B cell inhibitor comprising as an active ingredient a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of items [1] to

[0117] in the preceding paragraph. [9-2] The autoreactive B cell inhibitor according to the preceding item [9-1], wherein the autoreactive B cell inhibition is immunoglobulin production inhibition. [9-3] The autoreactive B cell inhibitor according to the preceding item [9-2], wherein the immunoglobulin is IgG or IgM.

[0020] [10-1] A T cell activation inhibitor comprising as an active ingredient a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of items [1] to

[0117] in the preceding paragraph. [10-2] The T cell activation inhibitor according to the preceding item [10-1], wherein the T cell activation inhibition is cytokine production inhibition. [10-3] The T cell activation inhibitor according to the preceding item [10-1] or [10-2], wherein the T cell activation inhibition is memory T cell inhibition.

[0021] [11-1] A method for inhibiting autoreactive B cells or inhibiting T cell activation, comprising administering an effective amount of a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of items [1] to

[0117] in the preceding paragraph to a patient.

[0022] [12-1] A PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[0117] , which is used for suppressing autoreactive B cells or suppressing T cell activation.

[0023] [13-1] Use of a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[0117] in the manufacture of an autoreactive B cell inhibitor or a T cell activation inhibitor.

Effect of the Invention

[0024] The PD-1 / CD19 bispecific antibody of the present invention allows the interaction between PD-1 and PD-L1, and it is expected to prevent autoimmune diseases, suppress symptom progression, suppress recurrence, and / or enhance or sustain the therapeutic effect.

Brief Description of the Drawings

[0025]

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Mode for Carrying Out the Invention

[0026] PD-1( P rogrammed Cell D eath -1In humans, it is a membrane protein consisting of the amino acid sequence represented by GenBank accession number NP_005009. When described as "PD-1" in this specification, unless otherwise specified, all its isoforms and those modified variants in which the epitope of the "first arm that specifically binds to PD-1" according to the present invention is conserved may be used. In the present invention, PD-1 is preferably human PD-1.

[0027] CD19 is a membrane protein expressed on B cells in humans, consisting of the amino acid sequence represented by GenBank accession number NP_001171569 or NP_001761, and is a molecule that transmits stimulation to B cells together with the B cell receptor complex. When described as "CD19" in this specification, unless otherwise specified, those modified variants in which the epitope of the "second arm that specifically binds to CD19" according to the present invention is conserved may be used. In the present invention, CD19 is preferably human CD19.

[0028] As used herein, "isolated" means being identified, separated and / or purified from contaminants containing a plurality or innumerable components extracted from a host cell, so as to become a substantially single pure component.

[0029] As used herein, "monoclonal antibody" means an antibody obtained from a substantially homogeneous population of antibodies that bind to the same specific antigen.

[0030] As used herein, "bispecific antibody" means an antibody having, in one molecule, binding specificities for two different antigen molecules or epitopes, and "bispecific monoclonal antibody" means, in addition, a bispecific antibody obtained from a substantially homogeneous population of antibodies.

[0031] The present invention relates to a bispecific antibody capable of specifically binding to PD-1 and CD19, respectively (which may be abbreviated as "PD-1 / CD19 bispecific antibody" herein). In the present invention, the PD-1 / CD19 bispecific antibody is preferably a PD-1 / CD19 bispecific monoclonal antibody, more preferably an isolated PD-1 / CD19 bispecific monoclonal antibody, and even more preferably an isolated human PD-1 / human CD19 bispecific monoclonal antibody. Here, the "isolated human PD-1 / human CD19 bispecific monoclonal antibody" means an isolated bispecific monoclonal antibody against human PD-1 and human CD19.

[0032] Here, the forms of bispecific antibodies include, for example, diabody, bispecific sc(Fv)2, bispecific F(ab′)2, bispecific minibody, bispecific hybrid antibody, covalently linked diabody (bispecific DART), bispecific (FvCys)2, bispecific F(ab′-zipper)2, bispecific (Fv-zipper)2, bispecific trispecific antibody, and bispecific mAb 2 and the like.

[0033] A diabody is a dimer of single-chain peptides in which VH and VL recognizing different antigens are linked by a peptide linker (see Proc. Natl. Acad. Sci. USA (1993), Vol. 90, No.14: p.6444-6448).

[0034] Bispecific sc(Fv)2 is a miniaturized antibody modified to be produced in a single-chain form in which two sets of VH / VL of two antibodies recognizing different antigens are continuous via a peptide linker (see J. Biological Chemistry (1994), Vol. 269: p.199-206).

[0035] Bispecific F(ab′)2 is a miniaturized antibody in which Fab′ fragments of antibodies recognizing two different antigens are covalently bound by a disulfide bond or the like.

[0036] A bispecific minibody is a small antibody fragment modified such that the CH3 domain of the constant region of an antibody is linked to scFvs that recognize different antigens, and is a small-sized antibody covalently bound by a disulfide bond or the like on the CH3 domain (see Biochemistry (1992), Vo.31, No.6, p.1579-1584).

[0037] A bispecific hybrid antibody is an intact antibody in which the heavy chain / light chain complexes of antibodies that recognize two different antigens are covalently bound by a disulfide bond or the like.

[0038] In the present invention, a preferred form of the bispecific antibody is a bispecific hybrid antibody.

[0039] The bispecific hybrid antibody can be produced, for example, from hybridomas prepared by the hybrid hybridoma method (see US4474893). It can also be produced by co-expressing and secreting a total of four types of cDNAs encoding the heavy and light chains of antibodies that recognize different antigens in mammalian cells.

[0040] The monoclonal antibodies used in the present invention can be prepared by the hybridoma method (see, for example, Kohler and Milstein et al., Nature (1975), Vol. 256, p.495-97, Hongo et al., Hybridoma (1995), Vol. 14, No. 3, p.253-260, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press (1988), Vol. 2) and Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, p.563-681 (Elsevier, N.Y., 1981)), recombinant DNA method (see, for example, US4816567), phage display method (see, for example, US5223409, US5403484 and US5571698 of Ladner et al., US5427908 and US5580717 of Dower et al., US5969108 and US6172197 of McCafferty et al. and US5885793, US6521404, US6544731, US6555313, US6582915 and US6593081 of Griffiths et al.).

[0041] When an antibody or a monoclonal antibody is administered to a human, it can be prepared in the form of a chimeric antibody, a humanized antibody or a fully human antibody in order to reduce or eliminate its antigenicity.

[0042] "Chimeric antibody" means an antibody in which the variable region sequence and the constant region sequence are derived from different mammals. For example, there is one in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. A chimeric antibody can be prepared by ligating a gene encoding an antibody variable region isolated by a known method from an antibody-producing hybridoma isolated by the above hybridoma method, recombinant DNA method or phage display method to a gene encoding a human-derived antibody constant region using a known method (see, for example, US4816567 of Cabilly et al.).

[0043] The term "humanized antibody" means an antibody in which the complementarity determining region (CDR) sequences derived from the germline of another mammal such as a mouse are transplanted onto a human framework sequence. Also in the case of a humanized antibody, from the antibody-producing hybridoma isolated by the above method, a gene encoding the antibody CDR region isolated by a known method is ligated to a gene encoding a human-derived antibody framework region using a known method, and can be produced (see, for example, US5225539 and US5530101 of Winter and US5585089 and US6180370 of Queen et al.).

[0044] The term "human antibody" or "fully human antibody" means an antibody in which both the variable region consisting of the framework region and the CDR region and the constant region are derived from human germline immunoglobulin sequences. The human antibodies used in the present invention can be produced by a method using a mouse transformed to produce human antibodies, for example, a Humab mouse (see, for example, US5545806, US5569825, US5625126, US5633425, US5789650, US5877397, US5661016, US5814318, US5874299 and US5770429 by Lonberg and Kay et al.), a KM mouse (see, for example, WO2002 / 43478 by Ishida et al.), a Xeno mouse (see, for example, US5939598, US6075181, US6114598, US6150584 and US6162963) or a Tc mouse (see, for example, Tomizuka et al., Proc. Natl. Acad. Sci. USA (2000), p.722-727). Also, it can be prepared using a SCID mouse (see, for example, US5476996 and US5698767 by Wilson et al.) in which human immune cells are reconstructed so that a human antibody response occurs by immunization. Furthermore, the human antibodies used in the present invention can also be produced by the phage display method described above.

[0045] As used herein, the term "antibody fragment" of a PD-1 / CD19 bispecific antibody refers to a portion of a full-length antibody that comprises an antigen-binding portion for PD-1 and an antigen-binding portion for CD19, such as F(ab')2. Here, the antigen-binding portion refers to the smallest unit of an antibody capable of binding to its antigen, and is composed, for example, of three CDRs each in VH and VL, and a framework region that positions the CDRs so that the combination of these CDRs recognizes the antigen of interest.

[0046] As used herein, the term "common light chain" refers to a light chain that can associate with two or more different heavy chains and exhibit binding ability to each of the heavy chains' antigens (see De Wildt RM et al., J. Mol. Biol. (1999), Vol. 285, pp. 895-901; De Kruif et al., J. Mol. Biol. (2009), Vol. 387, pp. 548-58; WO2004 / 009618; WO2009 / 157771; and WO2014 / 051433). A preferred example of such a common light chain is a light chain encoded by the germline gene of the human κ light chain IgVκ1-39*01 / IGJκ1*01 (nomenclature according to the IMGT database) (hereinafter, sometimes abbreviated as IGVK1-39 / JK1 common light chain). More preferred examples include such a light chain having a VL comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28, and even more preferred examples include such a light chain having a VL consisting of the amino acid sequence of SEQ ID NO: 25. A preferred example of the constant region of the common light chain is a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29. The amino acid sequences of the variable and constant regions of the common light chain used in the present invention are shown in FIG. 1, and the amino acid sequences of each CDR of the variable region are shown in FIG. 2.

[0047] As used herein, the term "isotype" refers to the antibody class (e.g., IgM or IgG) encoded by the heavy chain constant region gene. The preferred isotype for the PD-1 / CD19 bispecific antibody of the present invention is IgG, more preferably IgG1 or IgG4. Here, IgG1 antibodies with eliminated or weakened Fc receptor binding are preferred. Specifically, IgG1 antibodies with eliminated or weakened Fc receptor binding can be obtained by substituting, deleting, or inserting any amino acid in the heavy chain constant region. Examples include antibodies in which leucine at position 235 (EU numbering system) is substituted with glycine and / or glycine at position 236 (EU numbering system) is substituted with arginine in each of the two heavy chain constant regions or hinge regions of the bispecific antibody. Furthermore, antibodies lacking a C-terminal amino acid, such as lysine at position 447 (EU numbering system), are preferred to reduce antibody heterogeneity. Furthermore, when the bispecific antibody is IgG4, variants in which any amino acid in the heavy chain constant region is substituted, deleted, or inserted are more preferred to suppress intramolecular swapping. For example, an antibody in which serine at position 228 according to the EU numbering system, located in the hinge region, is substituted with proline is preferred. Herein, the amino acid positions assigned to the CDRs and frameworks of antibody variable regions may be defined according to the Kabat numbering system (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., (1987) and (1991)) and the amino acids in the constant regions are represented according to the EU numbering system based on Kabat amino acid positions (see Sequences of proteins of immunological interest, NIH Publication No. 91-3242).

[0048] The Fc region of a PD-1 / CD19 bispecific antibody of the present invention may have any amino acid substitution in that region to facilitate association of the two different heavy chains. Preferred embodiments include, for example, PD-1 / CD19 bispecific antibodies in which the leucine at position 351 (EU numbering system) is substituted with lysine and the threonine at position 366 is substituted with lysine in the constant region of the heavy chain having the VH of the first arm that specifically binds to PD-1, and the leucine at position 351 is substituted with aspartic acid and the leucine at position 368 is substituted with glutamic acid in the constant region of the heavy chain having the VH of the second arm that specifically binds to CD19. Other examples include PD-1 / CD19 bispecific antibodies in which the leucine at position 351, according to the EU numbering system, is substituted with aspartic acid and the leucine at position 368 is substituted with glutamic acid in the constant region of a heavy chain having a VH in a first arm that specifically binds to PD-1, and the leucine at position 351 is substituted with lysine and the threonine at position 366 is substituted with lysine in the constant region of a heavy chain having a VH in a second arm that specifically binds to CD19.

[0049] The first arm that specifically binds to PD-1 As used herein, a "first arm that specifically binds to PD-1" (hereinafter sometimes abbreviated as "first arm") refers to an antibody portion that contains at least the VH of an antibody that specifically binds to PD-1 and is capable of specifically binding to PD-1, regardless of whether it is contained in a portion of an antibody or antibody fragment or whether it exists as a standalone entity rather than as a portion. For example, such a first arm consists of the VH of an anti-PD-1 antibody and the VL of the common light chain that constitutes the anti-PD-1 antibody, and further includes the Fab portion of an antibody comprising the VH and VL. Here, "specifically binds to PD-1" means that the first arm binds to PD-1 at least 1 x 10 -5 M, preferably 1x10 -7 M, more preferably 1x10 -9It can directly bind to PD-1 with a binding activity having a higher affinity (dissociation constant (Kd value)) than M, and is used as a feature that does not substantially bind to other receptor members belonging to the so-called CD28 family receptors such as at least CD28, CTLA-4, and ICOS. In addition, the "antibody" in the "antibody specifically binding to PD-1" or "anti-PD-1 antibody" means a full-length antibody, that is, a complete-length antibody consisting of two heavy chains and two light chains linked by disulfide bonds, and preferably, it is a monoclonal antibody thereof.

[0050] Here, as the "first arm specifically binding to PD-1", for example, (a) HYJ 1 the amino acid sequence represented by LH [in the sequence, J 1 represents G (glycine) or A (alanine), and the other alphabets each represent the one-letter abbreviation of an amino acid.], VH-CDR1 consisting of (b) WJ 2 NTNTU 2 NPTX 2 the amino acid sequence represented by AQGFTG [in the sequence, J [[ID=1〕5]] 2 represents L (leucine) or I (isoleucine), U 2 represents E (glutamic acid) or G (glycine), X 2 represents F (phenylalanine) or Y (tyrosine), and the other alphabets each represent the same meaning as above.], VH-CDR2 consisting of and (c) GDJ 3 VVPTTIWNYYU 3 X 3 MZ 3 the amino acid sequence represented by V [in the sequence, J 3 represents M (methionine) or L (leucine), U 3 represents H (histidine) or Y (tyrosine), X 3 represents F (phenylalanine) or Y (tyrosine), Z 3 represents D (aspartic acid) or E (glutamic acid), and the other alphabets each represent the same meaning as above.], VH having VH-CDR3 consisting of are mentioned.

[0051] Also, as another aspect of the "first arm that specifically binds to PD-1", for example, (1b) a VH comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 6, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8, (2b) a VH comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 9, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 11, (3b) a VH comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 14, (4b) a VH comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 15, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17, and (5b) those having any one VH selected from VH comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 18, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 20.

[0052] Furthermore, in the present invention, the "first arm that specifically binds to PD-1" includes those in which any 1 to 5 amino acid residues in each VH-CDR of any one VH selected from the above (1b) to (5b) are substituted with other amino acids (preferably, their conservative amino acids), and which have a binding activity substantially equivalent to the binding activity of the original first arm that has not been substituted with such amino acids to PD-1. For example, in the case of VH-CDR1, 1 amino acid residue is substituted with another amino acid (preferably, its conservative amino acid), and in the case of VH-CDR2 or VH-CDR3, those in which 1 to 5 amino acid residues in each are substituted with other amino acids (preferably, their conservative amino acids) can be mentioned. Also, as shown in Figure 4, in each CDR of the anti-PD-1 antibody clones corresponding to the first arm that specifically binds to PD-1, each amino acid that differs between the clones or any plurality of combinations thereof can be mutually substituted between the clones. Here, substitution with conservative amino acids means the interchangeability of residues having similar side chains. For example, in the group of amino acids having aliphatic side chains, they are glycine, alanine, valine, leucine, and isoleucine; in the group of amino acids having aliphatic hydroxyl side chains, they are serine and threonine; in the group of amino acids having amide-containing side chains, they are asparagine and glutamine; in the group of amino acids having aromatic side chains, they are phenylalanine, tyrosine, and tryptophan; in the group of amino acids having basic side chains, they are lysine, arginine, and histidine; and in the group of amino acids having sulfur-containing side chains, they are cysteine and methionine. Examples of preferable substitution with conservative amino acids include substitution between valine, leucine, and isoleucine, substitution between phenylalanine and tyrosine, substitution between lysine and arginine, substitution between alanine and valine, and substitution between asparagine and glutamine.Also, here, the phrase "having a binding activity substantially equivalent to the binding activity of the original first arm not substituted with the amino acid to PD-1" means that the binding activity of the first arm substituted with the amino acid to PD-1 is 95% or more, preferably 98% or more, more preferably 99% or more of the binding activity of the original first arm not substituted with the amino acid.

[0053] Furthermore, the "first arm that specifically binds to PD-1" in the present invention includes each VH-CDR having the above-described specific amino acid sequence in its VH, and also includes those having an amino acid sequence of the framework of the VH encoded by a specific germline-type gene or a gene having a somatic mutation of the gene. For example, the VH represented by any one selected from the above (1b) to (5b) may be encoded by a VDJ recombinant gene having a germline-type V gene of IGHV7-4-1 and a germline-type J gene of JH6c or a gene having a somatic mutation of the gene. Here, the amino acid sequence encoded by each of the germline-type V genes IGHV7-4-1 corresponds to the amino acid sequence of SEQ ID NO: 21 (FIG. 3).

[0054] The framework of the VH of the first arm that specifically binds to PD-1 of the present invention may be encoded by the germline-type VDJ recombinant gene that has undergone somatic mutation. For example, for the FR1, FR2, and FR3 of VH shown by any one selected from the above (1b) to (5b), where the germline-type V gene is IGHV7-4-1, at the amino acid positions shown in FIG. 4, since the amino acid sequence encoded by the IGHV7-4-1 gene is different, somatic mutations have occurred at each of these positions. For example, for FR1, the lysine at position 13, alanine at position 16, or lysine at position 19 in the amino acid sequence of SEQ ID NO: 21 may be substituted with glutamine, valine, or methionine, respectively, or any combination of them may be substituted. For FR2, the valine at position 37 in the amino acid sequence of SEQ ID NO: 21 may be substituted with leucine. For FR3, the serine at position 77, cysteine at position 84 in the amino acid sequence of SEQ ID NO: 21 may be substituted with threonine, serine or asparagine, respectively, or any combination of them may be substituted. Further, for FR4 of VH shown by any one selected from the above (1b) to (5b), the lysine (Lys) in the amino acid sequence (Trp-Gly-Lys-Gly-Thr-Thr*-Val-Thr-Val-Ser-Ser) (SEQ ID NO: 41) of FR4 derived from the J gene JH6c may be substituted with glutamine or asparagine, and / or the *-marked threonine (Thr) may be substituted with leucine. Each of FR1, FR2, FR3, and FR4 having any combination of the above amino acid substitutions can also be used as a framework without substantially affecting the function of the first arm that specifically binds to PD-1.

[0055] Furthermore, the "first arm that specifically binds to PD-1" in the present invention includes each CDR having the specific amino acid sequence described above, and also includes those in which the amino acid sequence of the FR of the VH is encoded by a specific germline-type gene or the gene that has undergone somatic mutation thereof. For example, such a first arm includes those having a VH consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 5.

[0056] Furthermore, such a "first arm that specifically binds to PD-1" includes, for example, any one amino acid sequence selected from SEQ ID NOs: 1 to 5 and is at least 80% identical, preferably at least 90% identical, more preferably at least 95% identical, still more preferably at least 98% identical, and even more preferably at least 99% identical to the amino acid sequence, and has a VH consisting of the amino acid sequence, and the difference from the amino acid sequence of the VH of the original first arm does not substantially affect the binding activity to PD-1 (hereinafter, may be abbreviated as a homologous first arm). Here, the "% identity" used in the comparison of amino acid sequence identity is defined as the percentage of the amino acid sequence that is identical to the two aligned sequences, the reference amino acid sequence (here, the reference amino acid sequence after introducing gaps if necessary to achieve the maximum percentage of identity). Also, here, "the difference from the amino acid sequence of the VH of the original first arm does not substantially affect the binding activity to PD-1" means that the binding activity of the homologous first arm to PD-1 is 95% or more, preferably 98% or more, and more preferably 99% or more of the binding activity of the original first arm.

[0057] In yet another embodiment, the "first arm that specifically binds to PD-1" of the present invention includes (1) a first arm having a VH selected from any one of (1b) to (5b) above or a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 1 to 5 and a VL of the common light chain herein (preferably a VL consisting of the amino acid sequence of SEQ ID NO: 25), or (2) an anti-PD-1 antibody that cross-competes with the binding to PD-1 of a variable region of a monoclonal antibody that specifically binds to PD-1 and that is composed of the VH and VL. The term "anti-PD-1 antibody" also includes those having the variable region of the anti-PD-1 antibody (wherein the variable region includes the VH and VL that constitute it), and those having the variable region of an anti-PD-1 antibody whose binding to PD-1 cross-competes with (3) a first arm having a VH selected from (1b) to (5b) above or a VH consisting of any one of the amino acid sequences selected from SEQ ID NOS: 1 to 5 and the VL of the common light chain, or (4) a variable region of a monoclonal antibody that specifically binds to PD-1 and consists of the VH and VL. Here, "cross-competes for binding to PD-1" means that the binding of the first arm to PD-1 is inhibited, regardless of the extent, by binding to the same or a partially overlapping epitope as the first arm exemplified herein, or that the binding of an antibody that binds to the same or a partially overlapping epitope as the first arm exemplified herein is inhibited, regardless of the extent, by the first arm exemplified. Whether or not cross-competition occurs can be assessed by competitive binding assays. For example, this can be determined using Biacore analysis, ELISA assays, flow cytometry, enzyme-linked immunosorbent assays (ELISA), fluorescence energy transfer assays (FRET), or fluorescence micrometric assay technology (FMAT®).

[0058] For example, as something that cross-competes with the binding to PD-1 by the first arm having VH shown in the above (5b) and VL of the common light chain, for example, VH shown in any one selected from the above (1b) to (4b) and VL of the common light chain (preferably, VL having VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28), a first arm, and further, a first arm having VH consisting of the amino acid sequence selected from SEQ ID NOs: 1 to 4 and VL of the common light chain (preferably, VL consisting of the amino acid sequence of SEQ ID NO: 25) can be mentioned.

[0059] Also, for example, as something that cross-competes with the binding to PD-1 by the first arm having VH shown in any one selected from the above (1b) to (4b) or VH consisting of the amino acid sequence selected from SEQ ID NOs: 1 to 4 and VL of the common light chain, for example, VH shown in the above (5b) and VL of the common light chain (preferably, VL having VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28), a first arm, and further, a first arm having VH consisting of the amino acid sequence of SEQ ID NO: 5 and VL of the common light chain (preferably, VL consisting of the amino acid sequence of SEQ ID NO: 25) can be mentioned.

[0060] Here, the "first arm that specifically binds to PD-1" of the present invention preferably includes a first arm having a VH shown in (1b) to (5b) above. Furthermore, as described above, this preferred first arm also includes one in which any 1 to 5 amino acid residues in each CDR of the VH have been substituted with other amino acids (preferably, conservative amino acids), and the amino acid substitutions do not substantially affect the binding activity to PD-1. Furthermore, as described above, this preferred first arm also includes one in which the amino acid sequence of the VH framework is encoded by the germline V gene IGHV7-4-1 or the J gene JH6c, or a VH encoded by such a gene that has undergone somatic mutation. Furthermore, more preferably, this first arm includes one having a VH consisting of any one of the amino acid sequences selected from SEQ ID NOs: 1 to 5.

[0061] Furthermore, the "first arm that specifically binds to PD-1" of the present invention preferably comprises a VL of the common light chain herein, and a preferred example of such a common light chain is the IGVK1-39 / JK1 common light chain, more preferably a light chain having a VL comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28, and even more preferably a light chain having a VL consisting of the amino acid sequence of SEQ ID NO: 25. Furthermore, a preferred example of the constant region of the common light chain is a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29.

[0062] Furthermore, it is more preferable that the "first arm that specifically binds to PD-1" allows for the interaction between PD-1 and PD-L1, the interaction between PD-1 and PD-L2, or both of these interactions. Here, "allows for the interaction between PD-1 and PD-L1, the interaction between PD-1 and PD-L2, or both of these interactions" means that, even in the presence of a PD-1 / CD19 bispecific antibody of the invention at a concentration 20-fold in excess of soluble PD-L1 or PD-L2, the interaction between the PD-L1 and PD-1, the interaction between the PD-L2 and PD-1, or both of these interactions, is maintained at 50% or more, preferably 70% or more, and more preferably 80% or more of the level of the interaction in the absence of the PD-1 / CD19 bispecific antibody of the invention. Additionally, the definition "permitting interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both interactions" is sometimes used synonymously with the meaning "not substantially inhibiting interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both interactions."

[0063] Figure 6 shows the anti-PD-1 monoclonal antibody clones obtained to construct the PD-1 / CD19 bispecific antibodies of the present invention, along with the amino acid sequences of their VHs and their corresponding SEQ ID NOs. Figure 7 shows the amino acid sequences of each CDR in the VH of each anti-PD-1 monoclonal antibody clone and their corresponding SEQ ID NOs.

[0064] The second arm that specifically binds to CD19 As used herein, the "second arm that specifically binds to CD19" (hereinafter sometimes abbreviated as the "second arm") refers to an antibody or a part of an antibody fragment, or whether it exists not as a part but as an entity by itself. It means an antibody part that contains at least the VH of an antibody that specifically binds to CD19 and can specifically bind to CD19. For example, such a second arm consists of the VH of an anti-CD19 antibody and the VL of the common light chain that constitutes the anti-CD19 antibody. Further, the second arm also includes the Fab portion of the antibody containing the VH and VL. Here, "specifically binds to CD19" means that it can directly bind to CD19 with a binding activity having an affinity (dissociation constant (Kd value)) higher than at least 1×10 -5 M, preferably 1×10 -7 M, more preferably 1×10 -9 M, and is used as a feature that does not substantially bind to other proteins. Also, the "antibody" in the "antibody that specifically binds to CD19" or "anti-CD19 antibody" means a full-length antibody, that is, a complete-length antibody consisting of two heavy chains and two light chains linked by disulfide bonds, but preferably a monoclonal antibody thereof.

[0065] Here, examples of the "second arm that specifically binds to CD19" include (a) VH-CDR1 consisting of the amino acid sequence represented by SYWIJ 4 [in the sequence, J 4 represents G (glycine) or A (alanine), and the other alphabets each represent the one-letter abbreviation of an amino acid.], (b) VH-CDR2 consisting of the amino acid sequence represented by IIU 4 PGDSDTRYSPSFQG [in the sequence, U 4 represents W (tryptophan) or Y (tyrosine), and the other alphabets each represent the same meaning as above.], and (c) X 4 TIVZ 4 J 5 U 5 X 5 Z 5 AJ 6 DU 6The amino acid sequence represented by [in the sequence, X 4 represents K (lysine), Q (glutamine), H (histidine) or R (arginine), and Z 4 represents G (glycine) or A (alanine), and J 5 represents T (threonine) or V (valine), and U 5 represents V (valine), I (isoleucine) or T (threonine), and X 5 represents M (methionine), Y (tyrosine), G (glycine) or H (histidine), and Z 5 represents T (threonine), N (asparagine), L (leucine) or W (tryptophan), and J 6 represents F (phenylalanine) or S (serine), and U 6 represents I (isoleucine), F (phenylalanine) or Y (tyrosine), and the other alphabets each represent the same meaning as above.], and those having a VH having a VH-CDR3 consisting of the above are exemplified.

[0066] Also, as another aspect of the "second arm that specifically binds to CD19", for example, (1d) A VH comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 36, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 37, (2d) A VH comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 40, (3d) A VH comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, (4d) A VH comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 45, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and Those having any one VH selected from VH containing VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 47, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49 are exemplified.

[0067] Furthermore, in the present invention, the "second arm that specifically binds to CD19" includes, in each VH-CDR of any one VH selected from the above (1d) to (5d), any 1 to 5 amino acid residues thereof are substituted with other amino acids (preferably, conservative amino acids thereof), and those having a binding activity substantially equivalent to the binding activity of the original second arm that has not been substituted with such amino acids to CD19 are also included. For example, in the case of VH-CDR1, one amino acid residue is substituted with other amino acids (preferably, conservative amino acids thereof), and in the case of VH-CDR2 or VH-CDR3, those in which 1 to 5 amino acid residues are each substituted with other amino acids (preferably, conservative amino acids thereof) are exemplified. Also, as shown in FIG. 5, in each CDR of the anti-CD19 antibody clone corresponding to each second arm that specifically binds to CD19, each amino acid that differs between the clones or any plurality of combinations thereof are mutually substitutable between the clones. Here, "having a binding activity substantially equivalent to the binding activity of the original second arm that has not been substituted with such amino acids to CD19" means that the binding activity of the second arm substituted with such amino acids to CD19 is 95% or more, preferably 98% or more, more preferably 99% or more of the binding activity of the original second arm that has not been substituted with such amino acids. Note that examples of the "substitution with conservative amino acids" in each VH-CDR of the second arm include, for example, the examples of amino acid substitution in the first arm described above.

[0068] Furthermore, the "second arm that specifically binds to CD19" of the present invention also includes a VH that comprises CDRs having the specific amino acid sequences described above, and the amino acid sequence of the FR of the VH is encoded by a specific germline gene or a somatically mutated version of the gene. For example, any one of the VHs selected from (1d) to (5d) above can be encoded by a VDJ recombined gene whose germline V gene is IGHV5-51 or a somatically mutated version of the gene. Here, the amino acid sequence encoded by the germline V gene IGHV5-51 corresponds to the amino acid sequence set forth in SEQ ID NO: 22 (Figure 3).

[0069] The framework of the VH of the second arm of the present invention that specifically binds to CD19 may be encoded by a germline VDJ recombined gene that has undergone somatic mutation. For example, when the germline V gene is IGHV5-51, FR1 and FR3 of the VH shown in any one of (1d) to (5d) above differ from the amino acid sequence encoded by the IGHV5-51 gene at the amino acid positions shown in Figure 5 and have therefore undergone somatic mutation at those positions. For example, in FR1, the glutamic acid at position 1 in the amino acid sequence of SEQ ID NO: 22 may be substituted with glutamine, the proline at position 14 with serine, the tyrosine at position 27 with phenylalanine, or the threonine at position 30 with isoleucine, or any combination of these substitutions may be used. With regard to FR3, the isoleucine at position 76 in the amino acid sequence of SEQ ID NO: 22 may be substituted with phenylalanine, the serine at position 77 with threonine or asparagine, the threonine at position 78 with valine, the serine at position 84 with asparagine, the methionine at position 93 with isoleucine or leucine, or the alanine at position 97 with valine, or any combination thereof. FR1 and FR3 having any of the above combinations of amino acid substitutions can also be used as a framework without substantially affecting the function of the second arm to specifically bind to CD19.

[0070] Furthermore, the "second arm that specifically binds to CD19" of the present invention also includes a VH that comprises each CDR having the specific amino acid sequence described above, and the amino acid sequence of the FR of the VH is encoded by a specific germline gene or a gene that has undergone somatic mutation. For example, such a second arm may include a VH having any one of the amino acid sequences set forth in SEQ ID NOs: 30 to 34.

[0071] Furthermore, such a "second arm that specifically binds to CD19" also includes, for example, one having a VH consisting of an amino acid sequence that is at least 80% identical, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 98% identical, and even more preferably at least 99% identical to any one of the amino acid sequences selected from SEQ ID NOs: 30 to 34, and in which the difference from the amino acid sequence of the VH of the original second arm does not substantially affect the CD19-binding activity (hereinafter sometimes abbreviated as a "homologous second arm"). Here, "the difference from the amino acid sequence of the VH of the original second arm does not substantially affect the CD19-binding activity" means that the CD19-binding activity of the homologous second arm is 95% or more, preferably 98% or more, and more preferably 99% or more of the binding activity of the original second arm.

[0072] In yet another aspect, the "second arm that specifically binds to CD19" in the present invention includes (1) the binding of the second arm having VH represented by any one selected from the above (1d) to (5d) or VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30 to 34 and VL of the common light chain herein (preferably, VL consisting of the amino acid sequence of SEQ ID NO: 25) to CD19, or (2) the variable region of an anti-CD19 antibody that cross-competes with the binding of the variable region of a monoclonal antibody specifically binding to CD19 consisting of the VH and VL (wherein the variable region includes VH and VL constituting the same). Also included are those whose binding to CD19 is (3) the second arm having VH represented by any one selected from the above (1d) to (5d) or VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30 to 34 and VL of the common light chain, or (4) the variable region of an anti-CD19 antibody that is cross-competed by the variable region of a monoclonal antibody specifically binding to CD19 consisting of the VH and VL. Here, "cross-competing in the binding to CD19" means inhibiting the binding of the second arm to CD19, regardless of the degree, by binding to the same or partially overlapping epitope as the second arm exemplified herein, or the binding of an antibody that binds to the same or partially overlapping epitope as the exemplified second arm to CD19 is inhibited by the exemplified second arm, regardless of the degree. Here, whether cross-competing or not can be measured in the same manner according to the method described in the description of the "first arm that specifically binds to PD-1".

[0073] Here, the "second arm specifically binding to CD19" in the present invention preferably includes a second arm having a VH represented by the above (1d) to (5d). Further, as described above, in each CDR of the VH of this preferred second arm, any 1 to 5 amino acid residues thereof are substituted with other amino acids (preferably, their conservative amino acids), and those in which the amino acid substitution does not substantially affect the binding activity to CD19 are also included. Moreover, as described above, those having an amino acid sequence of the framework of VH encoded by the germline-type V gene IGHV5-51 or the VH encoded by the gene having somatic mutations thereof are also included. And more preferably, the second arm includes those having a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30 to 34.

[0074] Furthermore, in another aspect, the "second arm that specifically binds to CD19" in the present invention preferably includes a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30 to 34, in which the 114th amino acid in each amino acid sequence of SEQ ID NOs: 30 to 34 is substituted with arginine or may be substituted, and more preferably, a second arm having a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30 to 34, in which the 114th glutamine in each amino acid sequence of SEQ ID NOs: 30 to 34 is substituted with arginine. These correspond to a second arm having a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 62 to 66. By these amino acid substitutions, the isoelectric point (pI value) of the complex composed of the heavy chain and light chain having the second arm (hereinafter referred to as the second arm heavy chain / light chain complex) can be increased, whereby in the purification process of the PD-1 / CD19 bispecific antibody of the present invention, particularly, the separation of the bispecific antibody from the homodimer of the second arm heavy chain / light chain complex becomes easy. In the present invention, the isoelectric point of the second arm heavy chain / light chain complex is preferably about 8.3 to about 8.9, more preferably about 8.4 to about 8.8. Also, the isoelectric point of the complex composed of the heavy chain and light chain having the first arm (hereinafter referred to as the first arm heavy chain / light chain complex) is preferably about 7.4 to about 7.7, more preferably about 7.5 to about 7.6.

[0075] Furthermore, the "second arm that specifically binds to CD19" in the present invention preferably includes the VL of the common light chain in the present specification. Such a common light chain is preferably, for example, the IGVK1-39 / JK1 common light chain, and more preferably, a light chain having a VL including VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28. Even more preferably, it is a light chain having a VL consisting of the amino acid sequence of SEQ ID NO: 25, for example. Also, the constant region of the common light chain preferably includes a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29.

[0076] The correspondence between each clone of the anti-CD19 antibody for constructing the PD-1 / CD19 bispecific antibody of the present invention, the amino acid sequences of their VHs, and their SEQ ID Nos. is shown in FIG. 8, and the correspondence between the amino acid sequences of each CDR in the VH of each clone of the anti-CD19 antibody and their SEQ ID Nos. is shown in FIG. 9.

[0077] As a preferred embodiment of the PD-1 / CD19 bispecific antibody of the present invention, for example, the first arm that specifically binds to PD-1 is (A) In any one or more of VH-CDR1, VH-CDR2, and VH-CDR3 in VH represented by any one selected from the above (1b) to (5b), any 1 to 5 amino acid residues thereof may be substituted with other amino acids (preferably, conservative amino acids thereof), and (B) having a VL comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28, and the second arm that specifically binds to CD19 is (C) In any one or more of VH-CDR1, VH-CDR2, and VH-CDR3 selected from VH represented by any one selected from the above (1d) to (5d), any 1 to 5 amino acid residues thereof may be substituted with other amino acids (preferably, conservative amino acids), and (D) having a VL comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28. Examples of the PD-1 / CD19 bispecific antibody are characterized by this.

[0078] More preferably, for example, the first arm that specifically binds to PD-1 is (A) VH represented by any one selected from the above (1b) to (5b), and (B) a VL comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28; and the second arm specifically binding to CD19 is (C) VH selected from the above (1d) to (5d), and (D) A PD-1 / CD19 bispecific antibody characterized by having a VL comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28.

[0079] In another preferred embodiment of the PD-1 / CD19 bispecific antibody of the present invention, for example, the first arm that specifically binds to PD-1 is (A) a VH consisting of any one of the amino acid sequences selected from SEQ ID NOs: 1 to 5, or a VH consisting of an amino acid sequence that is at least 80% identical to the amino acid sequence of the VH; and (B) a VL consisting of the amino acid sequence of SEQ ID NO: 25, and the second arm specifically binding to CD19 is (C) a VH consisting of any one of the amino acid sequences selected from SEQ ID NOs: 30 to 34 and 62 to 66, or a VH consisting of an amino acid sequence that is at least 80% identical to the amino acid sequence of the VH; and (D) A PD-1 / CD19 bispecific antibody characterized by having a VL consisting of the amino acid sequence of SEQ ID NO: 25.

[0080] In this other embodiment, more preferably, for example, the first arm that specifically binds to PD-1 is (A) a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 1 to 5, and (B) a VL consisting of the amino acid sequence of SEQ ID NO: 25, and the second arm specifically binding to CD19 is (C) A VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30 to 34 and 62 to 66, and (D) A PD-1 / CD19 bispecific antibody characterized by having a VL consisting of the amino acid sequence of SEQ ID NO: 25.

[0081] Furthermore, as another aspect and more preferably, for example, the first arm that specifically binds to PD-1 (A) has a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 1 to 5, and (B) has a VL consisting of the amino acid sequence of SEQ ID NO: 25, and the second arm that specifically binds to CD19 (C) has a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 62 to 66, and (D) A PD-1 / CD19 bispecific antibody characterized by having a VL consisting of the amino acid sequence of SEQ ID NO: 25.

[0082] A preferred isotype of the PD-1 / CD19 bispecific antibody of the present invention is an IgG antibody, more preferably an IgG1 or IgG4 antibody, and even more preferably an IgG1 antibody. When the antibody is an IgG1 antibody, an IgG1 antibody in which the 235th leucine according to the EU numbering system is substituted with glycine and / or the 236th glycine is substituted with arginine on each of the two heavy chain constant regions or hinge regions is preferred. Furthermore, an antibody lacking the 447th lysine according to the EU numbering system at the C-terminus of the heavy chain of those bispecific antibodies is more preferred. Also, when the PD-1 / CD19 bispecific antibody is an IgG4 antibody, an antibody in which the 228th serine according to the EU numbering system located in its hinge region is substituted with proline is preferred.

[0083] Furthermore, when these PD-1 / CD19 bispecific antibodies are IgG1 antibodies, preferred embodiments include IgG1 antibodies in which the 351st leucine in the constant region of the heavy chain having VH of the first arm specifically binding to PD-1 is replaced with lysine and the 366th threonine is replaced with lysine according to the EU numbering system, and the 351st leucine in the constant region of the heavy chain having VH of the second arm specifically binding to CD19 is replaced with aspartic acid and the 368th leucine is replaced with glutamic acid. Also, IgG1 antibodies in which the 351st leucine in the constant region of the heavy chain having VH of the first arm specifically binding to PD-1 is replaced with aspartic acid and the 368th leucine is replaced with glutamic acid, and the 351st leucine in the constant region of the heavy chain having VH of the second arm specifically binding to CD19 is replaced with lysine and the 366th threonine is replaced with lysine are similarly preferred.

[0084] Preferred embodiments of the PD-1 / CD19 bispecific IgG1 antibody incorporating all of the above-described amino acid substitutions in the heavy chain constant region include, for example, an antibody in which the heavy chain having VH of the first arm specifically binding to PD-1 has a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23, and the heavy chain having VH of the second arm specifically binding to CD19 has a heavy chain constant region consisting of any one amino acid sequence selected from SEQ ID NO: 24 and SEQ ID NOs: 71 to 75. Some of their amino acid sequences are illustrated in FIG. 10.

[0085] Here, the heavy chain having a VH of the second arm that specifically binds to CD19 may further have, at its C-terminus, via an amide bond with its C-terminal amino acid, Gly (glycine), Gly-Lys-Lys-Ala (SEQ ID NO: 67), Gly-Lys-Ala-Lys-Ala (SEQ ID NO: 68), Gly-Arg-Arg-Ala (SEQ ID NO: 69), or Gly-Arg-Ala-Arg-Ala (SEQ ID NO: 70). By adding these amino acids or peptides, the isoelectric point (pI value) of the second arm heavy chain / light chain complex can be increased, thereby facilitating the separation of the bispecific antibody from the homodimer of the second arm heavy chain / light chain complex, similar to the case of amino acid substitution in the VH of the second arm described above. For the second arm heavy chain / light chain complex with the amino acid or peptide added to its C-terminus, its isoelectric point is preferably about 8.3 to about 8.9, more preferably about 8.4 to about 8.8.

[0086] Most preferably as an embodiment of the PD-1 / CD19 bispecific antibody of the present invention are clone CD19-1(Bi), clone CD19-2(Bi), clone CD19-3(Bi), clone CD19-4(Bi), and clone CD19-5(Bi) prepared in Example 12 herein, and clone CD19-6(Bi) prepared in Example 13.

[0087] Preferred characteristics of the PD-1 / CD19 bispecific antibody of the present invention include allowing the interaction between PD-1 and PD-L1, the interaction between PD-1 and PD-L2, or both of these interactions. Here, "allowing the interaction between PD-1 and PD-L1, the interaction between PD-1 and PD-L2, or both of these interactions" has the same meaning as the definition described in the description of "the first arm that specifically binds to PD-1".

[0088] Furthermore, preferred characteristics of the PD-1 / CD19 bispecific antibody of the present invention include those in which cytokine production is sufficiently reduced. Here, "cytokine production is sufficiently reduced" means that, for example, during intravenous administration of the PD-1 / CD19 bispecific antibody of the present invention by infusion or the like, or within 24 hours after administration, for example, the concentration of cytokines including IL-2, IFN-γ and / or TNF-α in blood or tissue does not increase, or if it increases, it is at a level that can be suppressed by steroid administration.

[0089] Furthermore, preferred characteristics of the PD-1 / CD19 bispecific antibody of the present invention include those having an inhibitory effect on the activation of T cells (for example, memory T cells). The inhibitory effect can be evaluated as an inhibitory effect on cytokine (for example, IL-2) production from peripheral blood mononuclear cells containing T cells.

[0090] Method for manufacturing and purifying a PD-1 / CD19 bispecific antibody The PD-1 / CD19 bispecific antibody and antibody fragments thereof of the present invention can also be produced by the methods disclosed in WO2014 / 051433, WO2013 / 157953 or WO2013 / 157954.

[0091] Specifically, an expression vector into which (1) a polynucleotide encoding a heavy chain having a VH of the first arm that specifically binds to PD-1, (2) a polynucleotide encoding a heavy chain having a VH of the second arm that specifically binds to CD19, and (3) a polynucleotide encoding a common light chain are inserted is transfected into mammalian cells for transformation, and the heavy chain and the common light chain can be co-expressed and secreted to produce the antibody.

[0092] The host cells expressing the PD-1 / CD19 bispecific antibodies of the present invention may be any host cells capable of transfecting with an expression vector and expressing the introduced expression vector. Preferred examples include insect cells such as SF-9 and SF-21 cells, and more preferred examples include mouse cells, including CHO cells, BHK cells, SP2 / 0 cells, and NS-0 myeloma cells, primate cells such as COS and Vero cells, MDCK cells, BRL 3A cells, hybridomas, tumor cells, immortalized primary cells, and embryonic retinal cells such as W138, HepG2, HeLa, HEK293, HT1080, or PER.C6. In selecting an expression system, mammalian cell expression vectors and hosts may be used to ensure appropriate glycosylation of the antibody. Human cell lines, preferably PER.C6, are advantageously used to obtain antibodies with glycosylation patterns consistent with those in humans.

[0093] Protein production in host cells transformed by gene introduction of an expression vector can be carried out with reference to, for example, Current Protocols in Protein Science (1995), Coligan JE, Dunn BM, Ploegh HL, Speicher DW, Wingfield PT, ISBN 0-471-11184-8, Bendig, 1988. Furthermore, general guidelines, procedures, and practical methods for maximizing productivity in host cell culture can be carried out with reference to Mammalian Cell Biotechnology: a Practical Approach (M. Butler, ed., IRL Press, 1991). Expression of antibodies in host cells is described in publications such as EP0120694, EP0314161, EP0481790, EP0523949, US4816567, and WO2000 / 63403.

[0094] Here, the culture conditions of the host cell can be optimized by known methods, and the protein production amount can be optimized. The culture can be carried out, for example, in a culture dish, a roller bottle or a reaction tank, by batch culture, fed-batch culture, continuous culture, or culture using hollow fibers. In order to produce a large-scale and continuous recombinant protein by cell culture, it is preferable to grow the cells in a suspension. Also, it is preferable to culture the cells under conditions without animal or human-derived serum or components of animal or human-derived serum.

[0095] The antibody expressed in the host cell and recovered from the cell or cell culture medium by a known method can be purified using a known method. The purification methods include immunoprecipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, hydrophobic interaction chromatography, etc. Furthermore, protein A or protein G affinity chromatography may be preferably used (see, for example, US4801687 and US5151504).

[0096] Anti-CD19 monoclonal antibody The present invention includes "monoclonal antibodies that specifically bind to CD19" (hereinafter sometimes abbreviated as "anti-CD19 monoclonal antibodies") and antibody fragments thereof for constructing the PD-1 / CD19 bispecific antibody of the present invention.

[0097] As one aspect of the anti-CD19 monoclonal antibody of the present invention, it is a monoclonal antibody that can specifically bind to CD19 by the association of VH and VL of the common light chain in the present invention. Here, "specifically binds to CD19" means at least 1x10 -5 M, preferably 1x10 -7 M, more preferably 1x10 -9The term "monoclonal antibody that specifically binds to CD19" refers to a feature of being able to directly bind to CD19 with a binding activity having a higher affinity (dissociation constant (Kd value)) than M, and not substantially binding to other proteins. Here, the "antibody" in "monoclonal antibody that specifically binds to CD19" refers to a full-length antibody, i.e., a full-length antibody consisting of two heavy chains and two light chains linked by disulfide bonds. Furthermore, "fragments of monoclonal antibodies that specifically bind to CD19" refer to portions of the full-length antibody that contain at least the antigen-binding portion, and include, for example, Fab, Fab', Fv, scFv, and F(ab')2.

[0098] Examples of anti-CD19 monoclonal antibodies of the present invention include those having any one VH selected from (1d) to (5d) above, which constitutes the VH of the "second arm that specifically binds to CD19," or a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30 to 34, and a variable region of the common light chain herein (preferably a VL consisting of the amino acid sequence of SEQ ID NO: 25).

[0099] Furthermore, the anti-CD19 monoclonal antibodies of the present invention also include those having a VH selected from the above (1d) to (5d), in which any one to five amino acid residues in each CDR of the VH have been substituted with other amino acids (preferably, conservative amino acids), and which have substantially the same binding activity to CD19 as that of an anti-CD19 monoclonal antibody having the original VH without the amino acid substitutions. For example, in the case of CDR1, one amino acid residue has been substituted with other amino acids (preferably, conservative amino acids), and in the case of CDR2 or CDR3, one to five amino acid residues have been substituted with other amino acids (preferably, conservative amino acids). Furthermore, as shown in Figure 5, in each CDR of anti-CD19 monoclonal antibody clones, each amino acid that differs between clones, or any combination thereof, can be mutually substituted between the clones. Here, "having substantially the same binding activity to CD19 as that of an anti-CD19 monoclonal antibody having the original VH without the amino acid substitution" means that the CD19 binding activity of the anti-PD-1 monoclonal antibody having the amino acid substitution is 95% or more, preferably 98% or more, and more preferably 99% or more of the binding activity of an anti-PD-1 monoclonal antibody having the original VH without the amino acid substitution.

[0100] Furthermore, the anti-CD19 monoclonal antibodies of the present invention also include those having a VH in which each CDR has the above-mentioned specific amino acid sequence, and the amino acid sequence of the framework of the VH is encoded by a specific germline gene or a gene that has undergone somatic mutation, such as those having a specific VH encoded by a specific germline gene or a gene that has undergone somatic mutation, as listed above in the description of the "second arm that specifically binds to CD19."

[0101] Furthermore, among the anti-CD19 monoclonal antibodies of the present invention, those containing each CDR in any one VH selected from the above (1d) to (5d) and having an amino acid sequence of the FR of the VH encoded by a specific germline gene or a somatic mutation thereof may include, for example, those having a VH consisting of an amino acid sequence selected from SEQ ID NOs: 30 to 34. Furthermore, such anti-CD19 monoclonal antibodies include, for example, those having a VH consisting of an amino acid sequence that is at least 80% identical, preferably at least 90% identical, more preferably at least 95% identical, still more preferably at least 98% identical, and even more preferably at least 99% identical to any one amino acid sequence selected from SEQ ID NOs: 30 to 34, and having a binding activity substantially equivalent to the binding activity to CD19 by the anti-CD19 monoclonal antibody having the original VH. Here, "having a binding activity substantially equivalent to the binding activity to CD19 by the anti-CD19 monoclonal antibody having the original VH" means that it is 95% or more, preferably 98% or more, and more preferably 99% or more of the binding activity to CD19 of the anti-CD19 monoclonal antibody having a VH consisting of any one of these amino acid sequences.

[0102] In yet another aspect, the anti-CD19 monoclonal antibodies of the present invention include (1) an anti-PD-1 monoclonal antibody that cross-competes with the binding of an anti-CD19 monoclonal antibody having any one VH selected from the above (1d) to (5d) or a VH consisting of an amino acid sequence selected from SEQ ID NOs: 30 to 34 and the VL of the common light chain herein (preferably, VL consisting of the amino acid sequence of SEQ ID NO: 25) to CD19, and (2) an anti-CD19 monoclonal antibody whose binding to CD19 is cross-competed by an anti-CD19 monoclonal antibody having any one VH selected from the above (1d) to (5d) or a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30 to 34 and the VL of the common light chain.

[0103] In yet another aspect, the anti-CD19 monoclonal antibody of the present invention includes a VH consisting of an amino acid sequence in which the 114th amino acid in any one of the amino acid sequences selected from SEQ ID NOs: 30 to 34 is substituted with arginine or may be substituted, and the variable region of the common light chain herein (preferably, VL consisting of the amino acid sequence of SEQ ID NO: 25). More preferably, it has a VH consisting of an amino acid sequence in which the 114th glutamine in any one of the amino acid sequences selected from SEQ ID NOs: 30 to 34 is substituted with arginine and the VL of the common light chain, and they respectively correspond to anti-CD19 monoclonal antibodies having a VH consisting of any one of the amino acid sequences selected from SEQ ID NOs: 62 to 66 and the VL of the common light chain.

[0104] Polynucleotide encoding a PD-1 / CD19 bispecific antibody The polynucleotide encoding the PD-1 / CD19 bispecific antibody is composed of (1) a polynucleotide encoding a heavy chain having a VH of the first arm that specifically binds to PD-1, (2) a polynucleotide encoding a heavy chain having a VH of the second arm that specifically binds to CD19, and (3) a polynucleotide encoding a common light chain. Here, the polynucleotide encoding a heavy chain having a VH of the first arm that specifically binds to PD-1 is composed of a polynucleotide encoding a VH of the first arm that specifically binds to PD-1 and a polynucleotide encoding a constant region of the heavy chain having the VH. Similarly, the polynucleotide encoding a heavy chain having a VH of the second arm that specifically binds to CD19 is composed of a polynucleotide encoding a VH of the second arm that specifically binds to CD19 and a polynucleotide encoding a constant region of the heavy chain having the VH.

[0105] The polynucleotide encoding the PD-1 / CD19 bispecific antibody may be any polynucleotide having polynucleotides encoding the respective parts constituting these PD-1 / CD19 bispecific antibodies, and may be genomic DNA, cDNA, synthetic DNA, RNA, or DNA-RNA hybrid. One to six codons encoding one amino acid are known. For example, for Phe, there are TTT or TTC; for Leu, there are TTA, TTG, CTT, CTC, CTA or CTG; for Ile, there are ATT, ATC or ATA; for Met, there is ATG; for Val, there are GTT, GTC, GTA or GTG; for Ser, there are TCT, TCC, TCA or TCG; for Pro, there are CCT, CCC, CCA or CCG; for Thr, there are ACT, ACC, ACA or ACG; for Ala, there are GCT, GCC, GCA or GCG; for Tyr, there are TAT or TAC; for His, there are CAT or CAC; for Gln, there are CAA or CAG; for Asn, there are AAT or AAC; for Lys, there are AAA or AAG; for Asp, there are GAT or GAC; for Glu, there are GAA or GAG; for Cys, there are TGT or TGC; for Trp, there is TGG; for Arg, there are CGT, CGC, CGA or CGG; for Ser, there are AGT or AGC; for Arg, there are AGA or AGG; and for Gly, there are GGT, GGC, GGA or GGG. Therefore, the polynucleotide encoding the PD-1 / CD19 bispecific antibody includes polynucleotides in which each codon corresponding to each amino acid is arbitrarily combined. Preferably, as the polynucleotide encoding the VH of the first arm that specifically binds to PD-1, for example, a polynucleotide consisting of any one of the base sequences selected from SEQ ID NOs: 50 to 54 encoding the VH of clones PD1-1 to PD1-5 can be mentioned. Preferably, as the polynucleotide encoding the VH of the second arm that specifically binds to CD19, for example, a polynucleotide consisting of any one of the base sequences selected from SEQ ID NOs: 56 to 60 and SEQ ID NOs: 76 to 80 encoding the VH of clones CD19-5, CD19-1, CD19-4, CD19-2 and CD19-3 can be mentioned. Preferably, as the polynucleotide encoding the variable region of the common light chain, a polynucleotide consisting of the base sequence of SEQ ID NO: 55 can be mentioned.

[0106] Pharmaceutical use The PD-1 / CD19 bispecific antibody of the present invention and the like are useful for preventing, suppressing symptom progression, suppressing recurrence, and / or treating autoimmune diseases or graft-versus-host disease (GVHD).

[0107] Autoimmune diseases that can be prevented, have symptom progression inhibited, and / or be treated with the PD-1 / CD19 bispecific antibody of the present invention and the like include, for example, Behçet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis (systemic scleroderma, progressive systemic sclerosis), scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa, microscopic polyangiitis), aortic inflammatory syndrome (Takayasu arteritis), rheumatoid arthritis, arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, large vessel vasculitis, ANCA-associated vasculitis (for example, granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis), Cogan syndrome, RS3PE syndrome, temporal arteritis, rheumatoid polymyalgia, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related diseases (for example, primary sclerosing cholangitis, autoimmune pancreatitis, etc.), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic fatty hepatitis, primary biliary cirrhosis, Goodpasture syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease (hyperthyroidism), Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, Addison's disease (chronic adrenal cortical insufficiency), idiopathic Addison's disease, type I diabetes, slowly progressive type I diabetes (latent autoimmune diabetes in adults), localized scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, gestational herpes, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo, vulgaris vitiligo, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease (for example, ulcerative colitis, Crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, transplantation immunity, familial Mediterranean fever, eosinophilic rhinitis, dilated cardiomyopathy, systemic mastocytosis, and inclusion body myositis, etc.

[0108] In the present invention, "treatment" means, for example, curing or improving a certain disease or its symptoms, "prevention" means preventing the onset of a certain disease or symptoms or delaying it for a certain period, and "symptom progression suppression" means suppressing the progression or aggravation of symptoms to stop the progression of the disease state. Note that the meaning of "prevention" includes recurrence suppression. "Recurrence suppression" means preventing the recurrence of a certain disease or symptoms or reducing the possibility of recurrence.

[0109] In another aspect, the PD-1 / CD19 bispecific antibody of the present invention is useful for the prevention, symptom progression suppression, recurrence suppression and / or treatment of self-reactive B cell-mediated diseases. Examples of self-reactive B cell-mediated diseases include systemic lupus erythematosus, Graves' disease, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary biliary cirrhosis and pernicious anemia. In this pharmaceutical use, the PD-1 / CD19 bispecific antibody of the present invention acts through an inhibitory effect on self-reactive B cells. Here, the inhibitory effect on self-reactive B cells includes an inhibitory effect on the production of immunoglobulins such as IgG and IgM. Furthermore, the PD-1 / CD19 bispecific antibody of the present invention has an inhibitory effect on memory T cell activation. Here, the inhibitory effect on memory T cell activation includes an inhibitory effect on cytokine production.

[0110] The PD-1 / CD19 bispecific antibodies and the like of the present invention are typically administered systemically or locally parenterally. Specific examples of such administration methods include injection, nasal administration, pulmonary administration, and transdermal administration. Injection administration includes, for example, intravenous injection, intramuscular injection, and intraperitoneal injection. Intravenous administration is preferably by infusion. The dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but typically ranges from 0.1 μg / kg to 300 mg / kg per dose per adult, and more preferably ranges from 0.1 mg / kg to 10 mg / kg per dose. The dosage is administered parenterally once or several times daily, or by continuous intravenous administration for 30 minutes to 24 hours per day. Of course, as mentioned above, the dosage varies depending on various conditions. Therefore, a smaller dosage than the above may be sufficient, or a larger dosage may be required.

[0111] Formulation When the PD-1 / CD19 bispecific antibodies of the present invention are formulated and used as injections or infusions for intravenous drip infusion, the injections or infusions may be in the form of an aqueous solution, suspension, or emulsion, or may be formulated as a solid together with a pharmaceutically acceptable carrier so that they can be dissolved, suspended, or emulsified by adding a solvent when used. Examples of solvents that can be used for injections or infusions for intravenous drip infusion include distilled water for injection, physiological saline, glucose solutions, and isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.).

[0112] Examples of pharmaceutically acceptable carriers include stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, antiseptics, pH adjusters, and antioxidants. Examples of stabilizers that can be used include various amino acids, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, polyethylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, and dibutylhydroxytoluene. Examples of solubilizing agents that can be used include alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., Polysorbate 20 (registered trademark), Polysorbate 80 (registered trademark), HCO-50, and the like. Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Examples of emulsifiers that can be used include gum arabic, sodium alginate, and tragacanth. Examples of soothing agents that can be used include benzyl alcohol, chlorobutanol, and sorbitol. Examples of buffers that can be used include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, and epsilon aminocaproic acid buffer. Examples of preservatives that can be used include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax. Examples of antiseptics that can be used include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. Examples of pH adjusters that can be used include hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid.As antioxidants, for example, (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, etc., (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, α-tocopherol, etc., and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid, sorbitol, tartaric acid, phosphoric acid, etc. can be used.

[0113] Infusions for injections or drips can be manufactured by sterilizing in the final step or by aseptic operation methods, for example, filtering with a filter, etc. to sterilize and then filling into a sterile container. Also, infusions for injections or drips can be used by dissolving a sterile powder (which may contain a powder of a pharmaceutically acceptable carrier) obtained by vacuum drying and freeze drying in an appropriate solvent at the time of use.

[0114] Combined or formulated agent Furthermore, the PD-1 / CD19 bispecific antibody of the present invention and the like may be used in combination with other agents used for the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of autoimmune diseases. In the present invention, when used in combination with other agents (combination administration), the administration form may be in the form of a formulation in which both components are formulated in one preparation, or in the form of separate preparations. By such combination, the preventive, symptom progression-suppressing, recurrence-suppressing, and / or therapeutic effects of other agents can be complemented, and the dosage or the number of administrations can be maintained or reduced. When the PD-1 / CD19 bispecific antibody of the present invention and the like and other agents are administered separately, they may be administered simultaneously for a certain period, and then only the PD-1 / CD19 bispecific antibody and the like or only other agents may be administered. Also, the PD-1 / CD19 bispecific antibody of the present invention and the like may be administered first, and then other agents may be administered after the completion of the administration, or other agents may be administered first, and then the PD-1 / CD19 bispecific antibody of the present invention and the like may be administered later, and each administration method may be the same or different. It is also possible to provide a kit of a preparation containing the PD-1 / CD19 bispecific antibody of the present invention and the like and a preparation containing other agents. Here, the dosage of other agents can be appropriately selected based on the clinically used dosage. Also, two or more arbitrary other agents may be administered in an appropriate ratio in combination. Further, the other agents include not only those found to date but also those to be found in the future.

[0115] For example, when applying the PD-1 / CD19 bispecific antibody of the present invention or the like to the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of type I diabetes, insulin preparations (e.g., human insulin, insulin glargine, insulin lispro, insulin detemir, and insulin aspart, etc.), sulfonylurea agents (e.g., glibenclamide, gliclazide, and glimepiride, etc.), rapid-acting insulin secretagogues (e.g., nateglinide, etc.), biguanide preparations (e.g., metformin, etc.), insulin resistance improvers (e.g., pioglitazone, etc.), α-glucosidase inhibitors (e.g., acarbose and voglibose, etc.), therapeutic agents for diabetic neuropathy (e.g., epalrestat, mexiletine, and imidapril, etc.), GLP-1 analog preparations (e.g., liraglutide, exenatide, lixisenatide, and semaglutide, etc.), and DPP-4 inhibitors (e.g., sitagliptin, vildagliptin, and alogliptin, etc.) and the like may be used in combination with any one or more agents selected therefrom.

[0116] In addition, for example, when applying the PD-1 / CD19 bispecific antibody of the present invention or the like for the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of multiple sclerosis, a steroid drug (for example, cortisone acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, fludrocortisone acetate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisolone butylacetate, prednisolone sodium phosphate, halopredone acetate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, triamcinolone, triamcinolone acetate, triamcinolone acetonide, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone palmitate, paramethasone acetate and betamethasone, etc.), interferon β-1a, interferon β-1b, glatiramer acetate, mitoxantrone, azathioprine, cyclophosphamide, cyclosporine, methotrexate, cladribine, adrenocorticotropic hormone (ACTH), corticotropin, mizoribine, tacrolimus, fingolimod and alemtuzumab, etc. It may be used in combination with any one or more drugs selected from the group consisting of.

[0117] In addition, for example, when applying the PD-1 / CD19 bispecific antibody of the present invention or the like for the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of systemic lupus erythematosus, a steroid drug (for example, the steroid drugs described above), an immunosuppressant (for example, cyclosporine, tacrolimus and fingolimod, etc.) and belimumab. It may be used in combination with any one or more drugs selected from the group consisting of.

[0118] For example, when applying the PD-1 / CD19 bispecific antibody of the present invention or the like for the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of rheumatoid arthritis, it may be used in combination with any one or more drugs selected from steroid drugs (for example, the steroid drugs described above), anti-rheumatic drugs (for example, methotrexate, sulfasalazine, bucillamine, leflunomide, mizoribine, tacrolimus, etc.) or anti-cytokine drugs (for example, infliximab, adalimumab, tocilizumab, etanercept, golimumab, certolizumab, etc.) and abatacept, etc.

[0119] When applying for the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of other autoimmune diseases, the PD-1 / CD19 bispecific antibody of the present invention or the like may be used in combination with any one or more of the other drugs described above.

[0120] The present invention will be further described in detail by the following examples, but the scope of the present invention is not limited thereto. Based on the description of the present invention, various changes and modifications are possible for those skilled in the art, and these changes and modifications are also included in the present invention.

Examples

[0121] Example 1: Immunization of MeMo (registered trademark) mice with recombinant human PD-1-Fc fusion protein As a method for obtaining the first arm that specifically binds to PD-1 of the present invention, a method of immunizing MeMo (registered trademark) mice (see WO2009 / 157771) with recombinant human PD-1 protein was selected. MeMo (registered trademark) mice are genetically modified mice in which gene fragments containing non-recombinant human heavy chain V gene regions, D gene regions and J gene regions and recombinant human κ light chain IgVκ1-39*01 / IGJκ1*01 germline gene types are linked to mouse constant region genes, and by directly immunizing the target protein of the antibody, antibodies consisting of a heavy chain with diversity and a common light chain can be produced.

[0122] Twelve 12- to 16-week-old MeMo (registered trademark) mice were each immunized at 14-day intervals with recombinant human PD-1-Fc fusion protein (R&D Systems, catalog number 1086-PD) emulsified with Gerbu adjuvant MM (Gerbu Biotechnik, catalog number #3001). The recombinant human PD-1-Fc fusion protein was administered subcutaneously on days 0, 14, and 28 of immunization, and at subsequent time points, the recombinant human PD-1-Fc fusion protein dissolved in PBS was administered subcutaneously. On days 21, 35, 56, 77, and 98 of immunization, the antibody titers in the serum were evaluated by flow cytometry using a human PD-1-overexpressing HEK293T cell line. When the human PD-1-overexpressing HEK293T cell line was stained with serum diluted 1000-fold, the lymphoid tissues of mice with an MFI value increased by 3-fold or more compared to the control human PD-1 non-expressing HEK293T cell line were used for the construction of a phage display library. Mice that met the criteria for proceeding with library construction were boost-immunized with the recombinant PD-1-Fc fusion protein for 3 days starting from the day of antibody titer evaluation, and the spleen and hilar lymph nodes were harvested. Mice with a serum antibody titer of 1 / 100 or more against human PD-1 and cynomolgus monkey PD-1 and whose antibody titers did not increase upon boost-immunization also had their spleen and hilar lymph nodes harvested. After extracting RNA from these lymphoid tissues, cDNA synthesis was performed.

[0123] Example 2: Construction of a phage display library for obtaining anti-PD-1 antibodies (protein immunization) Using the DNA prepared in Example 1, a PCR reaction was performed using primers specific for the immunoglobulin heavy chain variable region family. After the PCR product was digested with the restriction enzymes SfiI and XhoI, it was inserted into a phagemid vector digested with the same restriction enzymes [containing the gene encoding the common light chain (human κ light chain IgVκ1-39*01 / IGJκ1*01 germline gene type)] to construct the library.

[0124] Example 3: Screening of anti-PD-1 antibodies Phage selection based on PD-1 binding was performed using plates coated with human PD-1-Fc fusion protein, human PD-1-His tag fusion protein, cynomolgus monkey PD-1-His tag fusion protein, or mouse PD-1-His tag fusion protein. When human PD-1-Fc fusion protein was used, Fc-reactive clones were absorbed by adding human IgG (SIGMA, product number I4506) during phage incubation. Phage binding to human PD-1, cynomolgus monkey PD-1, and mouse PD-1 was enriched. Phage binding to cynomolgus monkey PD-1 was enriched by selection in HEK293T cell lines overexpressing cynomolgus monkey PD-1. Phage obtained by selection were transformed with the E. coli strain TG1, and clones were isolated to prepare master plates.

[0125] Furthermore, phage selection was performed from periplasmic extracts of the clones obtained from the above selection based on their binding to PD-1 on plates adsorbed with human PD-1-Fc fusion protein. The selection criterion was the signal (OD ) obtained in the negative control wells (PBS). 450 A clone that gave a signal three times or more higher than the normal value was considered positive.

[0126] Example 4: DNA sequence of candidate clones of anti-PD-1 antibodies The heavy chain variable region genes of the positive clones obtained by the screening in Example 3 were subjected to DNA sequencing. The analyzed DNA sequences were classified into superclusters (groups of clones with heavy chain CDR3s of the same length and with amino acid sequences of the CDR3s that are 70% or more identical) and clusters (groups of clones with identical heavy chain CDR3 amino acid sequences). Several hundred clones were obtained and classified into superclusters and clusters.

[0127] Example 5: Screening by evaluating the binding to PD-1-expressing cells From each classified supercluster, anti-PD-1 monoclonal antibody clones that met the following criteria were selected and isolated. (1) Somatic mutations are frequently introduced into the CDR region, (2) It has a germline gene of VH with a high usage frequency, and (3) High signals were obtained in the binding screening for human PD-1-Fc fusion protein.

[0128] Using the Fab fragments contained in those periplasmic extracts, the binding to human PD-1 overexpressing CHO-S cell line and cynomolgus PD-1 overexpressing CHO-S cell line was evaluated by detecting with anti-mouse IgG polyclonal antibody. Among the 117 clones (105 clusters) evaluated, binding to the human PD-1 expressing CHO-S cell line was observed in 22 clones including anti-PD-1 monoclonal antibody clones PD1-1, PD1-2, PD1-3 and PD1-4.

[0129] Example 6: Preparation of amino acid substitution variants of anti-PD-1 monoclonal antibodies Clones PD1-1 and PD1-4 contain a deamidation motif (Asn-Gly) in framework region 4 of their heavy chain variable region. For the purpose of obtaining a PD-1 arm with reduced deamidation risk, mutants with this deamidation motif converted were prepared. Clone PD1-5 was prepared and isolated in which asparagine (Asn) at position 119 according to the EU numbering system of clone PD1-4 was modified to glutamine by a known site-directed mutagenesis method. The binding of this clone to human PD-1 overexpressing CHO-S cells was also equivalent to that of clone PD1-4.

[0130] Example 7: Immunization of MeMo (registered trademark) mice with a CD19-expressing plasmid vector As a method for obtaining a second arm that specifically binds to CD19 of the present invention, a method of immunizing MeMo (registered trademark) mice (see WO2009 / 157771) with a human CD19 expression plasmid vector and a cynomolgus CD19 expression plasmid vector was selected.

[0131] MeMo (registered trademark) mice are genetically modified mice in which a gene fragment containing a non-recombinant human heavy chain V gene region, D gene region, and J gene region, as well as a recombinant human kappa light chain IgVκ1-39*01 / IGJκ1*01 germline gene, is linked to a mouse constant region gene. By immunizing MeMo (registered trademark) mice with a plasmid vector expressing the antibody target protein, antibodies consisting of diverse heavy chains and a common light chain can be produced.

[0132] Twelve MeMo® mice, aged 12–16 weeks, were immunized with a human CD19 expression plasmid vector and / or a cynomolgus monkey CD19 expression plasmid vector, either individually or alternately. The expression plasmid vectors were administered on days 0, 3, 6, 14, 17, 28, 31, 42, 49, 63, and / or 70 after immunization. Serum antibody titers were assessed by flow cytometry using a human CD19-expressing cell line. Lymphoid tissue from mice that showed a 3-fold or greater increase in MFI when stained with 1:100 diluted serum compared to a control human CD19-nonexpressing cell line was used for constructing a phage display library. After booster immunization, spleens and inguinal lymph nodes were harvested from mice that met the criteria for library construction. RNA was extracted from the lymphoid tissues and cDNA was synthesized by reverse transcription using IgG constant region-specific primers.

[0133] Example 8: Construction of a phage display library for obtaining anti-CD19 antibodies PCR was performed using primers specific to the immunoglobulin heavy chain variable region family from the DNA prepared in Example 7. The PCR products were digested with restriction enzymes and then inserted into a phagemid vector containing a gene encoding a common light chain (human κ light chain IgVκ1-39*01 / IGJκ1*01 germline gene) that had also been digested with the restriction enzymes, to construct the library.

[0134] Example 9: Screening of anti-CD19 antibodies Human CD19-Fc fusion protein (R&D systems, catalog number 9269-CD), cynomolgus CD19-Fc fusion protein (NovoPro Bioscience, catalog number 504385), human B cell line Raji or cynomolgus CD19-overexpressing HEK293T cell line were used to perform phage selection based on the binding ability to CDI9. Clones of Escherichia coli strain TG1 transformed with the phages obtained by the selection were obtained, and a master plate was prepared. As the selection criteria, those with a signal (OD 450 value or MFI) three times or more higher than that obtained with the negative control were defined as positive clones.

[0135] Example 10: DNA sequence of candidate clones of anti-CD19 antibodies The DNA sequences of the heavy chain variable region genes of the positive clones obtained by the screening of Example 9 were analyzed. The analyzed DNA sequences were classified into superclusters (a group in which the heavy chain CDR3 has the same length and the amino acid sequences of the heavy chain variable regions are 70% or more homologous to each other) and clusters (a group in which the amino acid sequences of the heavy chain CDR3 and the heavy chain variable region are identical to each other).

[0136] In the first screening, hundreds of clones were obtained and classified into superclusters, clusters, and four germline gene lineages. In the second screening, hundreds of clones were obtained and classified into superclusters, clusters, and eight germline gene lineages. Among them, 19 superclusters were different from those in the first screening.

[0137] Example 11: Screening by evaluating the binding to CD19 From each of the classified superclusters, anti-CD19 monoclonal antibody clones that met the following conditions were selected and isolated. (1) Somatic mutations were frequently introduced into the CDR regions, (2) They had a VH germline gene type that was frequently used, and (3) High signals were obtained in the binding screening for CD19.

[0138] The Fab fragments contained in these periplasmic extracts were used to assess binding to CD19.

[0139] Among the clones evaluated, several clones, including anti-CD19 monoclonal antibody clones CD19-1, CD19-2, CD19-3, CD19-4, and CD19-5, were found to bind to human CD19-expressing cell lines.

[0140] Example 12: Preparation of PD-1 / CD19 bispecific antibodies An expression vector expressing each heavy chain of the first arm that specifically binds to PD-1 was prepared by ligating DNA encoding the heavy chain variable region of each of the anti-PD-1 monoclonal antibody clones PD1-1 to PD1-5 selected in Examples 5 and 6 to DNA encoding the IgG1 heavy chain constant region. On the other hand, an expression vector expressing the heavy chain of the second arm that specifically binds to CD19 was prepared by ligating DNA encoding the heavy chain variable region of the anti-CD19 monoclonal antibody clones CD19-1 to CD19-5 selected in Example 11 to DNA encoding the IgG1 heavy chain constant region. Here, for the gene expressing these heavy chain constant regions, in the case of the first arm that specifically binds to PD-1, one expressing an Fc region having an L351D / L368E mutation (DE mutation) was used, and in the case of the second arm that specifically binds to CD19, one expressing an Fc region having an L351K / T366K mutation (KK mutation) was used. These expression vectors were constructed to contain a gene encoding the IGVK1-39 / JK1 common light chain so that they both express it. Furthermore, for the genes expressing these heavy chain constant regions, in order to abolish the Fc effector activity, the 235th leucine in the heavy chain constant region was substituted with glycine and the 236th glycine was substituted with arginine for expression, and furthermore, in order to avoid post-translational processing, one modified to delete the 447th lysine at the C-terminus of the heavy chain constant region was used. These expression vectors were both transfected into Free Style 293F cells to produce antibodies in the culture supernatant. The culture supernatant was collected and processed by protein A affinity chromatography to purify clone CD19-1(Bi), clone CD19-2(Bi), clone CD19-3(Bi), clone CD19-4(Bi), and clone CD19-5(Bi), which are the PD-1 / CD19 bispecific monoclonal antibodies of the present invention, respectively.

[0141] These PD-1 / CD19 bispecific antibody clones each correspond to the anti-CD19 monoclonal antibody clones used in their production, CD19-1, CD19-2, CD19-3, CD19-4, and CD19-5, in that they have a second arm that specifically binds to CD19 and is derived from those anti-CD19 monoclonal antibody clones. Further, these PD-1 / CD19 bispecific antibody clones all have a first arm that specifically binds to PD-1 and is derived from PD1-5.

[0142] Example 13: Preparation of variants of anti-CD19 monoclonal antibodies and corresponding PD-1 / CD19 bispecific antibodies In the production process scaled up for pharmaceutical development, the PD-1 / CD19 bispecific antibody clones prepared in Example 12 may not achieve sufficient separation and purification from by-products that are the first arm heavy chain / light chain complex, the second arm heavy chain / light chain complex, and / or their respective homodimers. Therefore, for the purpose of improving the separation of the bispecific antibody from the by-products during cation exchange chromatography purification, amino acid mutants of the anti-CD19 monoclonal antibody with an increased isoelectric point were prepared.

[0143] A mutant was prepared by substituting the glutamine at position 114 in SEQ ID NO: 30, which represents the amino acid sequence of the VH of clone CD19-5, with arginine by a known site-directed mutagenesis method. In the present invention, the mutant was named "CD19-6". The isoelectric point of each antibody was calculated using the gene and amino acid sequence analysis software Genetyx (Genetics Co., Ltd.).

[0144] Furthermore, each mutant in which glycine and the peptides represented by SEQ ID NOs: 67 to 70 were added to the C-terminus of the heavy chain having the VH of CD19-6 (however, having lysine as the C-terminal amino acid) (hereinafter, these are collectively referred to as "CD19-6 / C-terminal peptide adducts") was prepared by a known gene modification technique. In the present invention, the mutant in which the peptide represented by SEQ ID NO: 67 was added to the C-terminus of CD19-6 was named "CD19-7".

[0145] Following the same method as in Example 12, each expression vector described in the same example, into which each DNA encoding a variant of these anti-CD19 monoclonal antibodies and the DNA encoding clone PD1-5 were inserted, was transfected into Free Style 293F cells to produce antibodies in the culture supernatant. The culture supernatant was collected and processed by protein A affinity chromatography to purify each bispecific antibody of the present invention derived from each variant prepared in this example. Among these, the bispecific antibody derived from CD19-6 was named "CD19-6(Bi)", and the bispecific antibody clone derived from CD19-7 was named "CD19-7(Bi)".

[0146] Example 14: Examination of purification and separation of PD-1 / CD19 bispecific antibodies and by-products Regarding the PD-1 / CD19 bispecific antibodies prepared in Examples 12 and 13, respectively, the success or failure of the purification and separation from by-products that may be generated in their production was examined.

[0147] The culture supernatants containing the bispecific antibodies collected in Examples 12 and 13, respectively, were separately processed by protein A affinity chromatography and size exclusion chromatography to purify the bispecific antibodies, the anti-PD-1 antibody corresponding to the by-product, and the anti-CD19 antibody, respectively.

[0148] The solvent containing each of these purified antibodies was buffer-exchanged by ultrafiltration so that the pH became 6.0. Each purified antibody after buffer exchange was applied to a cation exchange column TSKgel SP-STAT Column (Tosoh, model number 0021964) equilibrated with Buffer A (pH 7.0). Each purified antibody bound to the column was eluted by a salt gradient using Buffer B (pH 7.0) containing 1 mol / L sodium chloride. The flow rate of the mobile phase was 0.5 mL / min, and the elution was a linear gradient from Buffer A to Buffer B. From 0 to 10 minutes after starting the application of each purified antibody, Buffer B was 0%, from 10 to 40 minutes, Buffer B was linearly increased from 0 to 100%, and from 40 to 50 minutes, Buffer B was 100%.

[0149] The retention times (minutes) of clones CD19-2 (isoelectric point: 8.32), CD19-5 (isoelectric point: 8.40), CD19-6 (isoelectric point: 8.49), and CD19-7 (isoelectric point: 8.75) on the cation exchange column were 15.167, 15.185, 15.749, and 17.521, respectively, and the retention times of clones CD19-6 and CD19-7 were prolonged. On the other hand, clones PD1-3 (isoelectric point: 7.67) and PD1-5 (isoelectric point: 7.52) did not bind to the cation exchange column and were eluted.

[0150] On the other hand, PD-1 / CD19 bispecific antibody clones CD19-2 (Bi) and CD19-5 (Bi) did not bind to the cation exchange column and were eluted, but clones CD19-6 (Bi) and CD19-7 (Bi) had retention times of 13.715 and 14.955 minutes, respectively.

[0151] As described above, by increasing the isoelectric point by amino acid substitution in the second arm or addition of a specific peptide to the C-terminus of its heavy chain, separation improvement between the bispecific antibody of the present invention, which is the target product, and the anti-PD-1 antibody and anti-CD19 antibody, which are by-products, was achieved during purification by cation exchange chromatography, and it became possible to provide the bispecific antibody of the present invention with extremely reduced contamination of the by-products.

[0152] Example 15: Evaluation of the binding of PD-1 / CD19 bispecific antibodies Using Biacore measurement with human IgG1-Fc fusion human PD-1 extracellular region recombinant protein (R&D systems, catalog number 1086-PD), the binding affinity of the PD-1 recombinant protein of the first arm of the PD-1 / CD19 bispecific monoclonal antibody obtained in Examples 12 and 13 was evaluated. For immobilization of the recombinant protein, Series S Sensor Chip CM5 sensor chip (GE Healthcare, catalog number 29-1049-88) was used.

[0153] Similarly, using Biacore measurement with human IgG1-Fc fusion human CD19 extracellular region recombinant protein (R&D systems, catalog number 9269-CD), the CD19 binding affinity of the second arm of the antibody was evaluated. The binding affinities (Kd values) of the first arm to PD-1 and the second arm to CD19 of each clone are shown in Figure 11. It was confirmed that the binding affinities of CD19-6(Bi) to PD-1 and CD19 were improved compared to the activity of CD19-5(Bi) with a structure not subjected to the amino acid substitution of Example 13.

[0154] Example 16: Confirmation of the binding of PD-1 / CD19 bispecific antibodies It was confirmed that the PD-1 / CD19 bispecific antibodies obtained in Examples 12 and 13 specifically bind to human PD-1, cynomolgus monkey PD-1, human CD19, and cynomolgus monkey CD19, respectively. Clone CD19-1(Bi) to CD19-6(Bi) was added to the human PD-1-overexpressing CHO-S cell line, cynomolgus monkey PD-1-overexpressing CHO-S cell line, CHO-S cell line, human CD19-overexpressing CHO-K1 cell line, cynomolgus monkey CD19-overexpressing CHO-K1 cell line, and CHO-K1 cell line, respectively, and incubated on ice for 20 minutes. After washing the cells, 100 μL of a PE-labeled goat anti-human IgG-Fc F(ab′)2 fragment antibody (ThermoFisher, catalog number H10104) was added and incubated on ice for 20 minutes. After washing the cells, the PD-1 binding property of the first arm and the CD19 binding property of the second arm of the antibody were evaluated by flow cytometry. The results of the assay are shown in FIGS. 12 to 16.

[0155] All clones bound to human PD-1, cynomolgus monkey PD-1, human CD19, and cynomolgus monkey CD19. Non-specific binding in this experimental system was not detected.

[0156] It was confirmed that the PD-1 / CD19 bispecific monoclonal antibody obtained in Example 12 specifically binds to PD-1 and CD19 simultaneously. First, clone CD19-1(Bi) to CD19-5(Bi) was added to the human CD19-overexpressing CHO-K1 cell line and the CHO-K1 cell line, respectively, and incubated on ice for 20 minutes. After washing the cells, 100 μL of a 6×His-tagged recombinant human PD-1 extracellular domain protein (R&D systems, catalog number 8986-PD) was added and incubated on ice for 20 minutes. After washing the cells, 100 μL of an AlexaFluor488-labeled mouse anti-His tag antibody (MBL, catalog number D291-A48) was added and incubated on ice for 20 minutes. After washing the cells, the binding amount of the recombinant human PD-1 extracellular domain protein was evaluated by flow cytometry. The results of the assay are shown in FIG. 17.

[0157] All clones bound to both PD-1 and CD19 simultaneously. No non-specific binding was detected in this experimental system.

[0158] Example 17: Evaluation of the binding characteristics of the first arm of PD-1 / CD19 bispecific antibodies To evaluate the effect of the first arm of the PD-1 / CD19 bispecific antibody obtained in Example 12 on PD-1 / PD-L1 binding, a competitive binding assay regarding the binding of the bispecific antibody clone and soluble PD-L1 recombinant protein to PD-1 was performed. First, clones CD19-1(Bi) to CD19-5(Bi), nivolumab, and anti-human PD-1 antibody J105 (Immunology Letters, 2002, Vol.83, Issue 3, p.215-220) were each added to human PD-1-overexpressing CHO-S cell line on ice. Further, soluble PD-L1 recombinant protein (R&D systems, catalog number 156-B7) biotinylated using a biotinylation kit (Dojindo Laboratories, catalog number LK03) was added on ice. After washing the cells, APC-labeled streptavidin (BioLegend, catalog number 405207) was added on ice. After washing the cells, the binding amount of the soluble PD-L1 recombinant protein was evaluated by flow cytometry. The results are shown in Figure 18.

[0159] Clones CD19-1(Bi) to CD19-5(Bi) allowed the binding of soluble PD-L1 recombinant protein to PD-1. On the other hand, nivolumab and J105 completely inhibited the binding of soluble PD-L1 recombinant protein to PD-1 under the same conditions.

[0160] Also, bispecific antibodies with anti-PD-1 monoclonal antibody clones PD1-1 to PD1-4 obtained in Example 5 as the first arm, respectively, were similarly evaluated and confirmed to allow the binding of soluble PD-L1 recombinant protein to PD-1.

[0161] Example 18: In vitro inhibitory effect of PD-1 / CD19 bispecific antibodies on activated B cells Using B cells isolated from peripheral blood mononuclear cells derived from healthy individuals (LONZA, product number CC-2702) with the B Cell Isolation Kit II, human (Miltenyi Biotec, product number 130-091-151), the inhibitory effect on human IgM production was evaluated. Human B cells were seeded in a cell culture plate, and anti-human CD79B antibody (LifeSpan Biosciences, product number LS-C134648), human CD40L recombinant protein (Enzo Life Sciences, product number ALX-522-110), and human IL-21 recombinant protein (R&D systems, product number 8879-IL) were added for activation treatment. Clone CD19-1(Bi)~CD19-5(Bi) or control antibody was treated, and IgM contained in the culture supernatant after activation treatment was quantified by ELISA method (ThermoFisher, product number BMS2098). The results are shown in Figure 19. Clone CD19-1(Bi)~CD19-5(Bi) all inhibited IgM production. The IgM production amount (ng / mL) in the figure was shown as the mean ± standard error (N = 4).

[0162] Example 19: In vivo inhibitory effect of PD-1 / CD19 bispecific antibodies on activated B cells Using NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ mice (hereinafter abbreviated as NSG mice) transplanted with peripheral blood mononuclear cells derived from healthy individuals (LONZA, product number CC-2702), the inhibitory effect on human IgG2 production was evaluated. 1×10 per NSG mouse 7Peripheral blood mononuclear cells derived from healthy individuals were transplanted. On the 3rd, 7th, 10th, 14th, and 17th days after transplantation, clone CD19-1(Bi) to CD19-4(Bi) or control antibody was intraperitoneally administered once a day at a dose of 3 mg / kg. On the 21st day after transplantation, blood was collected from the tail vein to prepare serum. On the other hand, for clone CD19-5(Bi) and CD19-6(Bi), they were intraperitoneally administered at the same dosage and usage on the 3rd, 7th, and 10th days after transplantation. At this time, the control antibody was also intraperitoneally administered in parallel. On the 14th day after transplantation, blood was collected from the tail vein to prepare serum. Human IgG2 contained in the serum was quantified by ELISA method (ThermoFisher, model number BMS2093). The results are shown in Figures 20 and 21. Clone CD19-1(Bi) to CD19-6(Bi) all inhibited IgG2 production. The IgG2 production amount (μg / mL) in the figure was shown as the mean value ± standard error (N = 4 - 8).

[0163] Example 20: Evaluation of the cross-competitiveness of PD-1 / CD19 bispecific antibodies against the binding to PD-1 To evaluate the cross-competitiveness of the binding of bispecific antibodies with each clone of anti-PD-1 monoclonal antibody PD1-1 to PD1-5 used in the preparation of PD-1 / CD19 bispecific antibody to PD-1 as the first arm, a competitive binding assay was performed.

[0164] First, a bispecific antibody with clone PD1-5 as the first arm was added to human PD-1-expressing CHO-S cell line on ice. Furthermore, bispecific antibodies with biotin-labeled clones PD1-1 to PD1-5 as the first arm were added respectively and incubated on ice. After washing the cells, PE-labeled streptavidin (BD Pharmingen, model number 554061) was added and incubated on ice. After washing the cells, the binding amount of the same biotin-labeled antibody was evaluated by flow cytometry.

[0165] It was confirmed that the bispecific antibody with clone PD1-5 as the first arm inhibited the binding of the same antibody with clones PD1-1 to PD1-4 as the first arm to PD-1 and cross-competed with their binding to PD-1.

[0166] Example 21: Evaluation of the in vitro effect of PD-1 / CD19 bispecific antibodies on cytokine release from human peripheral blood mononuclear cells For the purpose of analyzing the cytokine release activity of the PD-1 / CD19 bispecific antibody, addition experiments of the bispecific antibody clone of the present invention and mouse anti-human CD3 antibody OKT3 (BioLegend, catalog number 317304) to human peripheral blood mononuclear cells (hereinafter, human PBMC) were each performed.

[0167] To human PBMC (LONZA, catalog number CC-2702), clone CD19-6 (Bi) or OKT3 was added and cultured. IL-2 contained in the culture supernatant was quantified by flow cytometry using a Cytometric Bead Array (BD Biosciences, catalog number 551809). The results are shown in Figure 22. The IL-2 production amount (pg / mL) in the figure was shown as the mean ± standard error (N = 3).

[0168] Compared with OKT3 which significantly induced IL-2 production, IL-2 production by CD19-6 (Bi) was not observed.

[0169] Example 22: Evaluation of the Physicochemical Stability of a PD-1 / CD19 Bispecific Antibody The PD-1 / CD19 bispecific antibody of the present invention was confirmed to show good performance in any physicochemical stability evaluation including structural stability evaluation by differential scanning calorimetry (DSC), colloidal stability evaluation by measurement of the diffusion coefficient change rate (DLS), and chemical stability evaluation under stress conditions (for example, pH 3 - 4 / 5°C or pH 7 / 5°C / freeze-thaw 5 times) (for example, protein concentration change, molecular structure change, presence or absence of aggregation / agglutination, presence or absence of charge variant generation, structural change, and binding to CD19).

Industrial Applicability

[0170] The PD-1 / CD19 bispecific antibody or its antibody fragment of the present invention is useful for the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of autoimmune diseases or graft-versus-host disease (GVHD).

Claims

Claim 1 A pharmaceutical composition comprising, as an active ingredient, a bispecific antibody or its F(ab′)2 fragment having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19, and specifically binding to PD-1 and CD19 respectively, wherein (A) the first arm that specifically binds to PD-1 has a VH consisting of the amino acid sequence of SEQ ID NO: 5 and a VL consisting of the amino acid sequence of SEQ ID NO: 25, and (B) the second arm that specifically binds to CD19 has a VH consisting of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 62 and a VL consisting of the amino acid sequence of SEQ ID NO:

25. Claim 2. (A) The first arm that specifically binds to PD-1 has a VH consisting of the amino acid sequence of SEQ ID NO: 5 and a VL consisting of the amino acid sequence of SEQ ID NO: 25, and (B) the second arm that specifically binds to CD19 has a VH consisting of the amino acid sequence of SEQ ID NO: 62 and a VL consisting of the amino acid sequence of SEQ ID NO:

25. The pharmaceutical composition according to claim 1. Claim 3 The pharmaceutical composition according to claim 1 or 2, wherein the first arm that specifically binds to PD-1 permits the interaction between PD-1 and PD-L1. Claim 4 The pharmaceutical composition according to any one of claims 1 to 3, wherein the bispecific antibody is an IgG antibody. Claim 5 When the IgG antibody is IgG 1 antibody or IgG 4 antibody, the pharmaceutical composition according to claim 4. Claim 6 When the IgG antibody is IgG 1 The pharmaceutical composition according to claim 4, which is an antibody. Claim 7 The pharmaceutical composition according to claim 6, wherein the binding to the Fc receptor is lost or attenuated. Claim 8 The pharmaceutical composition according to claim 7, wherein the 235th leucine in the EU numbering system in the two heavy chain constant regions of the bispecific antibody is each substituted with glycine, and / or the 236th glycine is each substituted with arginine. Claim 9 The 351st leucine in the constant region of the heavy chain having the VH of the first arm that specifically binds to PD-1 is substituted with lysine, and the 366th threonine is substituted with lysine. The 351st leucine in the constant region of the heavy chain having the VH of the second arm that specifically binds to CD19 is substituted with aspartic acid, and the 368th leucine is substituted with glutamic acid. The pharmaceutical composition according to any one of claims 6 to 8. Claim 10 In the constant region of the heavy chain having a VH of the first arm that specifically binds to PD-1, the 351st leucine according to the EU numbering system is substituted with aspartic acid, and the 368th leucine is substituted with glutamic acid. In the constant region of the heavy chain having a VH of the second arm that specifically binds to CD19, the 351st leucine is substituted with lysine, and the 366th threonine is substituted with lysine. The pharmaceutical composition according to any one of claims 6 to 8.

11. The pharmaceutical composition according to any one of claims 6 to 10, wherein the 447th lysine according to the EU numbering system in the two heavy chain constant regions of the bispecific antibody is each absent.

12. The pharmaceutical composition according to any one of claims 1 to 7 and 10, wherein the heavy chain having a VH of the first arm that specifically binds to PD-1 comprises a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO:

23.

13. The pharmaceutical composition according to any one of claims 1 to 8, 10 and 12, wherein the heavy chain having a VH of the second arm that specifically binds to CD19 comprises a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO:

24.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the light chain having a VL of the first arm that specifically binds to PD-1 and / or the light chain having a VL of the second arm that specifically binds to CD19 comprises a light chain constant region consisting of the amino acid sequence of SEQ ID NO:

29.

15. A pharmaceutical composition comprising, as an active ingredient, a bispecific antibody having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19, or an F(ab′)2 fragment thereof, which specifically binds to PD-1 and CD19, respectively, wherein the bispecific antibody comprises (A) a heavy chain having a VH of the first arm that specifically binds to PD-1, (B) a light chain having a VL of the first arm that specifically binds to PD-1, (C) a heavy chain having a VH of the second arm that specifically binds to CD19, and (D) a light chain having a VL of the second arm that specifically binds to CD19, (a) the heavy chain having a VH of the first arm that specifically binds to PD-1 comprises a VH consisting of the amino acid sequence of SEQ ID NO: 5 and a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23, (b) a light chain having a VL of the first arm that specifically binds to PD-1 comprises a VL consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29; (c) a heavy chain having a VH of the second arm that specifically binds to CD19 comprises a VH consisting of any one amino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 62, and a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 24; and (d) The pharmaceutical composition, wherein the light chain having a VL of the second arm that specifically binds to CD19 comprises a VL consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain constant region consisting of the amino acid sequence of SEQ ID NO:

29.

16. A pharmaceutical composition comprising, as an active ingredient, a bispecific antibody which specifically binds to PD-1 and CD19, and which has a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19, wherein the bispecific antibody is (A) a heavy chain having a VH of the first arm that specifically binds to PD-1; (B) a light chain having a VL in the first arm that specifically binds to PD-1; (C) a heavy chain having a VH of the second arm that specifically binds to CD19; and (D) a light chain having a VL in the second arm that specifically binds to CD19; (a) a heavy chain having a VH of a first arm that specifically binds to PD-1 comprises a VH consisting of the amino acid sequence of SEQ ID NO: 5 and a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23; (b) a light chain having a VL of the first arm that specifically binds to PD-1 comprises a VL consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29; (c) a heavy chain having a VH of the second arm that specifically binds to CD19 comprises a VH consisting of the amino acid sequence of SEQ ID NO: 62 and a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 24; and (d) The pharmaceutical composition, wherein the light chain having a VL of the second arm that specifically binds to CD19 comprises a VL consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain constant region consisting of the amino acid sequence of SEQ ID NO:

29.

17. The pharmaceutical composition according to any one of claims 1 to 16, further comprising at least one pharmaceutically acceptable carrier.

18. A preventive, symptom progression inhibitor, recurrence inhibitor and / or therapeutic agent for autoimmune diseases, comprising the pharmaceutical composition according to any one of Claims 1 to 17.

19. The autoimmune diseases are Behçet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa, aortic inflammation syndrome, rheumatoid arthritis, arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, large vessel vasculitis, ANCA-associated vasculitis, Cogan's syndrome, RS3PE syndrome, temporal arteritis, rheumatoid polymyalgia, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG 4 -related diseases, Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic fatty hepatitis, primary biliary cirrhosis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease, Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, Addison's disease, idiopathic Addison's disease, type I diabetes, slowly progressive type I diabetes, localized scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, gestational herpes, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo, vulgar vitiligo, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease, celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, transplantation immunity, familial Mediterranean fever, eosinophilic rhinosinusitis, dilated cardiomyopathy, systemic mastocytosis or inclusion body myositis, the agent according to claim 18.

20. The agent according to Claim 18 or 19, which is administered together with any one or more agents selected from insulin preparations, sulfonylurea agents, rapid-acting insulin secretagogues, biguanide preparations, insulin resistance improvers, α-glucosidase inhibitors, therapeutic agents for diabetic neuropathy, GLP-1 analog preparations, DPP-4 inhibitors, steroid drugs, interferon β-1a, interferon β-1b, glatiramer acetate, mitoxantrone, azathioprine, cyclophosphamide, cyclosporine, methotrexate, cladribine, adrenocorticotropic hormone (ACTH), corticotropin, mizoribine, tacrolimus, fingolimod, alemtuzumab, immunosuppressive agents, belimumab, anti-rheumatic drugs, anti-cytokine drugs and abatacept.

21. A preventive, symptom progression inhibitor, recurrence inhibitor and / or therapeutic agent for graft-versus-host disease (GVHD), comprising the pharmaceutical composition according to any one of Claims 1 to 17.

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