Bispecific antibody

Abstract

To provide a new drug for preventing, inhibiting symptom progress of, inhibiting recurrence of, or treating autoimmune diseases.SOLUTION: It is found that a PD-1 / CD19 bispecific antibody or the like, as a substance which can solve the problem to be solved, may certainly be a new drug for preventing, inhibiting symptom progress of, inhibiting recurrence of, or treating autoimmune diseases. Also, the bispecific antibody certainly permits interaction between PD-1 and its ligand PD-L1.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to 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."), a pharmaceutical composition containing the same as an active ingredient, and their pharmaceutical therapeutic uses.

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 prophylactic 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 found a PD-1 / CD19 bispecific antibody that is easy to separate 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 wherein in any one or more of 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 be substituted with other amino acids, and / or in any one or more of 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 be substituted with other amino acids; the PD-1 / CD19 bispecific antibody or an antibody fragment thereof. [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 item [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 its antibody fragment described in the preceding item [1] or [2]. [4] (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) (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, (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 - 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 VH described in the preceding item [1] or [2], the PD - 1 / CD19 bispecific antibody or an antibody fragment thereof. [5] (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: 12, (b) VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and (c) VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 14, and (ii) The VH of the second arm that specifically binds to CD19 has (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 - 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 - 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) 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 group consisting of the PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to the preceding item [1] or [2]. [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) 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 VH of the PD - 1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1] or [2]. [7] (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 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) 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, any one selected from the VH described in the preceding [1] or [2], the PD - 1 / CD19 bispecific antibody or its antibody fragment. [8] (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: 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 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 described in the preceding [1] or [2]. [9] (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: 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 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) The PD-1 / CD19 bispecific antibody or antibody fragment thereof 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 antibody fragment thereof 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 antibody fragment thereof 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 an antibody fragment thereof according to the preceding [1] or [2].

[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 an antibody fragment thereof according to the preceding [1] or [2].

[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) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and (c) A PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to item [1] or [2] above, having a 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) A VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and (c) Having a 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) 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 PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to item [1] or [2] above, having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 37.

[19] (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: 12, (b) A VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and (c) Having a 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) 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 PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to item [1] or [2] above, having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 40.

[20] (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: 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 antibody fragment thereof according to item [1] or [2] above, having 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) having 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) having 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 antibody fragment thereof according to item [1] or [2] above.

[24] (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: 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) having 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 antibody fragment thereof according to item [1] or [2] above.

[25] (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: 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) 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 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) 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) 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) 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 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) 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) 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) 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 item [1] or [2] above, having 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) having 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) having 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 antibody fragment thereof according to item [1] or [2] above.

[29] (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: 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) having 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 antibody fragment thereof according to item [1] or [2] above.

[30] (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: 18, (b) 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 an 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 an 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 [1] or [2], which has a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49.

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

[32] .

[34] The framework 4 (hereinafter, "framework 4" may be abbreviated as FR4) region 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 that has undergone somatic mutations thereof (however, excluding the amino acid sequence included in the VH-CDR3 region). The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding

[33] .

[35] The FR region of VH of the first arm that specifically binds to PD-1 is encoded by the gene that may have undergone somatic mutations of the germline V gene IGHV7-4-1, and due to the somatic mutations, 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, respectively, 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

[33] or

[34] .

[36] The FR regions 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 undergone somatic mutations, and due to such 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 the FR2 region, and 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 regions 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 undergone somatic mutations, and due to such 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 the FR3 region, and 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 region 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 the lysine (Lys) in the amino acid sequence (Trp-Gly-Lys-Gly-Thr-Thr*-Val-Thr-Val-Ser-Ser) (SEQ ID NO: 61) of the FR4 region is replaced by glutamine or asparagine, and / or the threonine (* marked) is replaced by leucine or may be replaced, and 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 FR regions of the VH of the second arm that specifically binds to CD19, the FR1, FR2, and FR3 regions respectively correspond to the amino acid sequences encoded by the germline V gene IGHV5-51 or the gene that has undergone somatic mutations thereof, and 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 region 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 the FR1 region that may be replaced or replaced by any combination of them, the PD-1 / CD19 bispecific antibody or antibody fragment thereof described in the preceding item

[39] .

[41] The FR region of the VH of the second arm that specifically binds to CD19 consists of the FR2 region 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 region 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 the FR3 region that may be replaced or replaced by any combination of them, 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 is the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[43] .

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

[44] .

[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, in which the glutamine at position 114 is substituted with arginine, or an amino acid sequence that may be substituted, and is the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[45] .

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

[45] .

[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 is 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 antibody fragment thereof 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 antibody fragment thereof 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 antibody fragment thereof 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 antibody fragment thereof 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 antibody fragment thereof 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 antibody fragment thereof 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 antibody fragment thereof 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 antibody fragment thereof 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 antibody fragment thereof 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 antibody fragment thereof 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 antibody fragment thereof described in the preceding item [1], [2] or

[15] .

[63] 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: 33 or SEQ ID NO: 65. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1], [2], or

[16] .

[64] 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: 34 or SEQ ID NO: 66. The PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item [1], [2], or

[17] .

[65] 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 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

[18] .

[66] 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 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

[19] .

[67] 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 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

[20] .

[68] 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 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

[21] .

[69] 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 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

[22] .

[70] 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 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

[23] .

[71] 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 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

[24] .

[72] 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 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

[25] .

[73] 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 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

[26] .

[74] 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 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

[27] .

[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 its antibody fragment 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 its antibody fragment 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 its antibody fragment 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 its antibody fragment 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 its antibody fragment 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) A first arm specifically binding to PD-1, having 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) A PD-1 / CD19 bispecific antibody or an antibody fragment thereof, including a second arm specifically binding to CD19, having 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.

[83] 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, and specifically binds to PD-1 and CD19 respectively, wherein the first arm that specifically binds to PD-1 is (1) 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 the binding of the first arm that specifically binds to PD-1 to PD-1 or (2) the binding of the variable region of a monoclonal antibody that specifically binds to PD-1 consisting of the VH and VL to PD-1 cross-competes, and the PD-1 / CD19 bispecific antibody or an antibody fragment thereof.

[84] 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, and specifically binds to PD-1 and CD19 respectively, wherein the binding of the first arm that specifically binds to PD-1 to PD-1 is cross-competed by (1) a first arm that specifically binds to PD-1 consisting of 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 or (2) the variable region of a monoclonal antibody that specifically binds to PD-1 consisting of the VH and VL, and the PD-1 / CD19 bispecific antibody or an antibody fragment thereof.

[85] Further, the second arm that specifically binds to CD19 is (1) 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, and the binding of the second arm that specifically binds to CD19 to CD19 or (2) the binding of the variable region of a monoclonal antibody that specifically binds to CD19 consisting of the VH and VL to CD19 cross-competes, and the PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to the preceding item

[83] or

[84] .

[86] Furthermore, the binding of the PD-1 / CD19 bispecific antibody or its antibody fragment described in the preceding item

[83] or

[84] to CD19 by the second arm that specifically binds to CD19 is cross-competed by (1) a second arm that specifically binds to CD19 and 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, or (2) the variable region of a monoclonal antibody that specifically binds to CD19 and consists of the VH and VL.

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

[83] to

[89] , having a VL having a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28.

[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 antibody fragment thereof according to any one of the preceding items

[83] to

[89] .

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

[91] , which is an IgG antibody.

[93] The IgG antibody described in the preceding paragraph

[92] is an IgG 1 antibody or an IgG 4 antibody, and the PD-1 / CD19 bispecific antibody or an antibody fragment thereof described in the preceding paragraph

[92] .

[94] The IgG antibody described in the preceding paragraph

[92] is an IgG 1 antibody, and the PD-1 / CD19 bispecific antibody or an antibody fragment thereof described in the preceding paragraph

[92] .

[95] The IgG antibody described in the preceding paragraph

[92] is an IgG 4 antibody, and the PD-1 / CD19 bispecific antibody or an antibody fragment thereof described in the preceding paragraph

[92] .

[96] In the IgG 1 antibody described in the preceding paragraph

[94] , the 235th leucine in the EU numbering system in the two heavy chain constant regions is each replaced with glycine, and / or the 236th glycine is each replaced with arginine, and the PD-1 / CD19 bispecific antibody or an antibody fragment thereof described in the preceding paragraph

[94] .

[97] In the constant region of the heavy chain having VH of the first arm that specifically binds to PD-1, the 351st leucine and the 366th threonine according to the EU numbering system are both replaced with lysine, and in the constant region of the heavy chain having VH of the second arm that specifically binds to CD19, the 351st leucine is replaced with aspartic acid, and the 368th leucine is replaced with glutamic acid, and the PD-1 / CD19 bispecific antibody or an antibody fragment thereof described in the preceding paragraph

[94] or

[96] .

[98] In the constant region of the heavy chain having VH of the first arm that specifically binds to PD-1, the 351st leucine is replaced with aspartic acid, and the 368th leucine is replaced with glutamic acid, and in the constant region of the heavy chain having VH of the second arm that specifically binds to CD19, the 351st leucine and the 366th threonine are both replaced with lysine, and the PD-1 / CD19 bispecific antibody or an antibody fragment thereof described in the preceding paragraph

[94] or

[96] .

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

[94] and

[96] to

[98] , in which the 447th lysine according to the EU numbering system in the two heavy chain constant regions of the antibody is absent.

[0100] The IgG according to the preceding item

[95] 4 The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding item

[95] , in which the 228th serine according to the EU numbering system in the two heavy chain constant regions of the antibody is each replaced by proline.

[0101] The heavy chain having the VH of the second arm that specifically binds to CD19 has, at its C-terminus, further 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). The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items

[94] and

[96] to

[99] .

[0102] The heavy chain having the VH of the first arm that specifically binds to PD-1 includes a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23. 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] .

[0103] The heavy chain having the VH of the second arm that specifically binds to CD19 includes 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. 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] .

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

[0103] , 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.

[0105] 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, and specifically binds to PD-1 and CD19 respectively, (A) The heavy chain having a VH of the first arm that specifically binds to PD-1 comprises 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 heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23, (B) The 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) The 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, 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 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, and the PD-1 / CD19 bispecific antibody or an antibody fragment thereof.

[0106] A PD-1 / CD19 bispecific antibody or an 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 the interaction between PD-1 and PD-L1.

[0107] The isoelectric point of the complex consisting of the heavy and light chains of the first arm that specifically binds to PD-1 is from about 7.4 to about 7.7 (preferably from about 7.5 to about 7.6), and the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0106] .

[0108] The isoelectric point of the complex consisting of the heavy and light chains of the second arm that specifically binds to CD19 is from about 8.3 to about 8.8 (preferably from about 8.4 to about 8.6), and the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0107] .

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

[0108] , wherein cytokine production in blood or tissue is sufficiently reduced during or within 24 hours after administration.

[0110] The first arm that specifically binds to PD-1 permits the interaction between PD-1 and PD-L1, and the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to

[0108] , wherein cytokine production is sufficiently reduced.

[0111] The cytokine is at least IL-2, IFN-γ or TNF-α, and the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding item

[0109] or

[0110] .

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

[0111] , which suppresses B cell activation by binding to PD-1 and CD19 expressed on the same B cell, respectively.

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

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

[0115] The PD-1 / CD19 bispecific antibody or an 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 an 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 an 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 item [1-1], further comprising at least one pharmaceutically acceptable carrier.

[0012] [2-1] An agent for preventing, suppressing symptom progression, 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] The autoimmune disease is 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), Takayasu arteritis, rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous angiitis, hypersensitivity vasculitis, 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 antibody syndrome, eosinophilic fasciitis, IgG 4Agents according to item [2-1] above, which are related diseases (such as primary sclerosing cholangitis, autoimmune pancreatitis, etc.), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic steatohepatitis, 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 adrenocortical 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 (such as ulcerative colitis, Crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, transplantation immunity, familial Mediterranean fever, eosinophilic rhinitis, dilated cardiomyopathy, systemic mastocytosis or inclusion body myositis. [2-3] Agents for preventing, suppressing the progression of symptoms, suppressing recurrence and / or treating graft-versus-host disease (GVHD), which contain as an active ingredient a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of items [1] to

[0117] above. [2-4] 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.), therapeutic agents for diabetic neuropathy (e.g., epalrestat, mexiletine, imidapril, etc.), GLP-1 analog preparations (e.g., liraglutide, exenatide, lixisenatide, etc.) and DPP-4 inhibitors (e.g., sitagliptin, vildagliptin, alogliptin, etc.), and is administered together with any one or more agents selected therefrom, and a prophylactic, symptom progression-inhibiting, recurrence-inhibiting and / or therapeutic agent for type I diabetes, which contains, 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] . [2-5] It is characterized by being administered together with any one or more drugs selected from steroid drugs (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, 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 contains, 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] , a preventive, symptom progression inhibitor, relapse inhibitor, and / or therapeutic agent for multiple sclerosis. [2-6] It is characterized by being administered together with any one or more drugs selected from steroid drugs (for example, the steroid drugs described in the preceding item [2-5]), immunosuppressive agents (for example, cyclosporine, tacrolimus, fingolimod, etc.), and belimumab, and contains, 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] , a preventive, symptom progression inhibitor, relapse inhibitor, and / or therapeutic agent for systemic lupus erythematosus. [2-7] It is characterized by being administered together with any one or more drugs selected from steroid drugs (for example, the steroid drugs described in the preceding item [2-5]), anti-rheumatic drugs (for example, methotrexate, sulfasalazine, bucillamine, leflunomide, mizoribine, tacrolimus, etc.), anti-cytokine drugs (for example, infliximab, adalimumab, tocilizumab, etanercept, golimumab, certolizumab), and abatacept, and contains a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[0117] as an active ingredient, a preventive, symptom progression inhibitor, recurrence inhibitor, and / or therapeutic agent for rheumatoid arthritis. [2-8] It is characterized by being administered together with any one or more drugs listed in the preceding items [2-4] to [2-7], and contains a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[0117] as an active ingredient, a preventive, symptom progression inhibitor, recurrence inhibitor, and / or therapeutic agent for autoimmune diseases. [2-9] It is characterized by being administered to a patient who has been administered any one or more drugs listed in the preceding items [2-4] to [2-7], and is a preventive, symptom progression inhibitor, recurrence inhibitor, and / or therapeutic agent for each of the diseases described in the preceding items [2-4] to [2-8]. [2-10] It is characterized by being administered after the administration of any one or more drugs listed in the preceding items [2-4] to [2-7], and is a preventive, symptom progression inhibitor, recurrence inhibitor, and / or therapeutic agent for each of the diseases described in the preceding items [2-4] to [2-8]. [2-11] It is characterized by being administered before the administration of any one or more drugs listed in the preceding items [2-4] to [2-7], and is a preventive, symptom progression inhibitor, recurrence inhibitor, and / or therapeutic agent for each of the diseases described in the preceding items [2-4] to [2-8].

[0013] [3-1] An intravenous injection preparation containing a PD-1 / CD19 bispecific antibody or an 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 injection preparation described in the preceding item [3-1] for the prevention, symptom progression inhibition, recurrence inhibition, and / or treatment of autoimmune diseases. [3-3] An intravenous injection preparation according to the previous item [3-1] or [3-2], which is for intravenous drip. [4-1] An isolated polynucleotide or a fragment thereof encoding a heavy chain having a VH of a second arm that specifically binds to CD19, which constitutes a PD-1 / CD19 bispecific antibody selected from any one of the previous items [1] to

[0117] . [4-2] An isolated polynucleotide or a fragment thereof encoding a VH of a second arm that specifically binds to CD19, which constitutes a PD-1 / CD19 bispecific antibody selected from any one of the previous items [1] to

[0117] . [4-3] The isolated polynucleotide or a fragment thereof according to the previous item [4-1] or [4-2], wherein the VH of the second arm that specifically binds to CD19 is encoded by a polynucleotide consisting of a nucleotide sequence selected from SEQ ID NOs: 56 to 60 and SEQ ID NOs: 76 to 80. [4-4] An isolated polynucleotide or a fragment thereof containing a polynucleotide consisting of a nucleotide sequence selected from SEQ ID NOs: 56 to 60 and SEQ ID NOs: 76 to 80. [5-1] An expression vector containing the polynucleotide according to any one of the previous items [4-1] to [4-4]. [6-1] An animal cell into which the expression vector according to the previous item [5-1] has been introduced or which has been transformed by the introduction of the vector. [7-1] A method for preventing, suppressing symptom progression, suppressing recurrence and / or treating an autoimmune disease, which comprises administering an effective amount of a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the previous items [1] to

[0117] to a patient. [8-1] A PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the previous items [1] to

[0117] , which is used for preventing, suppressing symptom progression, suppressing recurrence and / or treating an autoimmune disease. [9-1] Use of a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of the previous items [1] to

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

[0014] [10-1] An isolated anti-CD19 monoclonal antibody or an antibody fragment thereof that cross-competes with the binding of an antibody specifically binding to CD19 and having 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 to CD19. [10-2] An isolated anti-CD19 monoclonal antibody or an antibody fragment thereof, wherein the binding of the antibody or the antibody fragment to CD19 is cross-competed by an antibody specifically binding to CD19 and having 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. [10-3] (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) 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) Any one VH selected from VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, (a) A VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, (b) A VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 27 and (c) A VL having a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28, an isolated anti-CD19 monoclonal antibody or an antibody fragment thereof. [10-4] (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) Any one VH selected from VH having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, having (a) VL 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) VL having VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28, wherein any one or more CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3 in the VH may have any 1 to 5 amino acid residues thereof substituted with other amino acids. An isolated anti-CD19 monoclonal antibody or an antibody fragment thereof. [10-5] 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, and SEQ ID NO: 34, or VH consisting of an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the VH, and VL consisting of the amino acid sequence of SEQ ID NO: 25. The isolated anti-CD19 monoclonal antibody or an antibody fragment thereof according to any one of the preceding items [10-1] to [10-4]. [10-6] VH consisting of an amino acid sequence in which the 114th glutamine in 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 is substituted with arginine, or an amino acid sequence that may be substituted, and VL consisting of the amino acid sequence of SEQ ID NO: 25. The isolated anti-CD19 monoclonal antibody or an antibody fragment thereof according to any one of the preceding items [10-1] to [10-4]. [10-7] 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 VL consisting of the amino acid sequence of SEQ ID NO: 25. The isolated anti-CD19 monoclonal antibody or an antibody fragment thereof according to any one of the preceding items [10-1] to [10-4]. [10-8] The CD19 is human CD19. The isolated anti-CD19 monoclonal antibody or an antibody fragment thereof according to any one of the preceding items [10-1] to [10-7]. [10-9] The anti-CD19 antibody is the isolated CD19 monoclonal antibody or antibody fragment thereof according to any one of the preceding items [10-1] to [10-8], which is an IgG antibody. [10-10] The IgG antibody according to the preceding item [10-9] is an IgG 1 antibody or an IgG 4 antibody, which is the isolated anti-CD19 monoclonal antibody or antibody fragment thereof according to the preceding item [10-9]. [10-11] The IgG antibody according to the preceding item [10-9] is an IgG 1 antibody, which is the isolated anti-CD19 monoclonal antibody or antibody fragment thereof according to the preceding item [10-9]. [10-12] The IgG antibody according to the preceding item [10-9] is an IgG 4 antibody, which is the isolated anti-CD19 monoclonal antibody or antibody fragment thereof according to the preceding item [10-9]. [10-13] (1) The isolated anti-CD19 monoclonal antibody or antibody fragment thereof according to any one of the preceding items [10-1] to [10-11], wherein the 351st leucine in the heavy chain constant region is substituted with aspartic acid and the 368th leucine is substituted with glutamic acid, or (2) the 351st leucine and the 366th threonine in the heavy chain constant region are both substituted with lysine. [10-14] The isolated anti-CD19 monoclonal antibody or antibody fragment thereof according to any one of the preceding items [10-1] to [10-13], further having 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 the C-terminus of the heavy chain. [10-15] The isolated anti-CD19 monoclonal antibody or antibody fragment thereof according to any one of the preceding items [10-1] to [10-14], wherein the heavy chain 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. A prophylactic, 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] of the preceding paragraph. [11-2] The prophylactic, symptom progression inhibitor, recurrence inhibitor and / or therapeutic agent according to the preceding paragraph [11-1], wherein the autoreactive B cell-mediated disease is systemic lupus erythematosus, Graves' disease, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosis or pernicious anemia. [12-1] 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] of the preceding paragraph. [12-2] The autoreactive B cell inhibitor according to the preceding paragraph [12-1], wherein the autoreactive B cell inhibition is immunoglobulin production inhibition. [12-3] The autoreactive B cell inhibitor according to the preceding paragraph [12-2], wherein the immunoglobulin is IgG or IgM. [13-1] 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] of the preceding paragraph. [13-2] The T cell activation inhibitor according to the preceding paragraph [13-1], wherein the T cell activation inhibition is cytokine production inhibition. [13-3] The T cell activation inhibitor according to the preceding paragraph [13-1] or [13-2], wherein the T cell activation inhibition is memory T cell inhibition. [14-1] A method for inhibiting autoreactive B cells or inhibiting T cell activation, comprising administering to a patient an effective amount of a PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of items [1] to

[0117] of the preceding paragraph. [15-1] A PD-1 / CD19 bispecific antibody or an antibody fragment thereof selected from any one of items [1] to

[0117] of the preceding paragraph, which is used for inhibiting autoreactive B cells or inhibiting T cell activation. 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 production of an autoreactive B cell inhibitor or a T cell activation inhibitor.

Advantages of the Invention

[0015] 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 the progression of symptoms, suppress recurrence, and / or enhance or sustain the therapeutic effect.

Brief Description of the Drawings

[0016]

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[0017] PD-1( P rogrammed Cell D eath -1 ) is a membrane protein consisting of the amino acid sequence represented by GenBank accession number NP_005009 in humans. When described as "PD-1" in this specification, unless otherwise specifically limited, 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 as included. In the present invention, PD-1 is preferably human PD-1.

[0018] ​CD19 is a membrane protein expressed on B cells, consisting of the amino acid sequence represented by GenBank accession number NP_001171569 or NP_001761 in humans, 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, it may be used as including those variants in which the epitope of the "second arm that specifically binds to CD19" according to the present invention is conserved. In the present invention, CD19 is preferably human CD19.

[0019] As used herein, "isolated" means becoming a substantially single pure component by being identified, separated and / or purified from contaminants containing a plurality or innumerable components extracted from a host cell.

[0020] As used herein, "monoclonal antibody" means an antibody obtained from a substantially homogeneous population of antibodies that bind to the same specific antigen.

[0021] As used herein, "bispecific antibody" means an antibody having binding specificities for two different antigen molecules or epitopes in one molecule, and "bispecific monoclonal antibody" means a bispecific antibody further obtained from a substantially homogeneous population of antibodies.

[0022] The present invention relates to a bispecific antibody that can specifically bind to PD-1 and CD19 respectively (which may be abbreviated as "PD-1 / CD19 bispecific antibody" in this specification). 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, "isolated human PD-1 / human CD19 bispecific monoclonal antibody" means an isolated bispecific monoclonal antibody against human PD-1 and human CD19.

[0023] Here, the forms of bispecific antibodies include, for example, diabodies, bispecific sc(Fv) 2 , bispecific minibodies, bispecific F(ab′) 2 , bispecific hybrid antibodies, covalently linked diabodies (bispecific DART), bispecific (FvCys) 2 , bispecific F(ab′-zipper) 2 , bispecific (Fv-zipper) 2 , bispecific trispecific antibodies and bispecific mAb 2 and the like.

[0024] A diabody is a dimer of single-chain peptides in which VHs and VLs that recognize different antigens are linked by a peptide linker (see Proc. Natl. Acad. Sci. USA (1993), Vol. 90, No.14: p.6444-6448).

[0025] Bispecific sc(Fv) 2 is a miniaturized antibody modified to be produced in a single-chain form in which two sets of VHs / VLs of two antibodies that recognize different antigens are continuous via a peptide linker (see J. Biological Chemistry (1994), Vol. 269: p.199-206).

[0026] Bispecific F(ab′) 2 is a miniaturized antibody in which Fab′ fragments of antibodies that recognize two different antigens are covalently linked by a disulfide bond or the like.

[0027] A bispecific minibody is a miniaturized antibody in which a small antibody fragment modified such that the CH3 domain of the constant region of an antibody is linked to an scFv that recognizes a different antigen is covalently linked by a disulfide bond or the like on the CH3 domain (see Biochemistry (1992), Vo.31, No.6, p.1579-1584).

[0028] 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 disulfide bonds or the like.

[0029] In the present invention, a preferred form of the bispecific antibody is a bispecific hybrid antibody.

[0030] Bispecific hybrid antibodies can be produced, for example, from hybridomas prepared by the hybrid hybridoma method (see US4474893). Also, four types of cDNAs encoding the heavy and light chains of antibodies that recognize different antigens can be co-expressed and secreted in mammalian cells for production.

[0031] The monoclonal antibodies used in the present invention can be prepared by the hybridoma method (see, for example, Kohler and Milstein et al., Natur (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)), the recombinant DNA method (see, for example, US4816567), and the 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.).

[0032] When an antibody or monoclonal antibody is administered to a human, it can be produced in the form of a chimeric antibody, humanized antibody, or fully human antibody in order to reduce or eliminate its antigenicity.

[0033] "Chimeric antibody" means an antibody in which the variable region sequence and the constant region sequence are derived from different mammals. For example, there are those in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. Chimeric antibodies 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 by Cabilly et al.).

[0034] "Humanized antibody" means an antibody in which a complementarity-determining region (CDR) sequence derived from the germline of another mammal such as a mouse is transplanted onto a human framework sequence. In the case of humanized antibodies, a gene encoding the antibody CDR region isolated by a known method from an antibody-producing hybridoma isolated by the above method can be ligated to a gene encoding a human-derived antibody framework region using a known method (see, for example, US5225539 and US5530101 by Winter, US5585089 and US6180370 by Queen et al.).

[0035] "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 prepared by methods using mice transformed to produce human antibodies, such as Humab mice (see, e.g., US5545806, US5569825, US5625126, US5633425, US5789650, US5877397, US5661016, US5814318, US5874299, and US5770429 by Lonberg and Kay et al.), KM mice (see, e.g., WO2002 / 43478 by Ishida et al.), Xeno mice (see, e.g., US5939598, US6075181, US6114598, US6150584, and US6162963), or Tc mice (see, e.g., Tomizuka et al., Proc. Natl. Acad. Sci. USA (2000), p.722-727). Further, it can also be prepared using SCID mice (see, e.g., 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 prepared by the phage display method described above.

[0036] As used herein, an "antibody fragment" of a PD-1 / CD19 bispecific antibody is a part of a full-length antibody and is an antibody having an antigen-binding portion for PD-1 and an antigen-binding portion for CD19, such as F(ab′) 2 and the like. Here, the antigen-binding portion means the minimum unit by which an antibody can bind to its antigen, and is composed of, for example, three CDRs each in VH and VL and the framework region that arranges the CDRs so that the target antigen can be recognized by the combination of these CDRs.

[0037] As used herein, the "common light chain" refers to a light chain that associates with two or more different heavy chains and can exhibit binding ability to each antigen (De Wildt RM et al., J. Mol. Biol. (1999), Vol. 285, p.895-901, De Kruif et al., J. Mol. Biol. (2009), Vol. 387, p.548-58, WO2004 / 009618, WO2009 / 157771 and WO2014 / 051433). Such a common light chain is preferably, for example, a light chain encoded by the germline gene of 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 preferably, for example, the 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. Even more preferably, for example, the light chain having a VL consisting of the amino acid sequence of SEQ ID NO: 25. Further, as the constant region of the common light chain, preferably, a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29 can be mentioned. The amino acid sequences of the VL and the constant region 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.

[0038] As used herein, "isotype" is used to refer to an antibody class (e.g., IgM or IgG) encoded by a heavy chain constant region gene. A preferred isotype as the PD-1 / CD19 bispecific antibody of the present invention is IgG, more preferably IgG 1 or IgG 4 Here, IgG 1 is preferably one in which the binding to the Fc receptor is lost or attenuated. Specifically, IgG in which the binding to the Fc receptor is lost or attenuated by substituting, deleting or inserting any amino acid in the heavy chain constant region 1Antibodies can be obtained. For example, antibodies in which the 235th leucine according to the EU numbering system is replaced with glycine and / or the 236th glycine is replaced with arginine on each of the two heavy chain constant regions or hinge regions of a bispecific antibody can be mentioned. Further, in order to reduce the heterogeneity of the antibody, an antibody lacking the C-terminal amino acid, for example, the 447th lysine according to the EU numbering system, is preferred. Furthermore, when the bispecific antibody is IgG 4 In the case of, a variant in which any amino acid in its heavy chain constant region is substituted, deleted or inserted so as to suppress swapping within the antibody molecule is more preferred. For example, an antibody in which the 228th serine according to the EU numbering system located in the hinge region is replaced with proline is preferred. In the present specification, the amino acid positions assigned to the CDR and framework of the variable region of the antibody 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))). Also, the amino acids in the constant region are represented according to the EU numbering system corresponding to the Kabat amino acid positions (see Sequences of proteins of immunological interest, NIH Publication No. 91-3242).

[0039] In the Fc region of the PD-1 / CD19 bispecific antibody of the present invention, any amino acid in the region may be substituted so that two different heavy chains are likely to associate. As a preferred embodiment, for example, the 351st leucine according to the EU numbering system of the constant region on 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, and 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, and a PD-1 / CD19 bispecific antibody is exemplified. Also, the 351st leucine according to the EU numbering system of the constant region on the heavy chain having the VH of the first arm that specifically binds to PD-1 is substituted with aspartic acid, and the 368th leucine is substituted with glutamic acid, and 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 lysine, and the 366th threonine is substituted with lysine, and a PD-1 / CD19 bispecific antibody is also exemplified.

[0040] The first arm that specifically binds to PD-1 As used herein, the "first arm that specifically binds to PD-1" (hereinafter sometimes abbreviated as "first arm") means an antibody or a part of an antibody fragment, or whether it exists as a single entity by itself rather than a part, at least includes the VH of an antibody that specifically binds to PD-1 and can specifically bind to PD-1. For example, such a first arm consists of the VH of an anti-PD-1 antibody and the VL of the common light chain constituting the anti-PD-1 antibody. Furthermore, the first arm also includes the Fab portion of the antibody containing the VH and VL. Here, "specifically binds to PD-1" means at least 1x10 -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 substantially does not bind to other receptor members belonging to the so-called CD28 family receptors such as CD28, CTLA-4, and ICOS. In addition, the "antibody" in the "antibody that specifically binds 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.

[0041] Here, as the "first arm that specifically binds 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 a 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 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.], and those having VH having VH-CDR3 are exemplified.

[0042] 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.

[0043] 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 having a binding activity substantially equivalent to the binding activity of the original first arm that has not been substituted with such amino acids. For example, in the case of VH-CDR1, 1 amino acid residue is substituted with other amino acids (preferably, their conservative amino acids), and in the case of VH-CDR2 or VH-CDR3, 1 to 5 amino acid residues each are substituted with other amino acids (preferably, their conservative amino acids). Also, as shown in FIG. 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 are mutually substitutable between the clones. Here, substitution by 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 preferred substitutions by conservative amino acids include substitutions between valine, leucine and isoleucine, substitutions between phenylalanine and tyrosine, substitutions between lysine and arginine, substitutions between alanine and valine, and substitutions between asparagine and glutamine.Here, "having a binding activity substantially equivalent to the binding activity of the original first arm that is 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, and more preferably 99% or more of the binding activity of the original first arm that is not substituted with the amino acid.

[0044] Furthermore, the "first arm that specifically binds to PD-1" in the present invention includes each VH-CDR having the specific amino acid sequence described above in its VH, and also includes those having an amino acid sequence of the framework of the VH encoded by a specific germline gene or a gene that has undergone somatic mutation of the germline gene. For example, the VH represented by any one selected from the above (1b) to (5b) can be encoded by a VDJ recombinant gene in which the germline V gene is IGHV7-4-1 and the germline J gene is JH6c or a gene that has undergone somatic mutation of the germline gene. Here, the amino acid sequence encoded by the germline V gene IGHV7-4-1 corresponds to the amino acid sequence of SEQ ID NO: 21 (FIG. 3).

[0045] The framework of the VH of the first arm that specifically binds to PD-1 of the present invention may be encoded by a germline-type VDJ recombinant gene that has undergone somatic mutations. 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 Figure 4, since the amino acid sequences encoded by the IGHV7-4-1 gene are different, somatic mutations have occurred at these respective positions. For example, for the FR1 region, 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 each substituted with glutamine, valine, or methionine, respectively, or substituted with any plurality of combinations thereof. For the FR2 region, valine at position 37 in the amino acid sequence of SEQ ID NO: 21 may be substituted with leucine. For the FR3 region, serine at position 77, cysteine at position 84 in the amino acid sequence of SEQ ID NO: 21 may be each substituted with threonine, serine, or asparagine, respectively, or substituted with any plurality of combinations. Also, for the FR4 region of VH shown by any one selected from the above (1b) to (5b), lysine (Lys) in the FR4 region amino acid sequence (Trp-Gly-Lys-Gly-Thr-Thr*-Val-Thr-Val-Ser-Ser) (SEQ ID NO: 41) 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 of the above amino acid substitution combinations may also be used as a framework without substantially affecting the function of the first arm that specifically binds to PD-1.

[0046] Furthermore, the "first arm that specifically binds to PD-1" in the present invention includes each CDR having the above-described specific amino acid sequence, 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 mutations thereof. For example, as such a first arm, those having a VH consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 5 may be mentioned.

[0047] 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 an amino acid sequence whose 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 amino acid sequences that are identical to the two aligned sequences, with 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.

[0048] In yet another aspect, the "first arm that specifically binds to PD-1" in the present invention includes: (1) the binding of a first arm having a VH represented by any one selected from the above (1b) to (5b) 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) to PD-1, or (2) the variable region of an anti-PD-1 antibody that cross-competes with the binding of the variable region of a monoclonal antibody specifically binding to PD-1 consisting of the VH and VL to PD-1 (wherein the variable region includes the VH and VL constituting the same). Also included are those in which the binding to PD-1 is (3) a first arm having a VH represented by any one selected from the above (1b) to (5b) 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, or (4) a variable region of an anti-PD-1 antibody that is cross-competed by the variable region of a monoclonal antibody specifically binding to PD-1 consisting of the VH and VL. Here, "cross-competes with the binding to PD-1" means inhibiting the binding of the first arm to PD-1 to any extent by binding to the same or partially overlapping epitope as the first arm exemplified herein, or the binding of an antibody that binds to the same or partially overlapping epitope as the exemplified first arm to PD-1 is inhibited to any extent by the exemplified first arm. Whether cross-competition occurs can be evaluated by a competitive binding assay. For example, it can be determined using BIAcore analysis, ELISA assay, flow cytometry, enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer measurement method (FRET), or fluorescence microplate assay technology (FMAT (registered trademark)).

[0049] 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), and further, the 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.

[0050] 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), and further, the 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.

[0051] Here, the "first arm that specifically binds to PD-1" in the present invention preferably includes a first arm having a VH represented by the above (1b) to (5b). Further, as described above, in each CDR of the VH of this preferred first arm, any 1 to 5 amino acid residues thereof are substituted with other amino acids (preferably, conservative amino acids thereof), and those in which the amino acid substitution does not substantially affect the binding activity to PD-1 are also included. Further, as described above, those having an amino acid sequence of the framework of VH encoded by the germline type V gene IGHV7-4-1 or J gene JH6c or genes thereof having somatic mutations thereof are also included. And more preferably, the first arm includes one having a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 1 to 5.

[0052] Furthermore, the "first arm that specifically binds to PD-1" in the present invention preferably includes a VL of the common light chain in the present specification. Such a common light chain is preferably, for example, an IGVK1-39 / JK1 common light chain. More preferably, for example, it is a light chain having a VL including 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. Even more preferably, for example, it is a light chain having a VL consisting of the amino acid sequence of SEQ ID NO: 25. Further, 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.

[0053] Also, the "first arm that specifically binds to PD-1" more preferably allows the interaction between PD-1 and PD-L1, the interaction between PD-1 and PD-L2, or both of these interactions. Here, "allows 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 situation where the PD-1 / CD19 bispecific antibody of the present invention is present in 20-fold excess of the concentration 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, more preferably 80% or more compared to the interaction when the PD-1 / CD19 bispecific antibody of the present invention is not present. Further, the definition of "allows the interaction between PD-1 and PD-L1, the interaction between PD-1 and PD-L2, or both of these interactions" may be used synonymously with the meaning of "does not substantially inhibit the interaction between PD-1 and PD-L1, the interaction between PD-1 and PD-L2, or both of these interactions".

[0054] The correspondence between each clone of the anti-PD-1 monoclonal antibody obtained for constructing the PD-1 / CD19 bispecific antibody of the present invention and the amino acid sequence of its VH and its SEQ ID NO. is shown in FIG. 6, and the correspondence between the amino acid sequence of each CDR in the VH of each clone of the anti-PD-1 monoclonal antibody and its SEQ ID NO. is shown in FIG. 7.

[0055] 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 as a single entity rather than a part, and at least includes the VH of an antibody that specifically binds to CD19 and can specifically bind to CD19. An antibody moiety, for example, such a second arm consists of the VH of an anti-CD19 antibody and the VL of the common light chain constituting the anti-CD19 antibody. Furthermore, 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.

[0056] Here, examples of the "second arm that specifically binds to CD19" include, for example, (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 are exemplified.

[0057] 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.

[0058] 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, 1 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 or any plurality of combinations thereof that differ between the clones 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 the "substitution with conservative amino acids" in each VH-CDR of the second arm includes, for example, the examples of amino acid substitution in the first arm described above.

[0059] Furthermore, in the present invention, the "second arm that specifically binds to CD19" includes, in its VH, each CDR having the specific amino acid sequence described above, and those in which the amino acid sequence of the FR of the VH has a VH encoded by a specific germline gene or a somatic mutation of the gene are also included. For example, any one of the VHs selected from the above (1d) to (5d) may be encoded by a VDJ recombinant gene whose germline V gene is IGHV5-51 or a somatic mutation of the gene. Here, the amino acid sequence encoded by the germline V gene IGHV5-51 corresponds to the amino acid sequence represented by SEQ ID NO: 22 (FIG. 3).

[0060] The framework of the VH of the second arm that specifically binds to CD19 of the present invention may be encoded by a germline VDJ recombinant gene that has undergone somatic mutation. For example, the FR1 and FR3 of the VH shown by any one selected from the above (1d) to (5d), in which the germline V gene is IGHV5-51, differ from the amino acid sequence encoded by the IGHV5-51 gene at the amino acid positions shown in FIG. 5, and thus have undergone somatic mutation at each of these positions. For example, for the FR1 region, the glutamic acid at position 1, proline at position 14, tyrosine at position 27, or threonine at position 30 in the amino acid sequence of SEQ ID NO: 22 may each be substituted with glutamine, serine, phenylalanine, or isoleucine, respectively, or any plurality of combinations thereof. For the FR3 region, the isoleucine at position 76, serine at position 77, threonine at position 78, serine at position 84, methionine at position 93, or alanine at position 97 in the amino acid sequence of SEQ ID NO: 22 may each be substituted with phenylalanine, threonine or asparagine, valine, asparagine, isoleucine or leucine, or valine, respectively, or any plurality of combinations thereof. Each of 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 that specifically binds to CD19.

[0061] Furthermore, the "second arm specifically binding to CD19" in the present invention includes each CDR having the specific amino acid sequence described above, and those having a VH whose amino acid sequence of the FR of the VH is encoded by a specific germline gene or the gene having a somatic mutation thereof. For example, as such a second arm, those having a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30 to 34 can be mentioned.

[0062] Furthermore, such a "second arm specifically binding to CD19" includes, for example, 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 the difference from the amino acid sequence of the VH of the original second arm does not substantially affect the binding activity to CD19 (hereinafter, may be 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 binding activity to CD19" means that the binding activity of the homologous second arm to CD19 is 95% or more, preferably 98% or more, and more preferably 99% or more of the binding activity of the original second arm.

[0063] 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 a VH represented by any one 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 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 the VH and VL constituting it). Also included are those in which the binding to CD19 is (3) the second arm having a VH represented by any one 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, 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, regardless of the degree, the binding of the second arm to CD19 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, regardless of the degree, by the exemplified second arm. Here, whether cross-competition occurs 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".

[0064] Here, the "second arm specifically binding to CD19" in the present invention preferably includes a second arm having 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, conservative amino acids thereof), and those in which the amino acid substitution does not substantially affect the binding activity to CD19 are also included. Further, as described above, those having an amino acid sequence of the framework of VH encoded by the germline 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 VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30 to 34.

[0065] Furthermore, in another aspect, the "second arm specifically binding 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, wherein 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, wherein the 114th glutamine in each amino acid sequence of SEQ ID NOs: 30 to 34 is substituted with arginine, and they respectively 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 the light chain having the second arm (hereinafter, 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, in particular, 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, and more preferably about 8.4 to about 8.8. Also, the isoelectric point of the complex composed of the heavy chain and the light chain having the first arm (hereinafter, the first arm heavy chain / light chain complex) is preferably about 7.4 to about 7.7, and more preferably about 7.5 to about 7.6.

[0066] Furthermore, the "second arm specifically binding to CD19" in the present invention preferably includes the VL of the common light chain herein, and such a common light chain is preferably, for example, the IGVK1-39 / JK1 common light chain, and more preferably, for example, 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, and even more preferably, for example, a light chain having a VL consisting of the amino acid sequence of SEQ ID NO: 25. 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.

[0067] 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 sequence numbers 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 sequence numbers is shown in FIG. 9.

[0068] On the other hand, 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 containing 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-CDR1, VH-CDR2, and VH-CDR3 in 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 containing 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 PD-1 / CD19 bispecific antibody is exemplified.

[0069] 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) It has 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 a second arm that specifically binds to CD19, (C) a VH represented by any one 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.

[0070] Also, as 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 (A) a VH consisting of any one amino acid sequence 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) has a VL consisting of the amino acid sequence of SEQ ID NO: 25, and a second arm that specifically binds to CD19 (C) a VH consisting of any one amino acid sequence 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.

[0071] More preferably, as this another embodiment, for example, the first arm that specifically binds to PD-1 (A) 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 a second arm that specifically binds to CD19 (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.

[0072] Furthermore, as another aspect and 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) having a VL consisting of the amino acid sequence of SEQ ID NO: 25, and the second arm that specifically binds to CD19 is (C) 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.

[0073] A preferred isotype as the PD-1 / CD19 bispecific antibody of the present invention is an IgG antibody, more preferably an IgG 1 or IgG 4 antibody, and even more preferably an IgG 1 antibody. When the antibody is an IgG 1 antibody, on each of the two heavy chain constant regions or hinge regions, an IgG in which the leucine at position 235 according to the EU numbering system is substituted with glycine, and / or the glycine at position 236 is substituted with arginine 1 antibody is preferred. Furthermore, an antibody lacking the amino acid at the C-terminus of the heavy chain of those bispecific antibodies, for example, lysine at position 447 according to the EU numbering system, is more preferred. Also, when the PD-1 / CD19 bispecific antibody is an IgG 4 antibody, an antibody in which the serine at position 228 according to the EU numbering system located in its hinge region is substituted with proline is preferred.

[0074] Furthermore, when these PD-1 / CD19 bispecific antibodies are IgG 1 antibodies, in a preferred embodiment, 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 replaced with lysine, and the 366th threonine is replaced with lysine, and the 351st leucine in the constant region of the heavy chain having the VH of the second arm that specifically binds to CD19 is replaced with aspartic acid, and the 368th leucine is replaced with glutamic acid in the IgG 1 antibody. Also, in the constant region of the heavy chain having the VH of the first arm that specifically binds to PD-1, the 351st leucine according to the EU numbering system is replaced with aspartic acid, and the 368th leucine is replaced with glutamic acid, and in the constant region of the heavy chain having the VH of the second arm that specifically binds to CD19, the 351st leucine is replaced with lysine, and the 366th threonine is replaced with lysine in the IgG 1 antibody is similarly preferred.

[0075] A preferred embodiment of the PD-1 / CD19 bispecific IgG incorporating all of the above-described amino acid substitutions in the heavy chain constant region 1 antibody is, for example, an antibody in which the heavy chain having the VH of the first arm that specifically binds 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 the VH of the second arm that specifically binds 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. Those amino acid sequences are illustrated in FIG. 10.

[0076] 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, and thereby, similar to the case of amino acid substitution in the VH of the second arm described above, in particular, the separation of the bispecific antibody from the homodimer of the second arm heavy chain / light chain complex becomes easier. Note that 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.

[0077] 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.

[0078] 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".

[0079] 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, for example, that 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.

[0080] 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.

[0081] Method for producing and purifying a PD-1 / CD19 bispecific antibody The PD-1 / CD19 bispecific antibody and its antibody fragments of the present invention can also be produced by the methods disclosed in WO2014 / 051433, WO2013 / 157953 or WO2013 / 157954.

[0082] 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 can be introduced into mammalian cells by gene transfer and transformed to co-express and secrete both the heavy chain and the common light chain for production.

[0083] Here, the host cell expressing the PD-1 / CD19 bispecific antibody of the present invention may be any host cell that can be transfected with an expression vector and express the introduced expression vector. Preferably, insect cells such as SF-9 and SF-21 cells, more preferably, 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, mammalian cells such as embryonic retinal cells like W138, HepG2, HeLa, HEK293, HT1080 or PER.C6. In addition, in the selection of the expression system, mammalian cell expression vectors and hosts may be used so that the antibody is properly glycosylated. Human cell lines, preferably PER.C6, are advantageously used to obtain antibodies that match the glycosylation pattern in humans.

[0084] The production of proteins in host cells transformed by gene transfection of an expression vector can be carried out, for example, with reference to 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 the productivity of host cell culture can be carried out with reference to Mammalian Cell Biotechnology: a Practical Approach (M. Butler, ed., IRL Press, 1991). The expression of antibodies in host cells is described, for example, in published gazettes such as EP0120694, EP0314161, EP0481790, EP0523949, US4816567 and WO2000 / 63403.

[0085] Here, the culture conditions of the host cells 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, roller bottle or reaction tank, by batch culture, fed-batch culture, continuous culture, or culture using hollow fibers. For large-scale and continuous production of recombinant proteins 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.

[0086] Antibodies expressed in host cells and recovered from the cells or cell culture medium by known methods can be purified using known methods. 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 in some cases (see, for example, US4801687 and US5151504).

[0087] 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.

[0088] 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 -9It can directly bind to CD19 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 proteins. Here, the "antibody" in the "monoclonal antibody that specifically binds to CD19" means a full-length antibody, that is, a complete antibody consisting of two heavy chains and two light chains linked by disulfide bonds. In addition, the "fragment of a monoclonal antibody that specifically binds to CD19" is a part of the full-length antibody and is an antibody containing at least an antigen-binding portion. For example, Fab, Fab′, Fv, scFv, and F(ab′) 2 and the like can be mentioned.

[0089] Examples of the anti-CD19 monoclonal antibody of the present invention include any one of the VH selected from the above (1d) to (5d) constituting the VH of the "second arm that specifically binds to CD19" or the VH consisting of any one of the amino acid sequences selected from SEQ ID NOs: 30 to 34 and the VL of the common light chain in the present specification (preferably, the VL consisting of the amino acid sequence of SEQ ID NO: 25).

[0090] Furthermore, the anti-CD19 monoclonal antibodies of the present invention include those having a VH in which any one of the CDRs selected from the above (1d) to (5d) has 1 to 5 amino acid residues each substituted with another amino acid (preferably a conservative amino acid), and having a VH having substantially the same binding activity to CD19 as that of the anti-CD19 monoclonal antibody having the original VH not substituted with the amino acid. For example, in the case of CDR1, one amino acid residue is substituted with another amino acid (preferably a conservative amino acid), and in the case of CDR2 or CDR3, 1 to 5 amino acid residues each are substituted with another amino acid (preferably a conservative amino acid). Also, as shown in FIG. 5, in each CDR of the anti-CD19 monoclonal antibody clone, each amino acid that differs between the clones or any plurality of combinations thereof are mutually substitutable between the clones. Here, "having substantially the same binding activity to CD19 as that of the anti-CD19 monoclonal antibody having the original VH not substituted with the amino acid" means that the binding activity of the anti-PD-1 monoclonal antibody substituted with the amino acid to CD19 is 95% or more, preferably 98% or more, more preferably 99% or more of the binding activity of the anti-PD-1 monoclonal antibody having the original VH not substituted with the amino acid.

[0091] Furthermore, the anti-CD19 monoclonal antibodies of the present invention include those containing each CDR having the specific amino acid sequence in the VH thereof, and having an amino acid sequence of the framework of the VH encoded by a specific germline gene or the gene having a somatic mutation thereof, for example, those having a specific VH encoded by a specific germline gene or the gene having a somatic mutation thereof as described in the above explanation regarding the "second arm specifically binding to CD19".

[0092] 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 the amino acid sequence of the FR of the VH encoded by a specific germline gene or the gene that has undergone somatic mutation thereof 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 to CD19 that is substantially equivalent to the binding activity of the anti-CD19 monoclonal antibody having the original VH. Here, "having a binding activity to CD19 that is substantially equivalent to the binding activity of 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.

[0093] 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, a 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.

[0094] 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 an amino acid sequence which may be substituted, and a VL of the common light chain herein (preferably, a 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 a 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 a VL of the common light chain.

[0095] 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.

[0096] 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 a DNA-RNA hybrid. One to six codons encoding one amino acid are known. For example, for Phe, it is TTT or TTC; for Leu, it is TTA, TTG, CTT, CTC, CTA or CTG; for Ile, it is ATT, ATC or ATA; for Met, it is ATG; for Val, it is GTT, GTC, GTA or GTG; for Ser, it is TCT, TCC, TCA or TCG; for Pro, it is CCT, CCC, CCA or CCG; for Thr, it is ACT, ACC, ACA or ACG; for Ala, it is GCT, GCC, GCA or GCG; for Tyr, it is TAT or TAC; for His, it is CAT or CAC; for Gln, it is CAA or CAG; for Asn, it is AAT or AAC; for Lys, it is AAA or AAG; for Asp, it is GAT or GAC; for Glu, it is GAA or GAG; for Cys, it is TGT or TGC; for Trp, it is TGG; for Arg, it is CGT, CGC, CGA or CGG; for Ser, it is AGT or AGC; for Arg, it is AGA or AGG; and for Gly, it is GGT, GGC, GGA or GGG respectively. 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 nucleotide 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 nucleotide 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. Also, preferably, as the polynucleotide encoding the variable region of the common light chain, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 55 can be mentioned.

[0097] 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).

[0098] Examples of autoimmune diseases that can be prevented, have symptom progression suppressed, and / or be treated by the PD-1 / CD19 bispecific antibody of the present invention and the like include 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), Takayasu arteritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous angiitis, hypersensitivity vasculitis, 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 antibody syndrome, eosinophilic fasciitis, IgG 4Related diseases (e.g., primary sclerosing cholangitis, autoimmune pancreatitis, etc.), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic steatohepatitis, 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 adrenocortical 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, herpes gestationis, 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 (e.g., 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.

[0099] In the present invention, "treatment" means, for example, curing or improving a certain disease or its symptoms, "prevention" means preventing or delaying the onset of a certain disease or symptoms for a certain period, and "symptom progression suppression" means suppressing the progression or deterioration 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.

[0100] In another aspect, the PD-1 / CD19 bispecific antibody of the present invention is useful for preventing, suppressing symptom progression, suppressing recurrence and / or treating 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 such pharmaceutical uses, 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.

[0101] The PD-1 / CD19 bispecific antibody of the present invention is usually administered systemically or locally in a parenteral form. Specific examples of such administration methods include injection administration, nasal administration, pulmonary administration, transdermal administration, and the like. Examples of injection administration include intravenous injection, intramuscular injection, intraperitoneal injection, etc. In the case of intravenous injection, administration by infusion is preferred. The dosage varies depending on age, body weight, symptoms, treatment effect, administration method, treatment time, etc., but is usually in the range of 0.1 μg / kg to 300 mg / kg per adult per administration, particularly preferably in the range of 0.1 mg / kg to 10 mg / kg, and is administered parenterally once to several times a day or continuously intravenously in the range of 30 minutes to 24 hours a day. Of course, as described above, the dosage varies depending on various conditions, so there may be cases where a sufficient amount is less than the above dosage, or there may be cases where administration beyond the range is necessary.

[0102] Formulation When the PD-1 / CD19 bispecific antibody of the present invention is formulated and used as an injection or an infusion for intravenous drip, the injection or the infusion may be in any form of an aqueous solution, a suspension or an emulsion, and may also be formulated as a solid preparation together with a pharmaceutically acceptable carrier so as to be dissolved, suspended or emulsified and used by adding a solvent at the time of use. As the solvent used for the injection or the infusion for intravenous drip, for example, distilled water for injection, physiological saline, glucose solution and isotonic solutions (for example, solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.) can be used.

[0103] Here, examples of pharmaceutically acceptable carriers include stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, antiseptics, pH adjusters, antioxidants, and the like. 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, dibutylhydroxytoluene, and the like. Examples of solubilizers that can be used include alcohols (such as ethanol, etc.), polyalcohols (such as propylene glycol, polyethylene glycol, etc.), nonionic surfactants (such as polysorbate 20 (registered trademark), polysorbate 80 (registered trademark), HCO-50, etc.), and the like. Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate, and the like. Examples of emulsifiers that can be used include gum arabic, sodium alginate, tragacanth, and the like. Examples of soothing agents that can be used include benzyl alcohol, chlorobutanol, sorbitol, and the like. Examples of buffers that can be used include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, tris buffer, glutamate buffer, epsilon-aminocaproic acid buffer, and the like. Examples of preservatives that can be used include methyl paraben, ethyl paraben, propyl paraben, butyl paraben, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax, and the like. Examples of antiseptics that can be used include benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like. Examples of pH adjusters that can be used include hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, and the like.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.

[0104] Injections or infusions for drip are sterilized in their final step or produced by aseptic operation methods, for example, filtering with a filter or the like and then filling into a sterile container. In addition, injections or infusions for drip can also be used by dissolving aseptic powders (which may contain powders of pharmaceutically acceptable carriers) by vacuum drying and freeze drying in an appropriate solvent at the time of use.

[0105] Combination or admixture Furthermore, the PD-1 / CD19 bispecific antibody of the present invention etc. 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, in the case of using in combination with other agents (combined use), the administration form may be in the form of a formulation in which both components are formulated in one preparation, or may be an administration form as separate preparations. By the combined use, 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 etc. and other agents are administered separately, they may be administered simultaneously for a certain period, and then only the PD-1 / CD19 bispecific antibody etc. or only other agents may be administered. Also, the PD-1 / CD19 bispecific antibody of the present invention etc. 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 etc. 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 etc. 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. Also, the other agents include not only those found so far but also those to be found in the future.

[0106] 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 1 diabetes, 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.), therapeutic agents for diabetic neuropathy (e.g., epalrestat, mexiletine, imidapril, etc.), GLP-1 analog preparations (e.g., liraglutide, exenatide, lixisenatide, etc.) and DPP-4 inhibitors (e.g., sitagliptin, vildagliptin, alogliptin, etc.) and the like may be used in combination with any one or more drugs selected therefrom.

[0107] Further, 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 multiple sclerosis, steroid drugs (e.g., cortisone acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, fludrocortisone acetate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisolone butylacetate, prednisolone phosphate sodium, halopredone acetate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, triamcinolone, triamcinolone acetate, triamcinolone acetonide, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone palmitate, paramethasone acetate, 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 the like may be used in combination with any one or more drugs selected therefrom.

[0108] 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, it may be used in combination with any one or more drugs selected from steroid drugs (for example, the steroid drugs described above), immunosuppressive agents (for example, cyclosporine, tacrolimus, fingolimod, etc.) and belimumab.

[0109] 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), antirheumatic drugs (for example, methotrexate, sulfasalazine, bucillamine, leflunomide, mizoribine, tacrolimus, etc.), anti-cytokine drugs (for example, infliximab, adalimumab, tocilizumab, etanercept, golimumab, and certolizumab, etc.) and abatacept, etc.

[0110] 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.

[0111] The present invention will be further described in detail by the following examples, but the scope of the present invention is not limited thereto. Various changes and modifications are possible for those skilled in the art based on the description of the present invention, and these changes and modifications are also included in the present invention.

Examples

[0112] 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 a MeMo (registered trademark) mouse (see WO2009 / 157771) with recombinant human PD-1 protein was selected. The MeMo (registered trademark) mouse is a genetically modified mouse in which a gene fragment containing a non-recombinant human heavy-chain V gene region, D gene region, and J gene region and a recombinant human κ light-chain IgVκ1-39*01 / IGJκ1*01 germline gene type is linked to a mouse constant region gene. By directly immunizing the target protein of the antibody, antibodies consisting of a diverse heavy chain and a common light chain can be produced.

[0113] 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). On days 0, 14, and 28 of immunization, the recombinant human PD-1-Fc fusion protein was administered subcutaneously, 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 titer in the serum was evaluated by flow cytometry using a human PD-1-overexpressing HEK293T cell line. 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 when staining the human PD-1-overexpressing HEK293T cell line in 1000-fold diluted serum were used for the construction of a phage display library. Mice that met the criteria for proceeding with library construction were boost-immunized with recombinant PD-1-Fc fusion protein for 3 days from the antibody titer evaluation day, and the spleen and hilar lymph nodes were collected. Mice with a serum antibody titer of 1 / 100 or more against human PD-1 and cynomolgus monkey PD-1 and whose antibody titer did not increase by boost-immunization also had their spleen and hilar lymph nodes collected. After extracting RNA from these lymphoid tissues, cDNA synthesis was performed.

[0114] 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 to the immunoglobulin heavy chain variable region family. After cutting the PCR product with restriction enzymes SfiI and XhoI, it was inserted into a phagemid vector cut 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.

[0115] Example 3: Screening of anti-PD-1 antibodies Phage selection based on binding to PD-1 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 using the human PD-1-Fc fusion protein, Fc-reactive clones were absorbed by adding human IgG (SIGMA, catalog number I4506) during the incubation of the phage. Binding phages that bind to human PD-1, cynomolgus monkey PD-1, and mouse PD-1 were enriched. By selection with a cynomolgus monkey PD-1-overexpressing HEK293T cell line, phages that bind to cynomolgus monkey PD-1 were enriched. Clones of Escherichia coli strain TG1 transformed with the phages obtained by selection were obtained, and a master plate was prepared.

[0116] Furthermore, phage selection was performed from the periplasmic extracts of the clones obtained from the above selection based on the binding to PD-1 on a plate adsorbed with human PD-1-Fc fusion protein. As the selection criterion, those with a signal (OD 450 value) 3 times or more that of the signal obtained in the negative control well (PBS) were regarded as positive clones.

[0117] Example 4: DNA sequencing of candidate clones of anti-PD-1 antibodies The DNA sequence of the heavy chain variable region gene of the positive clones obtained by the screening of Example 3 was performed. 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 same CDR3 are 70% or more homologous) and clusters (a group in which the amino acid sequences of the heavy chain CDR3 are identical). Hundreds of clones were obtained and they were classified into superclusters and clusters.

[0118] Example 5: Screening by evaluating the binding to PD-1-expressing cells From each of the classified superclusters, anti-PD-1 monoclonal antibody clones that satisfied the following conditions were selected and isolated. (1) Somatic mutations were frequently introduced into the CDR region, (2) They had a germline gene type of VH with a high usage frequency, and (3) High signals were obtained in the binding screening against the human PD-1-Fc fusion protein.

[0119] Using the Fab fragments contained in their periplasmic extracts, the binding to the human PD-1-overexpressing CHO-S cell line and the cynomolgus monkey PD-1-overexpressing CHO-S cell line was evaluated by detecting with an anti-mouse IgG polyclonal antibody. Among the 117 clones (105 types of clusters) evaluated, binding to the human PD-1-expressing CHO-S cell line was observed in 22 clones including the anti-PD-1 monoclonal antibody clones PD1-1, PD1-2, PD1-3 and PD1-4.

[0120] Example 6: Preparation of amino acid substitution variants of anti-PD-1 monoclonal antibodies Clone PD1-1 and PD1-4 contain a deamidation motif (Asn-Gly) in framework 4 of their heavy chain variable regions. 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.

[0121] Example 7: Immunization of MeMo (registered trademark) mice with a CD19 expression plasmid vector As a method for obtaining a second arm that specifically binds to CD19 of the present invention, a method of immunizing a MeMo (registered trademark) mouse (see WO2009 / 157771) with a human CD19 expression plasmid vector and a cynomolgus monkey CD19 expression plasmid vector was selected.

[0122] A MeMo (registered trademark) mouse is a mouse genetically modified such that a gene fragment containing a non-recombinant human heavy chain V gene region, D gene region, and J gene region and a recombinant human κ light chain IgVκ1-39*01 / IGJκ1*01 germline gene type gene is ligated to a mouse constant region gene. By immunizing a MeMo (registered trademark) mouse with a plasmid vector expressing an antibody target protein, antibodies consisting of a diverse heavy chain and a common light chain can be produced.

[0123] Twelve 12- to 16-week-old MeMo (registered trademark) mice were each immunized with a human CD19-expressing plasmid vector and / or a cynomolgus monkey CD19-expressing plasmid vector, either separately or alternately. The expression plasmid vectors were administered on days 0, 3, 6, 14, 17, 28, 31, 42, 49, 63, and / or 70 of immunization. Serum antibody titers were evaluated by flow cytometry using a human CD19-expressing cell line. When staining the human CD19-expressing cell line in 100-fold diluted serum, the lymphoid tissues of mice with an MFI value increased by 3-fold or more compared to the control human CD19 non-expressing cell line were used for the construction of a phage display library. Mice that met the criteria for proceeding with library construction were boosted and then the spleen and inguinal lymph nodes were harvested. After extracting RNA from these lymphoid tissues, cDNA synthesis was performed by reverse transcription reaction using IgG constant region-specific primers.

[0124] Example 8: Construction of a phage display library for obtaining anti-CD19 antibodies A PCR reaction was performed using primers specific for the immunoglobulin heavy chain variable region family from the DNA prepared in Example 7. After cutting the PCR product with a restriction enzyme, it was inserted into a phagemid vector (containing a gene encoding a common light chain (human κ light chain IgVκ1-39*01 / IGJκ1*01 germline type gene)) cut with the same restriction enzyme to construct the library.

[0125] Example 9: Screening of anti-CD19 antibodies Phage selection based on binding to CD19 was performed using human CD19-Fc fusion protein (R&D systems, catalog number 9269-CD), cynomolgus monkey CD19-Fc fusion protein (NovoPro Bioscience, catalog number 504385), human B cell line Raji, or cynomolgus monkey CD19-overexpressing HEK293T cell line. Clones of Escherichia coli strain TG1 transformed with the phage obtained by selection were obtained and a master plate was prepared. Note that as a selection criterion, the signal obtained in the negative control (OD 450Those that gave a signal three times or more with respect to the value or MFI were defined as positive clones.

[0126] Example 10: DNA sequencing 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).

[0127] In the first screening, several hundred clones were obtained, which were classified into superclusters, clusters, and four germline sequences. In the second screening, several hundred clones were obtained and classified into superclusters, clusters, and eight germline sequences. Among them, 19 superclusters were different from the first screening.

[0128] Example 11: Screening by evaluating the binding to CD19 From each of the classified superclusters, anti-CD19 monoclonal antibody clones that satisfied the following conditions were selected and isolated. (1) Somatic mutations were frequently introduced into the CDR region, (2) They had a VH germline gene type that was frequently used, and (3) A high signal was obtained in the binding screening for CD19.

[0129] Using the Fab fragments contained in their periplasmic extracts, the binding to CD19 was evaluated.

[0130] Among the evaluated clones, binding to human CD19-expressing cell lines was observed in a plurality of clones including anti-CD19 monoclonal antibody clones CD19-1, CD19-2, CD19-3, CD19-4, and CD19-5.

[0131] 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 each heavy chain variable region of anti-PD-1 monoclonal antibody clones PD1-1 to PD1-5 selected in Examples 5 and 6 to DNA encoding the IgG 1 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 regions of anti-CD19 monoclonal antibody clones CD19-1 to CD19-5 selected in Example 11 to DNA encoding the IgG 1 heavy chain constant region. Here, for the gene expressing those 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, those modified so that the 447th lysine at the C-terminus of the heavy chain constant region is deleted were 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 treated 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.

[0132] These PD-1 / CD19 bispecific antibody clones each correspond to the anti-CD19 monoclonal antibody clones CD19-1, CD19-2, CD19-3, CD19-4, and CD19-5 used in their production in that they have a second arm that specifically binds to CD19 and is derived from these 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.

[0133] 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 be sufficiently separated and purified 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, in order to improve the separation of the bispecific antibody from the by-products during purification by cation exchange chromatography, amino acid mutants of the anti-CD19 monoclonal antibody with an increased isoelectric point were prepared.

[0134] A mutant was prepared by substituting the 114th glutamine 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.).

[0135] Furthermore, mutants in which glycine and the peptides represented by SEQ ID NOs: 67 to 70 were each 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") were 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".

[0136] Following the same method as in Example 12, each expression vector described in the same example, into which each DNA encoding a mutant of these anti-CD19 monoclonal antibodies and the DNA encoding clone PD1-5 were inserted, was transfected into Free Style 293F cells to produce an antibody 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 mutant 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)".

[0137] 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 purification and separation from by-products that may be generated in their production was examined.

[0138] The culture supernatants containing the bispecific antibodies recovered in Examples 12 and 13, respectively, were separately processed by protein A affinity chromatography and size exclusion chromatography to purify the bispecific antibody, the anti-PD-1 antibody, and the anti-CD19 antibody corresponding to the by-product.

[0139] The solvent containing each of these purified antibodies was buffer-exchanged by ultrafiltration so as to have a pH of 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 the start of 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%.

[0140] 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.

[0141] 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 (minutes) of 13.715 and 14.955.

[0142] 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 the heavy chain, separation 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 improved 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.

[0143] Example 15: Evaluation of the binding of PD-1 / CD19 bispecific antibodies 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 by Biacore measurement using human IgG1-Fc fused human PD-1 extracellular region recombinant protein (R&D systems, catalog number 1086-PD). For immobilization of the recombinant protein, a Series S Sensor Chip CM5 sensor chip (GE Healthcare, catalog number 29-1049-88) was used.

[0144] Example 15: Evaluation of the binding of PD-1 / CD19 bispecific antibodies Similarly, the CD19 binding affinity of the second arm of the antibody was evaluated by Biacore measurement using human IgG1-Fc fused human CD19 extracellular region recombinant protein (R&D systems, catalog number 9269-CD). The binding affinity (Kd value) of the first arm of each clone to PD-1 and the binding affinity of the second arm to CD19 are shown in FIG. 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) having a structure not subjected to the amino acid substitution of Example 13.

[0145] 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 PD-1, human CD19, and cynomolgus CD19, respectively. Clones CD19-1(Bi) to CD19-6(Bi) were added to human PD-1-overexpressing CHO-S cell line, cynomolgus PD-1-overexpressing CHO-S cell line, CHO-S cell line, human CD19-overexpressing CHO-K1 cell line, cynomolgus CD19-overexpressing CHO-K1 cell line, and CHO-K1 cell line, respectively, and incubated on ice for 20 minutes. After washing the cells, PE-labeled goat anti-human IgG-Fc F(ab’) 2100 μL of the fragment antibody (ThermoFisher, product number H10104) was added and incubated on ice for 20 minutes. After washing the cells, the PD-1 binding of the first arm and the CD19 binding of the second arm of the antibody were evaluated by flow cytometry. The results of the assay are shown in Figures 12 to 16.

[0146] 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.

[0147] It was confirmed that the PD-1 / CD19 bispecific monoclonal antibody obtained in Example 12 specifically binds to PD-1 and CD19 simultaneously. First, clones CD19-1(Bi) to CD19-5(Bi) were 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 the 6×His-tagged recombinant human PD-1 extracellular domain protein (R&D systems, product number 8986-PD) was added and incubated on ice for 20 minutes. After washing the cells, 100 μL of the AlexaFluor488-labeled mouse anti-His-tag antibody (MBL, product number D291-A48) was added and incubated on ice for 20 minutes. After washing the cells, the amount of binding of the recombinant human PD-1 extracellular domain protein was evaluated by flow cytometry. The results of the assay are shown in Figure 17.

[0148] All clones bound to PD-1 and CD19 simultaneously. Non-specific binding in this experimental system was not detected.

[0149] 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 added to human PD-1-overexpressing CHO-S cell lines on ice, respectively. Further, a biotinylated 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.

[0150] Clones CD19-1(Bi) to CD19-5(Bi) allowed the binding of the soluble PD-L1 recombinant protein to PD-1. On the other hand, nivolumab and J105 completely inhibited the binding of the soluble PD-L1 recombinant protein to PD-1 under the same conditions.

[0151] In addition, bispecific antibodies with anti-PD-1 monoclonal antibody clones PD1-1 to PD1-4 obtained in Example 5 as the first arm, respectively, were also confirmed to allow the binding of the soluble PD-L1 recombinant protein to PD-1 by the same evaluation.

[0152] Example 18: In vitro inhibitory effect of PD-1 / CD19 bispecific antibodies on activated B cells B cells isolated from peripheral blood mononuclear cells derived from healthy donors (LONZA, product number CC-2702) using the B Cell Isolation Kit II, human (Miltenyi Biotec, product number 130-091-151) were used to evaluate the inhibitory effect on human IgM production. Human B cells were seeded in cell culture plates, 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. Clones CD19-1(Bi)~CD19-5(Bi) or control antibody were 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. Clones 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).

[0153] Example 19: In vivo inhibitory effect of PD-1 / CD19 bispecific antibodies on activated B cells NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ mice (hereinafter abbreviated as NSG mice) transplanted with peripheral blood mononuclear cells derived from healthy donors (LONZA, product number CC-2702) were used to evaluate the inhibitory effect on human IgG 2 production. 1×10 7 peripheral blood mononuclear cells derived from healthy donors were transplanted per NSG mouse. On the 3rd, 7th, 10th, 14th, and 17th days after transplantation, clones CD19-1(Bi)~CD19-4(Bi) or control antibody were 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 clones CD19-5(Bi) and CD19-6(Bi), they were intraperitoneally administered at the same dosage 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 IgG contained in the serum 2It was quantified by ELISA method (ThermoFisher, model number BMS2093). The results are shown in Figures 20 and 21. Clones CD19-1(Bi) to CD19-6(Bi) all suppressed IgG 2 production. Note that the IgG 2 production amount (μg / mL) in the figure was shown as the mean ± standard error (N = 4 - 8).

[0154] 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 antibodies PD1-1 to PD1-5 used in the preparation of PD-1 / CD19 bispecific antibodies to PD-1 as the first arm, a competitive binding assay was performed.

[0155] 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.

[0156] 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.

[0157] 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, model number 317304) to human peripheral blood mononuclear cells (hereinafter, human PBMC) were each performed.

[0158] Human PBMCs (LONZA, catalog number CC-2702) were added with clone CD19-6 (Bi) and OKT3 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 is shown as the mean ± standard error (N = 3).

[0159] In contrast to OKT3 which significantly induced IL-2 production, no IL-2 production was observed with CD19-6 (Bi).

[0160] Example 22: Evaluation of the Physicochemical Stability of a PD-1 / CD19 Bispecific Antibody The PD-1 / CD19 bispecific antibody of the present invention has shown good performance in any physicochemical stability evaluation, including structural stability evaluation by differential scanning calorimetry (DSC), colloidal stability by measuring the change rate of diffusion coefficient (DLS), and chemical stability (e.g., protein concentration change, molecular structure change, presence or absence of association / aggregation, presence or absence of charge variant generation, structural change, and binding to CD19) under stress conditions (e.g., pH 3-4 / 5°C or pH 7 / 5°C / freeze-thaw 5 times).

Industrial Applicability

[0161] 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

1. 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, wherein 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 therefrom, and 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) 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) having any one VH selected from VH having a VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, a first arm that specifically binds to PD - 1 and a second arm that specifically binds to CD19, each (a) a VL - CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, (b) a VL - CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and (c) having a VL having a VL - CDR3 consisting of the amino acid sequence of SEQ ID NO: 28, said bispecific antibody or its F(ab′)2 fragment (provided that it has a first arm that specifically binds to PD - 1 and a second arm that specifically binds to CD19, and is a bispecific antibody that specifically binds to PD - 1 and CD19 respectively, (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, excluding said bispecific antibody.).

2. (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: 6, (b) a VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 7, and (c) has 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) (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) has a VH having 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) has a VH having 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) has a VH having a 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) The bispecific antibody or its F(ab')2 fragment according to claim 1, which is any one selected from VH having 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 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, (ii) The VH of the second arm that specifically binds to CD19 has (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) The bispecific antibody or its F(ab')2 fragment according to claim 1, which is any one selected from VH having VH - CDR3 consisting of the amino acid sequence of SEQ ID NO:

49.

4. (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: 12, (b) VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, and (c) having a 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) (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, and the bispecific antibody or its F(ab′)2 fragment according to claim 1, which is any one selected from the above.

5. (i) the VH of the first arm that specifically binds to PD - 1 has (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) 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 VH having a VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, VH having a VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, VH having a VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 45 and VH having a VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and VH having a VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 47, VH having a VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and VH having a VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, wherein the bispecific antibody or its F(ab′)2 fragment according to claim 1 is any one selected therefrom. **Claim 6** The VH of the first arm that specifically binds to PD - 1 has 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, and the VH of the second arm that specifically binds to CD19 has VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 36 and VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 37, VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 and VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 40, VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 42 and VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, VH - CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, VH - CDR2 consisting of the amino acid sequence of SEQ ID NO: 45 and VH - CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and 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, wherein the bispecific antibody or its F(ab′)2 fragment according to claim 1 is any one selected therefrom. **Claim 7** The FR1, FR2, and FR3 regions of the VH of the first arm that specifically binds to PD-1 each correspond to the amino acid sequences encoded by the gene, which may have somatic mutations of the germline V gene IGHV7-4-1, and the FR4 region consists of the amino acid sequence encoded by the gene, which may have somatic mutations of the germline J gene JH6c (however, excluding the amino acid sequence included in the VH-CDR3 region). The bispecific antibody or its F(ab′)2 fragment according to any one of claims 1 to 6.

8. The FR1, FR2, and FR3 regions of the VH of the second arm that specifically binds to CD19 each correspond to the amino acid sequences encoded by the gene, which may have somatic mutations of the germline V gene IGHV5-51. The bispecific antibody or its F(ab′)2 fragment according to any one of claims 1 to 7.

9. 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 90% identical to the amino acid sequence of the VH. The bispecific antibody or its F(ab′)2 fragment according to any one of claims 1 to 8.

10. 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 90% identical to the amino acid sequence of the VH. The bispecific antibody or its F(ab′)2 fragment according to any one of claims 1 to 9.

11. 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. The bispecific antibody or its F(ab′)2 fragment according to claim 1.

12. 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. The bispecific antibody or its F(ab')2 fragment according to claim 1 or 2.

13. 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. The bispecific antibody or its F(ab')2 fragment according to claim 1 or 3.

14. 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 bispecific antibody or its F(ab')2 fragment according to claim 1 or 4.

15. 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 bispecific antibody or its F(ab')2 fragment according to claim 1 or 5.

16. 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 bispecific antibody or its F(ab')2 fragment according to claim 1 or 6.

17. The bispecific antibody or its F(ab')2 fragment according to any one of claims 1 to 16, wherein the first arm that specifically binds to PD-1 and / or the second arm that specifically binds to CD19 each has a VL consisting of the amino acid sequence of SEQ ID NO:

25.

18. 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, (A) a first arm that specifically binds to PD-1, having 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) a second arm that specifically binds to CD19, having 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 bispecific antibody or its F(ab')2 fragment (however, excluding the bispecific antibody 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 the amino acid sequence of SEQ ID NO: 62 and a VL consisting of the amino acid sequence of SEQ ID NO: 25).

19. The bispecific antibody or its F(ab′)2 fragment according to any one of claims 1 to 18, wherein the first arm specifically binding to PD-1 allows the interaction between PD-1 and PD-L1.

20. The bispecific antibody or its F(ab′)2 fragment according to any one of claims 1 to 19, wherein the bispecific antibody is an IgG antibody.

21. When the IgG antibody is IgG １ antibody or IgG ４ antibody, the bispecific antibody or its F(ab′)2 fragment according to claim 20.

22. When the IgG antibody is IgG １ The bispecific antibody or its F(ab')2 fragment according to claim 20, which is an antibody.

23. The bispecific antibody or its F(ab′)2 fragment according to claim 22, wherein the binding to the Fc receptor is abolished or attenuated.

24. The bispecific antibody or its F(ab′)2 fragment according to claim 23, wherein the 235th leucine in the EU numbering system in the constant regions of the two heavy chains of the bispecific antibody is each substituted with glycine, and / or the 236th glycine is each substituted with arginine.

25. The 351st leucine in the constant region of the heavy chain having VH of the first arm specifically binding to PD-1 is substituted with lysine, and the 366th threonine is substituted 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 substituted with aspartic acid, and the 368th leucine is substituted with glutamic acid. The bispecific antibody or its F(ab′)2 fragment according to any one of claims 22 to 24.

26. The 351st leucine in the constant region of the heavy chain having VH of the first arm specifically binding to PD-1 is substituted with aspartic acid, and the 368th leucine is substituted with glutamic acid 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 substituted with lysine, and the 366th threonine is substituted with lysine. The bispecific antibody or its F(ab′)2 fragment according to any one of claims 22 to 24.

27. The bispecific antibody or its F(ab′)2 fragment according to any one of claims 22 to 26, wherein the 447th lysine in the two heavy chain constant regions of the bispecific antibody is each absent according to the EU numbering system.

28. The heavy chain having VH of the first arm that specifically binds to PD-1, and comprising a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23, the bispecific antibody according to any one of claims 1 to 23, or an F(ab')2 fragment thereof.

29. The heavy chain having VH of the second arm that specifically binds to CD19, and comprising 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, the bispecific antibody according to any one of claims 1 to 24, 26, and 28, or an F(ab')2 fragment thereof.

30. The light chain having VL of the first arm that specifically binds to PD-1 and / or the light chain having VL of the second arm that specifically binds to CD19, and comprising a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29, the bispecific antibody according to any one of claims 1 to 29, or an F(ab')2 fragment thereof.

31. A bispecific antibody or an F(ab')2 fragment thereof 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, (A) the heavy chain having VH of the first arm that specifically binds to PD-1, comprising 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 heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23, (B) the light chain having VL of the first arm that specifically binds to PD-1, comprising 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) the heavy chain having VH of the second arm that specifically binds to CD19, comprising 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 The double - specificity antibody or its F(ab')2 fragment having a light chain with a VL that specifically binds to CD19, which light chain 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 (provided that it is a double - specificity antibody having a first arm that specifically binds to PD - 1 and a second arm that specifically binds to CD19 and specifically binds to PD - 1 and CD19 respectively, excluding the double - specificity antibody 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 the amino acid sequence of SEQ ID NO: 62 and a VL consisting of the amino acid sequence of SEQ ID NO: 25).

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