JP2022060182A - Pharmaceutical composition comprising bispecific antibody as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new drug for prevention, suppression of symptom progression, suppression of recurrence or treatment of autoimmune diseases.
As a result of diligent studies, the inventors of the present invention have focused on PD-1 / CD19 bispecific antibodies and the like as substances that can solve such problems, and the bispecific antibodies are autoimmune diseases. It was confirmed that it could be a new drug for prevention, suppression of progression of symptoms, suppression of recurrence or treatment of the disease. It was also confirmed that the bispecific antibody has a characteristic that allows interaction with PD-1 and its ligand, PD-L1.
[Selection diagram] None

Description

The present invention is a bispecific antibody capable of specifically binding to PD-1 and CD19 (hereinafter, may be abbreviated as "PD-1 / CD19 bispecific antibody") or an antibody thereof. The present invention relates to pharmaceutical compositions containing fragments (hereinafter, these may be abbreviated as "PD-1 / CD19 bispecific antibodies, etc.") as active ingredients, and their pharmaceutical therapeutic uses.

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 analysis of PD-1 knockout mice, PD-1 signals are autoimmune dilated myocardial disease, lupus-like syndrome, autoimmune encephalomyelitis, systemic lupus erythematosus, transplanted piece-to-host disease, type I diabetes and It is known to play an important role in suppressing autoimmune diseases such as rheumatoid arthritis. Therefore, it has been pointed out that a substance that enhances PD-1 signal can be a preventive or therapeutic agent for autoimmune diseases and the like.

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

There have been several reports on PD-1 bispecific antibodies aimed at the prevention or treatment of autoimmune diseases (Patent Documents 1 to 3), one of which is the T cell receptor complex. CD3 which is a member of.

There is also a report on bispecific antibodies targeting PD-1 and CD19 for the purpose of cancer treatment (Patent Document 4), but prevention of autoimmune diseases, suppression of symptom progression, suppression of recurrence or treatment. There are no reports of targeted PD-1 / CD19 bispecific antibodies.

International Publication No. 2003/011911 International Publication No. 2004/0722 86 International Publication No. 2013/022091 Chinese Patent Application Publication No. 106939050

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

As a result of diligent 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 a problem, and completed the present invention.

Furthermore, the inventors of the present invention confirmed that the PD-1 / CD19 bispecific antibody has a characteristic that allows interaction with PD-1 and its ligand, PD-L1. In addition, the inventors of the invention have substituted one or more amino acid substitutions on the heavy chain of the second arm that specifically binds to CD19 constituting the PD-1 / CD19 bispecific antibody. By addition, PD-1 / CD19 bispecific antibodies were found that are easy to separate from by-products in the purification process.

That is, the present invention is as follows.

[1] A bispecific antibody or antibody thereof that has a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19 and CD19, respectively. The first arm, which is a fragment and specifically binds to PD-1,
(A) (a) Complementarity determining regions 1 of heavy chain variable regions consisting of the amino acid sequence of SEQ ID NO: 6 (hereinafter, "complementarity determining regions 1 of heavy chain variable regions" may be abbreviated as VH-CDR1. .),
(B) Complementarity determining regions 2 of heavy chain variable regions consisting of the amino acid sequence of SEQ ID NO: 7 (hereinafter, "complementarity determining regions 2 of heavy chain variable regions" may be abbreviated as VH-CDR2) and (C) Complementarity determining regions 3 of heavy chain variable regions consisting of the amino acid sequence of SEQ ID NO: 8 (hereinafter, "complementarity determining regions 3 of heavy chain variable regions" may be abbreviated as VH-CDR3). Heavy chain variable regions (hereinafter, "heavy chain variable regions" 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) 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) VH- consisting of the amino acid sequence of SEQ ID NO: 18. CDR1,
It has any one VH selected from (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.
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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
It has any one VH selected from (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, wherein PD- Remaining any 1-5 amino acids in any one or more VH-CDRs selected from VH-CDR1, VH-CDR2 and VH-CDR3 in the first arm that specifically binds to 1. The group may be substituted with another amino acid and / or any one or more selected from VH-CDR1, VH-CDR2 and VH-CDR3 in the second arm that specifically binds to CD19. The PD-1 / CD19 bispecific antibody or antibody fragment thereof, wherein any 1 to 5 amino acid residues thereof may be replaced with other amino acids in VH-CDR.
[2] The first arm that specifically binds to PD-1
(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) VH- consisting of the amino acid sequence of SEQ ID NO: 18. CDR1,
It has any one VH selected from (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.
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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
(B) The VH according to the preceding paragraph [1], which has any one VH selected from VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and VH having VH-CDR3 consisting of the amino acid sequence of (c) SEQ ID NO: 49. PD-1 / CD19 bispecific antibody or antibody fragment thereof.
[3] (i) The VH of the first arm that specifically binds to PD-1
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 6.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) 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) 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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
(B) Any one selected from VH-CDR2 having 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, the preceding paragraph [1] or [2]. ] Described PD-1 / CD19 bispecific antibody or antibody fragment thereof.
[4] (i) The VH of the first arm that specifically binds to PD-1 is
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 9.
It has (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
(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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
(B) Any one selected from VH-CDR2 having 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, the preceding paragraph [1] or [2]. ] Described PD-1 / CD19 bispecific antibody or antibody fragment thereof.
[5] (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: 12.
It has (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.
(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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
(B) Any one selected from VH-CDR2 having 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, the preceding paragraph [1] or [2]. ] Described PD-1 / CD19 bispecific antibody or antibody fragment thereof.
[6] (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.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17.
(Ii) The VH of the second arm that specifically binds to CD19
(A) (a) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
(B) Any one selected from VH-CDR2 having 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, the preceding paragraph [1] or [2]. ] Described PD-1 / CD19 bispecific antibody or antibody fragment thereof.
[7] (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: 18.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) 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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
(B) Any one selected from VH-CDR2 having 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, the preceding paragraph [1] or [2]. ] Described PD-1 / CD19 bispecific antibody or antibody fragment thereof.
[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.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8.
(Ii) The VH of the second arm that specifically binds to CD19
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 35,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[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.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8.
(Ii) The VH of the second arm that specifically binds to CD19
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 38,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[10] (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.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8.
(Ii) The VH of the second arm that specifically binds to CD19
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 41,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[11] (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.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8.
(Ii) The VH of the second arm that specifically binds to CD19
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 44,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[12] (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.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8.
(Ii) The VH of the second arm that specifically binds to CD19
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 47,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[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.
It has (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
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 35,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[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.
It has (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
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 38,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[15] (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.
It has (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
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 41,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[16] (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.
It has (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
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 44,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[17] (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.
It has (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
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 47,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[18] (i) The VH of the first arm that specifically binds to PD-1 is
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 12.
It has (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.
(Ii) The VH of the second arm that specifically binds to CD19
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 35,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[19] (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: 12.
It has (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.
(Ii) The VH of the second arm that specifically binds to CD19
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 38,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[20] (i) The VH of the first arm that specifically binds to PD-1 is
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 12.
It has (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.
(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,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[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.
It has (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.
(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,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[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.
It has (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.
(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,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[23] (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: 15.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) 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: 35,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[24] (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: 15.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) 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: 38,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[25] (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: 15.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) 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,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[26] (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: 15.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) 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,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[27] (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: 15.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) 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,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[28] (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: 18.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) 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: 35,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[29] (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: 18.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) 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: 38,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[30] (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: 18.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) 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,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[31] (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: 18.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) 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,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[32] (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: 18.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) 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,
PD-1 / CD19 bispecific according to the preceding paragraph [1] or [2], having (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. Sex antibodies or antibody fragments thereof.
[33] Of the framework area of VH of the first arm specifically coupled to PD-1 (hereinafter, “framework area” may be abbreviated as FR), framework area 1 (hereinafter, referred to as FR). The framework region 2 (hereinafter, may be abbreviated as FR2) and the framework region 3 (hereinafter, may be abbreviated as FR3) are the germ cell lineage type V. PD-1 / CD19 double according to any one of the preceding paragraphs [1] to [32], each corresponding to the amino acid sequence encoded by the gene IGHV7-4-1 or the gene encoded by the somatic cell mutation thereof. Specific antibody or antibody fragment thereof.
[34] The framework region 4 of VH of the first arm that specifically binds to PD-1 (hereinafter, “framework region 4” may be abbreviated as FR4) is a germline type J gene. PD-1 / CD19 double according to the preceding paragraph [33], which consists of an amino acid sequence encoded by the gene encoded by JH6c or its somatic hypermutation (excluding the amino acid sequence contained in the VH-CDR3 region). Specific antibody or antibody fragment thereof.
[35] The FR of the first arm VH that specifically binds to PD-1 is encoded by the gene, which may be subject to somatic mutations in the germline V gene IGHV7-4-1. Somatic hypermutation replaces lysine at position 13 in the amino acid sequence of SEQ ID NO: 21 with glutamine, alanine at position 16 with valine, or lysine at position 19 with methionine, or any of them. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [33] or [34], which comprises FR1 substituted or optionally substituted with the combination of.
[36] The FR of the first arm VH that specifically binds to PD-1 is encoded by the gene, which may be subject to a somatic mutation in the germline V gene IGHV7-4-1. 13. PD-1 / CD19 bispecific antibody or antibody fragment thereof.
[37] The FR of the first arm VH that specifically binds to PD-1 is encoded by the gene, which may be subject to somatic mutations in the germline V gene IGHV7-4-1. By somatic hypermutation, serine at position 77 in the amino acid sequence of SEQ ID NO: 21 was replaced with threonine, or cysteine at position 84 was replaced with serine or asparagine, respectively, or with any combination thereof. The PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to any one of the above items [33] to [36], which comprises FR3 which may be substituted.
[38] FR4 of the first arm VH that specifically binds to PD-1 may be subject to a somatic mutation of the germline J gene JH6c (provided that it encodes the VH-CDR3 region). The lysine (Lys) in the FR4 amino acid sequence (Trp-Gly-Lys-Gly-Thr-Thr * -Val-Thr-Val-Ser-Ser) (SEQ ID NO: 61) is encoded by the gene region ) Is substituted or may be substituted with glutamine or asparagine and / or threonin (Thr) marked with * with leucine, PD-1 / according to any one of the preceding paragraphs [33] to [37]. CD19 bispecific antibody or antibody fragment thereof.
[39] Of the FRs of the second arm VH that specifically bind to CD19, FR1, FR2 and FR3 are encoded by the germline V gene IGHV5-51 or its somatic mutated gene. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the above items [1] to [38], which corresponds to each of the amino acid sequences.
[40] The FR of the second arm VH that specifically binds to CD19 is encoded by the gene, which may have undergone a somatic mutation in the germline V gene IGHV5-51, and the somatic mutation. Will replace glutamine at position 1 in the amino acid sequence of SEQ ID NO: 22 with glutamine, proline at position 14 with serine, tyrosine at position 27 with phenylalanine, or threonine at position 30 with isoleucine, respectively. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [39], which comprises FR1 substituted or may be substituted with any plurality of combinations of the above.
[41] The FR of the second arm VH that specifically binds to CD19 consists of FR2 encoded by the somatic mutation of the germline V gene IGHV5-51. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to [39] or [40].
[42] The FR of the second arm VH that specifically binds to CD19 is encoded by the gene, which may have undergone a somatic mutation in the germline V gene IGHV5-51, and the somatic mutation. Therefore, isoleucine at position 76 in the amino acid sequence of SEQ ID NO: 22 is phenylalanine, serine at position 77 is threonine or asparagine, threonine at position 78 is valine, serine at position 84 is asparagine, and methionine at position 93 is isoleucine. Alternatively, consisting of FR3, wherein leucine or alanine at position 97 is replaced with valine, respectively, or is substituted or optionally substituted with any plurality of combinations thereof, according to the preceding paragraphs [39] to [41]. The PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to any one of the above.
[43] The VH of the first arm that specifically binds to PD-1 is 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 PD-1 / CD19 double according to any one of the preceding paragraphs [1] to [42], which comprises an amino acid sequence that is at least 80%, 90%, 95%, 98% or 99% identical to the amino acid sequence of the VH. Specific antibody or antibody fragment thereof.
[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. , PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding paragraphs [1] to [43].
[45] 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 and SEQ ID NO: 34, or the VH. PD-1 / CD19 bispecificity according to any one of the preceding paragraphs [1] to [44], which comprises an amino acid sequence that is at least 80%, 90%, 95%, 98% or 99% identical to the amino acid sequence of. An antibody or antibody fragment thereof.
[46] 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 and SEQ ID NO: 34. The PD-1 / CD19 bispecificity according to any one of the preceding paragraphs [1] to [45], wherein the 114th glutamine in the sequence consists of an amino acid sequence in which arginine is substituted or may be substituted. An amino acid or an antibody fragment thereof.
[47] The VH of the second arm specifically bound to CD19 is 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: The PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to any one of the preceding items [1] to [45], which comprises any one of the amino acid sequences selected from No. 65 and SEQ ID NO: 66.
[48] The VH of the first arm that specifically binds to PD-1 consists of any one of the amino acid sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. , VH of the second arm specifically bound to CD19 is 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. The PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to the above item [1] or [2], which comprises any one of the amino acid sequences selected from SEQ ID NO: 66.
[49] A bispecific antibody or antibody fragment thereof having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19.
The VH of the first arm that specifically binds to PD-1 is 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 the VH of the said VH. The VH of the second arm, which consists of an amino acid sequence that is at least 80%, 90%, 95%, 98% or 99% identical to the amino acid sequence and specifically binds to CD19, has SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. , Any one of the amino acid sequences selected from 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 at least 80% of the amino acid sequence of the VH. A PD-1 / CD19 bispecific antibody or antibody fragment thereof consisting of 90%, 95%, 98% or 99% identical amino acid sequences.
[50] 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 is SEQ ID NO: 30, SEQ ID NO: 31, [1] or [1] above, which comprises any one amino acid sequence selected from 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. 2] The PD-1 / CD19 bispecific antibody or fragment thereof.
[51] 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 is SEQ ID NO: 30, SEQ ID NO: 31, [1] or [1] above, which comprises any one amino acid sequence selected from 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. 2] The PD-1 / CD19 bispecific antibody or fragment thereof.
[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 is SEQ ID NO: 30, SEQ ID NO: 31, [1] or [1] above, which comprises any one amino acid sequence selected from 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. 2] The PD-1 / CD19 bispecific antibody or fragment thereof.
[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 is SEQ ID NO: 30, SEQ ID NO: 31, [1] or [1] above, which comprises any one amino acid sequence selected from 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. 2] The PD-1 / CD19 bispecific antibody or fragment thereof.
[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 is SEQ ID NO: 30, SEQ ID NO: 31, [1] or [1] above, which comprises any one amino acid sequence selected from 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. 2] The PD-1 / CD19 bispecific antibody or fragment thereof.
[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 is SEQ ID NO: 30 or SEQ ID NO: 62. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [8], which comprises an amino acid sequence.
[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 is SEQ ID NO: 31 or SEQ ID NO: 63. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [9], which comprises an amino acid sequence.
[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 is SEQ ID NO: 32 or SEQ ID NO: 64. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [10], which comprises an amino acid sequence.
[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 is SEQ ID NO: 33 or SEQ ID NO: 65. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [11], which comprises an amino acid sequence.
[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 is SEQ ID NO: 34 or SEQ ID NO: 66. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [12], which comprises an amino acid sequence.
[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 is SEQ ID NO: 30 or SEQ ID NO: 62. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [13], which comprises an amino acid sequence.
[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 is SEQ ID NO: 31 or SEQ ID NO: 63. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [14], which comprises an amino acid sequence.
[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 is SEQ ID NO: 32 or SEQ ID NO: 64. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [15], which comprises an amino acid sequence.
[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 is SEQ ID NO: 33 or SEQ ID NO: 65. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [16], which comprises an amino acid sequence.
[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 is SEQ ID NO: 34 or SEQ ID NO: 66. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [17], which comprises an amino acid sequence.
[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 is SEQ ID NO: 30 or SEQ ID NO: 62. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [18], which comprises an amino acid sequence.
[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 is SEQ ID NO: 31 or SEQ ID NO: 63. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [19], which comprises an amino acid sequence.
[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 is SEQ ID NO: 32 or SEQ ID NO: 64. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [20], which comprises an amino acid sequence.
[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 is SEQ ID NO: 33 or SEQ ID NO: 65. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [21], which comprises an amino acid sequence.
[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 is SEQ ID NO: 34 or SEQ ID NO: 66. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [22], which comprises an amino acid sequence.
[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 is SEQ ID NO: 30 or SEQ ID NO: 62. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [23], which comprises an amino acid sequence.
[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 is SEQ ID NO: 31 or SEQ ID NO: 63. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [24], which comprises an amino acid sequence.
[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 is SEQ ID NO: 32 or SEQ ID NO: 64. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [25], which comprises an amino acid sequence.
[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 is SEQ ID NO: 33 or SEQ ID NO: 65. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [26], which comprises an amino acid sequence.
[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 is SEQ ID NO: 34 or SEQ ID NO: 66. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [27], which comprises an amino acid sequence.
[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 is SEQ ID NO: 30 or SEQ ID NO: 62. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [28], which comprises an amino acid sequence.
[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 is SEQ ID NO: 31 or SEQ ID NO: 63. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [29], which comprises an amino acid sequence.
[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 is SEQ ID NO: 32 or SEQ ID NO: 64. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [30], which comprises an amino acid sequence.
[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 is SEQ ID NO: 33 or SEQ ID NO: 65. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [31], which comprises an amino acid sequence.
[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 is SEQ ID NO: 34 or SEQ ID NO: 66. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the above item [1], [2] or [32], which comprises an amino acid sequence.
[80] A first arm that specifically binds to PD-1 and / or a second arm that specifically binds to CD19, respectively.
(A) Complementarity determining regions 1 of the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 26 (hereinafter, "complementarity determining regions 1 of the light chain variable region" may be abbreviated as VL-CDR1).
(B) Complementarity determining regions 2 of the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 27 (hereinafter, "complementarity determining regions 2 of the light chain variable region" may be abbreviated as VL-CDR2). And (c) complementarity determining regions 3 of the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 28 (hereinafter, "complementarity determining regions 3 of the light chain variable region" may be abbreviated as VL-CDR3). PD-1 / according to any one of the preceding paragraphs [1] to [79], which has a light chain variable region (hereinafter, "light chain variable region" may be abbreviated as "VL"). CD19 bispecific antibodies or antibody fragments thereof.
[81] The first arm specifically bound to PD-1 and / or the second arm specifically bound to CD19 each have a VL consisting of the amino acid sequence of SEQ ID NO: 25, [1] to [1] above. 80] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the above.
[82] A bispecific antibody or antibody thereof that has a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19 and CD19, respectively. It ’s a fragment,
(A) The first arm that specifically binds to PD-1 is a VH consisting of any one of the amino acid sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The second arm having VL consisting of the amino acid sequence of SEQ ID NO: 25 and (B) specifically binding to CD19 is SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: PD-1 / CD19 having a VH consisting of any one of the amino acid sequences selected from 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. A heavily specific antibody or antibody fragment thereof.
[83] A bispecific antibody or an antibody thereof that has 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. The first arm that is a fragment and specifically binds to the PD-1 is selected from (1) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. Binding to PD-1 of the first arm that specifically binds to PD-1, having VH consisting of the amino acid sequence and VL consisting of the amino acid sequence of SEQ ID NO: 25, or (2) PD-1 consisting of the VH and VL. A PD-1 / CD19 bispecific antibody or antibody fragment thereof that cross-competites with the binding of a variable region of a monoclonal antibody that specifically binds to PD-1 to PD-1.
[84] A bispecific antibody or an antibody 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. Binding to PD-1 by the first arm, which is a fragment and specifically binds to PD-1, is from (1) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. A first arm specifically bound to PD-1, having a VH consisting of any one of the selected amino acid sequences and a VL consisting of the amino acid sequence of SEQ ID NO: 25, or (2) PD-1 consisting of the VH and VL. The PD-1 / CD19 bispecific antibody or antibody fragment thereof cross-competited by the variable region of the monoclonal antibody that specifically binds to.
[85] Further, the second arm specifically bound to the CD19 is (1) 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: A second arm specifically bound to CD19 having a VH consisting of any one of the amino acid sequences selected from 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. PD-1 / CD19 dual according to the preceding paragraph [83] or [84], which cross-competes for binding or (2) binding to CD19 of a variable region of a monoclonal antibody specifically binding to CD19 consisting of the VH and VL. Specific antibodies or antibody fragments thereof.
[86] Further, the binding to CD19 by the second arm that specifically binds to the CD19 is as follows: (1) SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 62, A second that specifically binds to CD19, having a VH consisting of any one of the amino acid sequences selected from 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 PD-1 / CD19 bispecific antibody according to the preceding paragraph [83] or [84], which is cross-competited by the arm or (2) the variable region of the monoclonal antibody that specifically binds to CD19 consisting of the VH and VL. That antibody fragment.
[87] 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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
(B) Having any one VH selected from VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 48 and VH having VH-CDR3 consisting of the amino acid sequence of (c) SEQ ID NO: 49, the preceding paragraph [83] or [ 84] The PD-1 / CD19 bispecific antibody or antibody fragment thereof.
[88] The VH of the second arm specifically bound to CD19 is 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: Any one of the amino acid sequences selected from 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, according to the preceding paragraph [83]. Alternatively, the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to [84].
[89] The VH of the second arm specifically bound to CD19 is 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: The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [83] or [84], which comprises any one of the amino acid sequences selected from No. 65 and SEQ ID NO: 66.
[90] The first arm that specifically binds to PD-1 and / or the second arm that specifically binds to CD19
(A) VL-CDR1, consisting of the amino acid sequence of SEQ ID NO: 26,
The item according to any one of the preceding paragraphs [83] to [89], which has VL-CDR2 having the amino acid sequence of SEQ ID NO: 27 and VL having VL-CDR3 consisting of the amino acid sequence of (c) SEQ ID NO: 28. PD-1 / CD19 bispecific antibody or antibody fragment thereof.
[91] The first arm specifically binding to PD-1 and / or the second arm specifically binding to CD19 has a VL consisting of the amino acid sequence of SEQ ID NO: 25, [83]-[89] above. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the above.
[92] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the above items [1] to [91], wherein the PD-1 / CD19 bispecific antibody is an IgG antibody.
[93] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding item [92], wherein the IgG antibody according to the preceding item [92] is an IgG ₁ antibody or an IgG ₄ antibody.
[94] The IgG antibody according to the previous item [92] is an IgG ₁ antibody. The PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to the previous item [92].
[95] The IgG antibody according to the previous item [92] is an IgG ₄ antibody. The PD-1 / CD19 bispecific antibody or an antibody fragment thereof according to the previous item [92].
[96] The leucine at position 235 and / or the glycine at position 236 in the EU numbering system in the two heavy chain constant regions of the IgG ₁ antibody described in the preceding paragraph [94] are each replaced with arginine. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [94].
[97] Both leucine at position 351 and threonine at position 366 by the EU numbering system in heavy chains with VHs of the first arm specifically bound to PD-1 were replaced with lysine, specific to CD19. The PD according to the preceding paragraph [94] or [96], wherein the leucine at position 351 in the constant region in the heavy chain having VH of the second arm bound to is replaced with aspartic acid, and leucine at position 368 is replaced with glutamate. -1 / CD19 bispecific antibody or antibody fragment thereof.
[98] The leucine at position 351 and leucine at position 368 are replaced with aspartic acid by the EU numbering system in the constant region of the heavy chain with VH of the first arm specifically bound to PD-1. The PD according to the preceding paragraph [94] or [96], wherein both leucine at position 351 and threonine at position 366 in the constant region in a heavy chain having a VH of the second arm specifically bound to CD19 were replaced with lysine. -1 / CD19 bispecific antibody or antibody fragment thereof.
[99] The above paragraph, each of which lacks the 447th lysine by the EU numbering system in the two heavy chain constant regions of the IgG ₁ antibody according to any one of the preceding paragraphs [94] and [96] to [98]. [94] and the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of [96] to [98].
[100] The PD-1 / CD19 dual according to the previous item [95], wherein the serine at position 228 by the EU numbering system in the two heavy chain constant regions in the IgG ₄ antibody described in the previous item [95] was replaced with proline, respectively. Specific antibodies or antibody fragments thereof.
[101] A heavy chain with a VH of the second arm that specifically binds to CD19 is further attached to its C-terminus to Gly (glycine), Gly-Lys-Lys-Ala (SEQ ID NO: 67), Gly-Lys-. Previous paragraphs [94] and [96]-[with 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). 99] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the above.
[102] The heavy chain having VH of the first arm specifically bound to PD-1 contains a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23, [1] to [94], [96] above. , [98], [99] and [101] according to any one of the PD-1 / CD19 bispecific antibodies or antibody fragments thereof.
[103] The heavy chain having the VH of the second arm specifically bound to CD19 is 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.
[104] A light chain having a VL of the first arm that specifically binds to PD-1 and / or a light chain having a VL of the second arm that specifically binds to CD19 comprises the amino acid sequence of SEQ ID NO: 29. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding paragraphs [1] to [103], which comprises a light chain constant region.
[105] A bispecific antibody or antibody thereof that has a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19 and CD19, respectively. It ’s a fragment,
(A) Any one of the heavy chains having the VH of the first arm that specifically binds to PD-1 is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. It contains VH consisting of the amino acid sequence and a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23.
(B) A light chain having a VL of the first arm that specifically binds to PD-1 comprises a VL consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29.
(C) The heavy chain having the VH of the second arm specifically bound to CD19 is 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: It contains a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 24 and VH consisting of any one of the amino acid sequences selected from No. 64, SEQ ID NO: 65 and SEQ ID NO: 66, and specifically binds to (D) CD19. The PD-1 / CD19 bispecific antibody or antibody thereof, wherein the light chain having the VL of the second arm comprises a light chain constant region consisting of the VL consisting of the amino acid sequence of SEQ ID NO: 25 and the amino acid sequence of SEQ ID NO: 29. piece.
[106] PD-1 according to any one of the preceding paragraphs [1] to [105], wherein the first arm specifically bound to PD-1 allows interaction between PD-1 and PD-L1. / CD19 bispecific antibody or antibody fragment thereof.
[107] The isoelectric point of the complex consisting of the heavy chain and the light chain of the first arm specifically bound to PD-1 is about 7.4 to about 7.7 (preferably about 7.5 to about 7.6). The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of [1] to [106].
[108] The isoelectric point of the complex consisting of the heavy chain and the light chain of the second arm specifically bound to CD19 is about 8.3 to about 8.8 (preferably about 8.4 to about 8.6). ] To [107], the PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the above items.
[109] PD-1 / CD19 dual according to any one of the preceding paragraphs [1] to [108], wherein cytokine production in blood or tissue was sufficiently reduced during or within 24 hours after administration. Specific antibodies or antibody fragments thereof.
[110] The first arm specifically bound to PD-1 allowed the interaction between PD-1 and PD-L1 and the cytokine production was sufficiently reduced, [1] to [108] above. The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the above.
[111] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to the preceding paragraph [109] or [110], wherein the cytokine is at least IL-2, IFN-γ or TNF-α.
[112] PD-1 / according to any one of the above items [1] to [111], which suppresses B cell activation by binding to PD-1 and CD19 expressed on the same B cell, respectively. CD19 bispecific antibody or antibody fragment thereof.
[113] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to [111], which suppresses the activation of T cells (preferably memory T cells).
[114] PD-1 / CD19 bispecific according to the previous section [113], which suppresses T cell activation by binding to CD19 expressed on B cells and PD-1 expressed on T cells, respectively. Sex antibodies or antibody fragments thereof.
[115] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the preceding paragraphs [1] to [114], wherein PD-1 and CD19 are human PD-1 and human CD19, respectively. ..
[116] The PD-1 / CD19 bispecific antibody or antibody fragment thereof according to any one of the above items [1] to [115], wherein the PD-1 / CD19 bispecific antibody is a monoclonal antibody.
[117] The PD-1 / CD19 bispecific antibody according to any one of the above items [1] to [116] or an antibody thereof, wherein the PD-1 / CD19 bispecific antibody is an isolated antibody. piece.
[1-1] A pharmaceutical composition containing a PD-1 / CD19 bispecific antibody selected from any one of the above items [1] to [117] or an antibody fragment thereof as an active ingredient.
[1-2] The pharmaceutical composition according to the preceding paragraph [1-1], further comprising at least one pharmaceutically acceptable carrier.

[2-1] Prevention of autoimmune diseases and progression of symptoms containing PD-1 / CD19 bispecific antibody selected from any one of the above items [1] to [117] or an antibody fragment thereof as an active ingredient. Suppression, recurrence suppression and / or therapeutic agent.
[2-2] Autoimmune diseases include Bechet's disease, systemic erythematosus, chronic discoid erythematosus, polysclerosis (systemic scleroderma, progressive systemic sclerosis), scleroderma, polymyositis, Dermatitis, periarteritis nodule (nodular polyarteritis, microscopic polyangiitis), aortitis syndrome (hyperan arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed coupling Tissue disease, Sjogren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, irritable vasculitis, rheumatoid vasculitis, macroangiitis, ANCA-related vasculitis (eg, polyangiitis granulomatosis and favor) Acidocytic polyangiitis granulomatosis), Cogan syndrome, RS3PE syndrome, temporal arteritis, rheumatic polymyopathy, fibromyopathy, antiphospholipid antibody syndrome, autoimmune myelitis, IgG ₄ Related diseases (eg, primary sclerosing cholangitis, autoimmune pancreatitis, etc.), Gillan Valley syndrome, severe myasthenia, chronic atrophic gastric inflammation, autoimmune hepatitis, non-alcoholic steatosis, primary biliary Liver cirrhosis, Good Pasture syndrome, rapidly progressive glomerulonephritis, scleroderma anemia, autoimmune hemolytic anemia, malignant anemia, autoimmune neutrophilia, idiopathic thrombocytopenic purpura, Basedow's disease ( Graves' disease (hyperthyroidism)), Hashimoto's disease, autoimmune adrenal dysfunction, primary hypothyroidism, Azison's disease (chronic hypocortical dysfunction), idiopathic Azison's disease, type I diabetes, slowly progressive Type I diabetes (adult latent autoimmune diabetes), localized scleroderma, psoriasis, psoriatic arthritis, bullous scleroderma, scleroderma, scleroderma, gestational herpes, linear IgA bullous dermatosis, Acquired epidermal vesicular disease, circular alopecia, leukoplakia, leukoplakia vulgaris, optic neuromyelitis, chronic inflammatory demyelinating polyneuritis, multifocal motor neuropathy, sarcoidosis, giant cell artitis, muscle atrophic lateral scleroderma Disease, Harada disease, autoimmune optic neuropathy, idiopathic asperemia, habitual abortion, inflammatory bowel disease (eg, ulcerative colitis, Crohn's disease), celiac disease, tonic spondylitis, severe asthma, chronic 蕁The agent according to the preceding paragraph [2-1], which is measles, transplant immunity, familial Mediterranean fever, eosinophilic scleroderma, dilated scleroderma, systemic obesity cell disease or encapsulated myopathy.
[2-3] Graft-versus-host disease (GVHD) containing PD-1 / CD19 bispecific antibody selected from any one of the preceding paragraphs [1] to [117] or an antibody fragment thereof as an active ingredient. Prevention, suppression of symptom progression, suppression of recurrence and / or therapeutic agents.
[2-4] Insulin preparations (eg, human insulin, insulin glargine, insulin lispro, insulin detemil, insulin aspart, etc.), sulfonylurea agents (eg, glybenclamide, glycladide, glymepyrid, etc.), haste insulin secretagogues (eg, glymepyrid). Nateglunide, etc.), biguanide preparations (eg, metformin, etc.), insulin sensitizers (eg, pyoglitazone, etc.), α-glucosidase inhibitors (eg, acarbose and boglibose, etc.), diabetic neuropathy therapeutic agents (eg, epalrestat, etc.) With any one or more agents selected from mexiletins and imidaprils, etc.), GLP-1 analog preparations (eg, liraglutide, exenatide, lixisenatide and semaglutide, etc.) and DPP-4 inhibitors (eg, sitagliptin, vildagliptin, allogliptin, etc.). Prevention of type I diabetes, characterized by being administered, comprising PD-1 / CD19 bispecific antibody selected from any one of the preceding paragraphs [1] to [117] or an antibody fragment thereof as an active ingredient. , Suppression of symptom progression, suppression of recurrence and / or therapeutic agent.
[2-5] Steroid drugs (eg, cortisone acetate, hydrocortisone, sodium hydrocortisone phosphate, hydrocortisone sodium succinate, fludrocortisone acetate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisolone butyl acetate, prednisolone sodium phosphate, acetate. Haloprednisolone, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, triamsinolone, triamsinolone acetate, triamsinolone acetonide, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone palmitate, parameterzone acetate, betamethasone, etc.), interferon β One or more selected from interferon β-1b, glatiramer acetate, mitoxantron, azathiopurine, cyclophosphamide, cyclosporin, methotrexate, cladribine, corticostimulatory hormone (ACTH), corticotropin, misolibin, tachlorimus, fingerimodo and alemtuzumab. It is characterized by being administered together with the above-mentioned drug, and contains a PD-1 / CD19 bispecific antibody selected from any one of the above items [1] to [117] or an antibody fragment thereof as an active ingredient. Prevention of sclerosis, suppression of progression of symptoms, suppression of recurrence and / or therapeutic agents.
[2-6] Administered with any one or more drugs selected from steroid drugs (eg, the steroid drug according to the preceding paragraph [2-5]), immunosuppressive drugs (eg, cyclosporine, tacrolimus, fingolimod, etc.) and berimumab. Prevention of systemic elitematodes, which comprises a PD-1 / CD19 bispecific antibody selected from any one of the above items [1] to [117] or an antibody fragment thereof as an active ingredient. Suppression of symptom progression, suppression of recurrence and / or therapeutic agent.
[2-7] A steroid drug (for example, the steroid drug described in the preceding paragraph [2-5]), an anti-rheumatic drug (for example, methotrexate, sulfasalazine, bushilamine, reflunomide, misolibin, tacrolimus, etc.), an anti-cytocytomous drug (for example, infliximab, etc.). PD selected from any one of the preceding paragraphs [1] to [117], characterized by being administered with any one or more agents selected from adalimumab, tocilizumab, etanelcept, golimumab, sertrizumab, etc.) and abatacept. -A 1 / CD19 bispecific antibody or an antibody fragment thereof as an active ingredient for preventing, suppressing the progression of symptoms, suppressing recurrence and / or treating rheumatoid arthritis.
[2-8] It is characterized in that it is administered together with any one or more of the drugs listed in the preceding paragraphs [2-4] to [2-7], and is selected from any one of the preceding paragraphs [1] to [117]. PD-1 / CD19 bispecific antibody or antibody fragment thereof as an active ingredient, which is a prophylactic, symptom progression inhibitory, recurrence inhibitory and / or therapeutic agent for autoimmune diseases.
[2-9] The preceding paragraphs [2-4] to [2] are characterized in that they are administered to a patient to whom any one or more of the agents listed in the preceding paragraphs [2-4] to [2-7] is administered. -8] A prophylactic, symptom progression-suppressing, recurrence-suppressing and / or therapeutic agent for each of the described diseases.
[2-10] The preceding paragraph [2-4] to [2-8], characterized in that it is administered after administration of any one or more of the agents listed in the preceding paragraphs [2-4] to [2-7]. A prophylactic, symptom progression inhibitory, recurrence inhibitory and / or therapeutic agent for each of the described disorders.
[2-11] The preceding paragraphs [2-4] to [2-8], characterized in that they are administered before the administration of any one or more of the agents listed in the preceding paragraphs [2-4] to [2-7]. ] A prophylactic, symptom-progressing, recurrence-suppressing and / or therapeutic agent for each of the listed diseases.

[3-1] A vein containing a PD-1 / CD19 bispecific antibody or antibody fragment thereof selected from any one of the preceding paragraphs [1] to [117] and at least one pharmaceutically acceptable carrier. Preparation for internal injection.
[3-2] The intravenous injection preparation according to the preceding item [3-1] for prevention, suppression of symptom progression, suppression of recurrence and / or treatment of autoimmune diseases.
[3-3] The pharmaceutical product for intravenous injection according to the preceding paragraph [3-1] or [3-2], which is for infusion.

[4-1] Use of the pharmaceutical composition according to the preceding paragraph [1-1] or [1-2] for prevention of autoimmune diseases, suppression of symptom progression, suppression of recurrence and / or treatment.
[4-2] Autoimmune diseases include Bechet's disease, systemic erythematosus, chronic discoid erythematosus, polysclerosis (systemic scleroderma, progressive systemic sclerosis), scleroderma, polymyositis, Dermatitis, periarteritis nodule (nodular polyarteritis, microscopic polyangiitis), aortitis syndrome (hyperan arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed coupling Tissue disease, Sjogren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, irritable vasculitis, rheumatoid vasculitis, macroangiitis, ANCA-related vasculitis (eg, polyangiitis granulomatosis and favor) Acidocytic polyangiitis granulomatosis), Cogan syndrome, RS3PE syndrome, temporal arteritis, rheumatic polymyopathy, fibromyopathy, antiphospholipid antibody syndrome, autoimmune myelitis, IgG ₄ Related diseases (eg, primary sclerosing cholangitis, autoimmune pancreatitis, etc.), Gillan Valley syndrome, severe myasthenia, chronic atrophic gastric inflammation, autoimmune hepatitis, non-alcoholic steatosis, primary biliary Liver cirrhosis, Good Pasture syndrome, rapidly progressive glomerulonephritis, scleroderma anemia, autoimmune hemolytic anemia, malignant anemia, autoimmune neutrophilia, idiopathic thrombocytopenic purpura, Basedow's disease ( Graves' disease (hyperthyroidism)), Hashimoto's disease, autoimmune adrenal dysfunction, primary hypothyroidism, Azison's disease (chronic hypocortical dysfunction), idiopathic Azison's disease, type I diabetes, slowly progressive Type I diabetes (adult latent autoimmune diabetes), localized scleroderma, psoriasis, psoriatic arthritis, bullous scleroderma, scleroderma, scleroderma, gestational herpes, linear IgA bullous dermatosis, Acquired epidermal vesicular disease, circular alopecia, leukoplakia, leukoplakia vulgaris, optic neuromyelitis, chronic inflammatory demyelinating polyneuritis, multifocal motor neuropathy, sarcoidosis, giant cell artitis, muscle atrophic lateral scleroderma Disease, Harada disease, autoimmune optic neuropathy, idiopathic asperemia, habitual abortion, inflammatory bowel disease (eg, ulcerative colitis, Crohn's disease), celiac disease, tonic spondylitis, severe asthma, chronic urticaria The use according to the preceding paragraph [4-1], which is measles, transplant immunity, familial Mediterranean fever, eosinophilic scleroderma, dilated scleroderma, systemic obesity cell disease or encapsulated myopathy.
[4-3] Use of the pharmaceutical composition according to the preceding paragraph [1-1] or [1-2] for prevention of graft-versus-host disease (GVHD), suppression of symptom progression, suppression of recurrence and / or treatment.
[4-4] In the prevention, suppression of symptom progression, suppression and / or treatment of type I diabetes, insulin preparations (eg, human insulin, insulin glargine, insulin lispro, insulin detemil, insulin aspart, etc.), sulfonylurea agents (eg, for example). , Glybenclamide, glycladide and glymepyride, etc.), fast-acting insulin secretagogues (eg, nateglinide, etc.), biguanide preparations (eg, metformin, etc.), insulin resistance improvers (eg, pioglycazone, etc.), α-glucosidase inhibitors (eg, etc.) , Acarbose and Boglibose, etc.), therapeutic agents for diabetic neuropathy (eg, epalrestat, mexiretin, imidapril, etc.), GLP-1 analog preparations (eg, liraglutide, exenatide, lixsenatide, semaglutide, etc.) and DPP-4 inhibitors (eg, etc.) Use of the pharmaceutical composition according to the preceding paragraph [1-1] or [1-2], which is administered together with any one or more drugs selected from (citagliptin, vildagliptin, allogliptin, etc.).
[4-5] In the prevention, suppression of progression, suppression and / or treatment of multiple sclerosis, steroid drugs (eg, cortisone acetate, hydrocortisone, sodium hydrocortisone phosphate, sodium hydrocortisone succinate, fludrocortisone acetate, prednisolone). , Prednisolone acetate, prednisolone sodium succinate, prednisolone butyl acetate, prednisolone sodium phosphate, haloprednisolone acetate, methylprednisolone, methylprednisolone acetate, sodium methylprednisolone succinate, triamsinolone, triamcinolone acetate, dexamethasone, dexamethasone, dexamethasone Dexamethasone sodium, dexamethasone palmitate, prednisolone acetate and betamethasone, etc.), interferon β-1a, interferon β-1b, glatiramer acetate, mitoxanthrone, azathiopurine, cyclophosphamide, cyclosporin, methotrexate, cladribine, corticostimulatory hormone (ACTH) ), Corticotropin, misolibin, tachlorimus, fingolimod and alemtuzumab, the use of the pharmaceutical composition according to the preceding paragraph [1-1] or [1-2], which is administered with any one or more agents.
[4-6] In the prevention, suppression of progression of symptoms, suppression and / or treatment of systemic lupus erythematosus, steroid drugs (eg, steroid drugs according to the previous section [2-5]), immunosuppressants (eg, cyclosporine, tacrolimus and / or). Use of the pharmaceutical composition according to the preceding paragraph [1-1] or [1-2], which is administered together with any one or more drugs selected from fingolimod etc.) and belimumab.
[4-7] In the prevention, suppression of symptom progression, suppression and / or treatment of rheumatoid arthritis, steroid drugs (for example, the steroid drugs described in the previous section [2-5]) and anti-rheumatic drugs (eg, methotrexate, sulfasalazine, bushiramine). , Reflunomid, misolivin and tacrolimus, etc.), anti-cytogenic drugs (eg, infliximab, adalimumab, tosirizumab, etanercept, golimumab and sertrizumab, etc.) and one or more agents selected from abatacept, the preceding paragraph [1-1]. ] Or the use of the pharmaceutical composition according to [1-2].
[4-8] In the prevention, suppression of symptom progression, suppression and / or treatment of autoimmune diseases, the preceding paragraph is administered together with any one or more of the agents listed in the preceding paragraphs [4-4] to [4-7]. Use of the pharmaceutical composition according to [1-1] or [1-2].

[5-1] Autoimmunity comprising administering to a patient an effective amount of a PD-1 / CD19 bispecific antibody or antibody fragment thereof selected from any one of the preceding paragraphs [1] to [117]. Disease prevention, symptom progression suppression, recurrence suppression and / or treatment methods.

[6-1] PD-1 / CD19 dual selected from any one of the preceding paragraphs [1] to [117], which is used for prevention of autoimmune diseases, suppression of symptom progression, suppression of recurrence and / or treatment. Specific antibodies or antibody fragments thereof.

[7-1] PD-1 / selected from any one of the preceding paragraphs [1] to [117] in the manufacture of drugs for the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of autoimmune diseases. Use of CD19 bispecific antibodies or antibody fragments thereof.

[8-1] Self-reactive B cell-mediated disease comprising PD-1 / CD19 bispecific antibody selected from any one of the preceding paragraphs [1] to [117] or an antibody fragment thereof as an active ingredient. Prevention, suppression of symptom progression, suppression of recurrence and / or therapeutic agents.
[8-2] Autoimmune B-cell-mediated diseases include systemic lupus erythematosus, Graves disease, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglobulinemia, and primary disease. The prophylactic, symptom progression inhibitory, recurrence inhibitory and / or therapeutic agent according to the preceding paragraph [8-1], which is bile duct sclerosis or malignant anemia.

[9-1] A self-reactive B cell inhibitor comprising a PD-1 / CD19 bispecific antibody selected from any one of the above items [1] to [117] or an antibody fragment thereof as an active ingredient.
[9-2] The self-reactive B cell inhibitor according to the preceding item [9-1], wherein the self-reactive B cell inhibition is the inhibition of immunoglobulin production.
[9-3] The self-reactive B cell inhibitor according to the previous item [9-2], wherein the immunoglobulin is IgG or IgM.

[10-1] A T cell activation inhibitor comprising a PD-1 / CD19 bispecific antibody selected from any one of the above items [1] to [117] or an antibody fragment thereof as an active ingredient.
[10-2] The T cell activation inhibitor according to the preceding item [10-1], wherein the suppression of T cell activation is suppression of cytokine production.
[10-3] The T cell activation inhibitor according to the above item [10-1] or [10-2], wherein the suppression of T cell activation is memory T cell inhibition.

[11-1] Self-reactivity comprising administering to a patient an effective amount of a PD-1 / CD19 bispecific antibody or antibody fragment thereof selected from any one of the preceding paragraphs [1] to [117]. A method for suppressing sex B cells or T cell activation.

[12-1] A PD-1 / CD19 bispecific antibody selected from any one of the preceding items [1] to [117], which is used for self-reactive B cell suppression or T cell activation suppression. That antibody fragment.

[13-1] PD-1 / CD19 bispecific antibody selected from any one of the preceding items [1] to [117] in the production of a self-reactive B cell inhibitor or T cell activation inhibitor. Or the use of that antibody fragment.

The PD-1 / CD19 bispecific antibody of the present invention allows the interaction between PD-1 and PD-L1 to prevent autoimmune diseases, suppress symptom progression, suppress recurrence and / or enhance the therapeutic effect. Or it can be expected to last.

The amino acid sequences of the variable region and the constant region of the common light chain are shown. The amino acid sequence of each CDR of the variable region of the common light chain is shown. The amino acid sequences encoded by the germline V genes IGHV7-4-1 and IGHV5-51, respectively, are shown. Sequence alignment of VH of each clone of an antibody that specifically binds to PD-1 (hereinafter, abbreviated as anti-PD-1 antibody) and germline genes IGHV7-4-1 and JH6c. show. In the figure, "-" in the amino acid sequence of each clone represents an amino acid that is the same as the amino acid sequence of the corresponding germ cell lineage gene IGHV7-4-1 or JH6c, and the part where the abbreviation of the amino acid is described is , Represents an amino acid that differs from the amino acid sequence of the same germline type gene. A sequence alignment of the VH of each clone of an antibody that specifically binds to CD19 (hereinafter, may be abbreviated as "anti-CD19 antibody") and the amino acid sequence encoded by the germline gene IGHV5-51. show. Each notation in the figure has the same meaning as that in FIG. The amino acid sequence of VH of each clone of anti-PD-1 antibody is shown. The amino acid sequence of each CDR in VH of each clone of anti-PD-1 antibody is shown. The amino acid sequence of VH of each clone of anti-CD19 antibody is shown. The amino acid sequence of each CDR in VH of each clone of anti-CD19 antibody is shown. The amino acid sequence of each heavy chain constant region of PD-1 / CD19 bispecific antibody is shown. PD-1 / CD19 bispecific antibody Biacore measurement results confirming the binding activity of each clone to PD-1 and CD19 are shown. PD-1 / CD19 bispecific antibody Flow cytometry showing the binding of each clone CD19-1 (Bi) to CD19-3 (Bi) to CD19 is shown. PD-1 / CD19 bispecific antibody Flow cytometry showing the binding of each clone CD19-4 (Bi) and CD19-5 (Bi) to CD19 is shown. PD-1 / CD19 bispecific antibody Flow cytometry showing the binding of each clone CD19-1 (Bi) to CD19-3 (Bi) to PD-1 is shown. PD-1 / CD19 bispecific antibody Flow cytometry showing the binding of each clone CD19-4 (Bi) and CD19-5 (Bi) to PD-1 is shown. PD-1 / CD19 bispecific antibody Flow cytometry confirming the binding of each clone CD19-6 (Bi) to PD-1 and CD19 is shown. PD-1 / CD19 bispecific antibody Flow cytometry showing the simultaneous binding of each clone to PD-1 and CD19 is shown. PD-1 / CD19 bispecific antibody Flow cytometry showing the effect of each clone on PD-1 / PD-L1 interaction is shown. The results of the action of each clone of PD-1 / CD19 bispecific antibody on IgM production during in vitro activation of human B cells are shown. In addition, "Ctrl" in the figure represents a control group. The results of the action of PD-1 / CD19 bispecific antibody clones (CD19-1 (Bi) to CD19-4 (Bi)) on IgG ₂ production during in vivo activation of human B cells are shown. In addition, "Ctrl" in the figure represents a control group. The results of the actions of PD-1 / CD19 bispecific antibody clones CD19-5 (Bi) and CD19-6 (Bi) on IgG ₂ production during in vivo activation of human B cells are shown, respectively. In addition, "Ctrl" in the figure represents a control group. The results of the action of PD-1 / CD3 bispecific antibody clone CD19-6 (Bi) on cytokine production from human peripheral blood mononuclear cells are shown. In addition, "Medium" in the figure represents a control group.

PD-1 ( Programmed Cell Death -1 ) is a membrane-type protein consisting of the amino acid sequence represented by GenBank registration number NP_005009 in humans. When referred to herein as "PD-1", unless otherwise specified, all isoforms thereof and the epitope of the "first arm specifically bound to PD-1" according to the invention are conserved. In addition, it may be used as including those variants. In the present invention, PD-1 is preferably human PD-1.

In humans, CD19 is a membrane protein expressed on B cells consisting of the amino acid sequence represented by GenBank registration number NP_001171569 or NP_001761, and is a molecule that transmits stimulation to B cells together with the B cell receptor complex. When the term "CD19" is used herein, unless otherwise specified, the epitope of the "second arm that specifically binds to CD19" according to the present invention is conserved and used as including variants thereof. May be done. In the present invention, the CD19 is preferably a human CD19.

As used herein, "isolation" is defined as substantially a single pure substance by being identified, separated and / or purified from a contaminant containing multiple or innumerable components extracted from a host cell. It means that it becomes an ingredient.

As used herein, the term "monoclonal antibody" means an antibody obtained from a substantially homogeneous population of antibodies that binds to the same particular antigen.

As used herein, the term "bispecific antibody" means an antibody having binding specificity to two different antigen molecules or epitopes in one molecule, and the term "bispecific monoclonal antibody" further means substantially. Means a bispecific antibody obtained from an antibody population that is uniformly uniform.

The present invention is a bispecific antibody capable of specifically binding to PD-1 and CD19, respectively (in the present specification, it may be abbreviated as "PD-1 / CD19 bispecific antibody"). Regarding. 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. Yes, more preferably isolated human PD-1 / human CD19 bispecific monoclonal antibodies. Here, the "isolated human PD-1 / human CD19 bispecific monoclonal antibody" means an isolated bispecific monoclonal antibody against human PD-1 and human CD19.

Here, the morphology of bispecific antibodies includes, for example, diabodies, bispecific sc (Fv) ₂ , bispecific F (ab') ₂ , bispecific minibodies, and bispecific. Hybrid antibody, covalent diabody (bispecific DART), bispecific (FvCys) ₂ , bispecific F (ab'-zipper) ₂ , bispecific (Fv-zipper) ₂ , 2 There are multispecific triple chain antibodies and bispecific mAbs ² .

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

Bispecific sc (Fv) ₂ is a small molecule modified so that two sets of VH / VL of two antibodies that recognize different antigens are produced in a continuous single chain form via a peptide linker. It is a peptide antibody (see J. Biological Chemistry (1994), Vol. 269: p. 199-206).

Bispecific F (ab') ₂ is a low molecular weight antibody in which Fab'fragments of an antibody that recognizes two different antigens are covalently bonded by a disulfide bond or the like.

The bispecific minibody is a low molecular weight antibody fragment in which a small molecule antibody fragment modified so that the constant region CH3 domain of an antibody is linked to scFv that recognizes different antigens is covalently bonded by a disulfide bond or the like on the CH3 domain. It is a molecularized antibody (see Biochemistry (1992), Vo.31, No.6, p.1579-1584).

The bispecific hybrid antibody is an intact antibody in which a heavy chain / light chain complex of an antibody that recognizes two different antigens is covalently bonded by a disulfide bond or the like.

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

The bispecific hybrid antibody can be produced, for example, from a hybridoma produced by the hybrid hybridoma method (see US4474893). In addition, a total of four types of cDNA encoding the heavy chain and light chain of an antibody that recognizes different antigens can be co-expressed and secreted into mammalian cells for production.

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

Antibodies or monoclonal antibodies, when administered to humans, can be made in the form of chimeric antibodies, humanized antibodies or fully human antibodies in order to reduce or eliminate their antigenicity.

The "chimeric antibody" means an antibody in which the variable region sequence and the constant region sequence are derived from different mammals. For example, the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. .. The chimeric antibody is a gene encoding an antibody variable region isolated by a known method from an antibody-producing hybridoma isolated by the above-mentioned hybridoma method, recombinant DNA method or phage display method, using a known method. , Can be made by ligating to a gene encoding a human-derived antibody constant region (see, eg, US4816567 of Cabilly et al.).

By "humanized antibody" is meant an antibody in which a complementarity determining region (CDR) sequence from another mammalian germ cell type, such as a mouse, is transplanted onto a human framework sequence. Also in the case of humanized antibodies, a gene encoding an antibody CDR region isolated by a known method from an antibody-producing hybridoma isolated by the above method can be obtained from a human-derived antibody framework region using a known method. Can be ligated and made (see, eg, US5225539 and US5530101 in Winter and US5585089 and US6180370 by Queen et al.).

By "human antibody" or "fully humanized antibody" is meant an antibody derived from a human germ cell type immunoglobulin sequence, both in the variable and constant regions consisting of the framework and CDR regions. The human antibodies used in the present invention are mice transformed to produce human antibodies, such as Humab mice (eg, US5545806, US5569825, US5625126, US5633425, US5789650, US5877397, US5661016, US5814318 by Lonberg and Kay et al. , US5874299 and US5770429), KM mice (see, eg, WO2002 / 43478 of Ishida et al.), Xeno mice (see, eg, US5939598, US6075181, US6114598, US6150584 and US6162963) or Tc mice (eg, Tomizuka et al., Proc. Natl. It can be produced by the method using Acad. Sci. USA (2000), p.722-727). It can also be prepared using SCID mice with reconstructed human immune cells (see, eg, US5476996 and US5698767 by Wilson et al.) So that immunization causes a human antibody response. Furthermore, the human antibody used in the present invention can also be produced by the phage display method described above.

As used herein, an "antibody fragment" of a PD-1 / CD19 bispecific antibody is an antibody that is part of a full-length antibody and has an antigen-binding moiety to PD-1 and an antigen-binding moiety to CD19. , For example, F (ab') ₂ . Here, the antigen-binding portion means the minimum unit that an antibody can bind to the antigen, and for example, the target antigen can be recognized by the combination of three CDRs each in VH and VL and those CDRs. It consists of a framework area where the CDR is placed.

As used herein, the term "common light chain" means a light chain that is capable of associating with two or more different heavy chains and exhibiting 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 / See 051433). As such a common light chain, for example, a human κ light chain IgVκ1-39 * 01 / IGJκ1 * 01 (nomenclature according to the IMGT database) a light chain encoded by a germline gene (hereinafter, IGVK1-39 /) is preferable. It may be abbreviated as JK1 common light chain), and more preferably, for example, 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 the amino acid of SEQ ID NO: 28. The light chain having a VL comprising the sequence VL-CDR3, 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, a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29 is preferable. The amino acid sequences of the variable region 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.

As used herein, "isotype" is used to refer to an antibody class (eg, IgM or IgG) encoded by a heavy chain constant region gene. The preferred isotype of the PD-1 / CD19 bispecific antibody of the invention is IgG, more preferably IgG ₁ or IgG ₄ . Here, as IgG ₁ , those in which the binding to the Fc receptor is lost or attenuated are preferable. Specifically, by substituting, deleting or inserting an arbitrary amino acid in the heavy chain constant region, an IgG ₁ antibody in which the binding to the Fc receptor is abolished or attenuated can be obtained. For example, antibodies in which leucine at position 235 and / or glycine at position 236 have been replaced with arginine by the EU numbering system on each of the two heavy chain constant regions or hinge regions of bispecific antibodies. Can be mentioned. Further, in order to reduce the heterogeneity of the antibody, an antibody lacking a C-terminal amino acid, for example, lysine at position 447 according to the EU numbering system is preferable. Further, when the bispecific antibody is IgG ₄ , variants in which any amino acid in the heavy chain constant region is substituted, deleted or inserted so as to suppress swapping in the antibody molecule are more preferable. For example, an antibody in which serine at position 228 by the EU numbering system is replaced with proline, which is located in the hinge region, is preferable. In addition, in this specification, the amino acid positions assigned to the CDR of the variable region of the antibody and the framework may be defined according to the Kabat numbering system (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md.). , (1987) and (1991)). Also, amino acids in the constant region are represented according to the EU numbering system according to the amino acid position of Kabat (see Sequences of proteins of immunological interest, NIH Publication No. 91-3242).

The Fc region of the PD-1 / CD19 bispecific antibody of the invention may be substituted with any amino acid in that region to facilitate association of two different heavy chains. In a preferred embodiment, for example, leucine at position 351 is replaced with lysine and threonine at position 366 by the EU numbering system of a constant region on a heavy chain with a VH of the first arm that specifically binds to PD-1. PD- in which the 351st leucine in the constant region in the heavy chain with the VH of the second arm substituted with lysine and specifically bound to CD19 was replaced with aspartic acid and the 368th leucine was replaced with glutamate. Examples include 1 / CD19 bispecific antibodies. In addition, leucine at position 351 was replaced with aspartic acid and leucine at position 368 was replaced with glutamate by the EU numbering system in the constant region of the heavy chain with VH of the first arm specifically bound to PD-1. PD-1 / CD19 dual in which leucine at position 351 and threonine at position 366 in the constant region of the heavy chain with VH of the second arm specifically bound to CD19 was replaced with lysine. Specific antibodies can also be mentioned.

First arm that specifically binds to PD-1 In the present specification, the "first arm that specifically binds to PD-1" (hereinafter, may be abbreviated as "first arm") is an antibody. Alternatively, it contains at least VH of an antibody that specifically binds to PD-1, and PD- It means an antibody moiety that can specifically bind to 1, for example, such a first arm consists of the VH of the anti-PD-1 antibody and the VL of the common light chain that constitutes the anti-PD-1 antibody. Further, the first arm also contains the Fab portion of the antibody containing the VH and VL. Here, "specifically binds to PD-1" means an affinity higher than at least 1x10 ^-5 M, preferably 1x10 ^-7 M, and more preferably 1x10 ^-9 M (dissociation constant (Kd value)). It can bind directly to PD-1 with its binding activity, and at least does not substantially bind to other receptor members belonging to the so-called CD28 family receptors such as CD28, CTLA-4 and ICOS. Used as a feature. Further, the "antibody" in the "antibody that specifically binds to PD-1" or "anti-PD-1 antibody" is a full-length antibody, that is, two heavy chains linked by a disulfide bond and two light chains. Means a full-length antibody, preferably a monoclonal antibody thereof.

Here, the "first arm that specifically binds to PD-1" is, for example, (a) an amino acid sequence represented by HYJ ¹ LH [in the sequence, J ¹ is G (glycine) or A (alanine). And the other alphabets represent the one-letter abbreviations of each amino acid. ] VH-CDR1, (b) WJ ² NTNTU ² NPTX ² Amino acid sequence represented by AQGFTG [In the sequence, J ² represents L (leucine) or I (isoleucine), and U ² represents E (glutamic acid). Or G (glycine), X ² represents F (phenylalanine) or Y (tyrosine), and the other alphabets have the same meanings as above. ] VH-CDR2 consisting of, and (c) GDJ ³ VVPTTIWNYYU ³ X ³ MZ ³ V amino acid sequence [in the sequence, J ³ represents M (methionine) or L (leucine), U ³ represents H. Represents (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 represent, respectively. It has the same meaning as above. ] With VH having VH-CDR3.

Further, as another aspect of the "first arm that specifically binds to PD-1," for example,
(1b) 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 containing VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 8.
(2b) 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 containing VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 11.
(3b) 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 containing VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 14.
(4b) 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 containing VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17 and (5b) VH of SEQ ID NO: 18. Examples thereof include those having any one VH selected from VH-CDR1 consisting of an amino acid sequence, 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.

Further, the "first arm that specifically binds to PD-1" in the present invention includes any of the above (1b) to (5b) in each VH-CDR in any one of the VHs. 1 to 5 amino acid residues of the original amino acid residue is replaced with another amino acid (preferably its conservative amino acid), and the binding activity and substance of the original first arm not replaced with the amino acid to PD-1. Also included are those having the same binding activity. For example, in the case of VH-CDR1, one amino acid residue is replaced with another amino acid (preferably its conservative amino acid), and in the case of VH-CDR2 or VH-CDR3, 1 to 5 each. Amino acid residues are substituted with other amino acids (preferably the conservative amino acids thereof). Also, as shown in FIG. 4, in each CDR of the anti-PD-1 antibody clone corresponding to the first arm specifically bound to PD-1, each amino acid different between each clone or any of them. Multiple combinations are interchangeable between the clones. Here, substitution with conservative amino acids means the interchangeability of residues with similar side chains, eg, in the group of amino acids with aliphatic side chains, with glycine, alanin, valine, leucine and isoleucine. In the group of amino acids having an aliphatic hydroxyl side chain, they are serine and threonine, in the group of amino acids having an amide-containing side chain, they are asparagine and glutamine, and in the group of amino acids having an aromatic side chain, they are. , Phenylalanine, tyrosine and tryptophan, lysine, arginine and histidine in the group of amino acids with basic side chains, and cysteine and methionine in the group of amino acids with sulfur-containing side chains. Examples of preferred conservative amino acid substitutions are between valine, leucine and isoleucine, between phenylalanine and tyrosine, between lysine and arginine, between alanine and valine and between asparagine and glutamine. The replacement of is mentioned. Further, here, the above-mentioned "having substantially the same binding activity to PD-1 by the original first arm not substituted with the amino acid" means that the amino acid is substituted first. The binding activity of the 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 not substituted with the amino acid. Means.

Further, the "first arm that specifically binds to PD-1" in the present invention contains each VH-CDR having the above-mentioned specific amino acid sequence in the VH, and the amino acid sequence of the framework of the VH is included. Also included are those with a specific germline gene or a VH encoded by that gene that has undergone a somatic mutation thereof. For example, the VH indicated by any of the above (1b) to (5b) is a VDJ group in which the germline V gene is IGHV7-4-1 and the germline J gene is JH6c. It can be encoded by the gene that has undergone a conversion gene or its somatic mutation. Here, the amino acid sequences encoded by the germline V gene IGHV7-4-1 correspond to the amino acid sequence of SEQ ID NO: 21 (FIG. 3).

The framework of the first arm VH that specifically binds to PD-1 of the present invention may be encoded by the germline VDJ recombinant gene that has undergone somatic mutation. For example, FR1, FR2, FR3 of VH shown in any of the above (1b) to (5b), wherein the germline V gene is IGHV7-4-1, are at the amino acid positions shown in FIG. , IGHV7-4-1 gene is different from the amino acid sequence encoded by it, so it has undergone somatic mutation at each position. For example, for FR1, lysine at position 13 in the amino acid sequence of SEQ ID NO: 21 is replaced with glutamine, alanine at position 16 is replaced with valine, or lysine at position 19 is replaced with methionine, or any plurality of them. It may be replaced with a combination of. For FR2, valine at position 37 in the amino acid sequence of SEQ ID NO: 21 may be replaced with leucine. For FR3, serine at position 77 in the amino acid sequence of SEQ ID NO: 21 may be replaced with threonine, cysteine at position 84 may be replaced with serine or asparagine, or any combination may be substituted. Regarding FR4 of VH shown in any of the above (1b) to (5b), the amino acid sequence of FR4 derived from J gene JH6c (Trp-Gly-Lys-Gly-Thr-Thr * -Val). -Lysine (Lys) in Thr-Val-Ser-Ser (SEQ ID NO: 41) may be replaced with glutamine or asparagine, and / or threonine (Thr) marked with * may be replaced with leucine. FR1, FR2, FR3 and FR4, respectively, which have any of the above amino acid substitution combinations, also have no substantial effect on the function of the first arm that specifically binds to PD-1, and can be used as a framework. can.

Furthermore, the "first arm that specifically binds to PD-1" in the present invention contains each CDR having the above-mentioned specific amino acid sequence, and the FR amino acid sequence of the VH is a specific germ cell. It also includes a lineage gene or one encoded by the gene that has undergone a somatic cell mutation thereof. For example, such a first arm may have a VH consisting of an amino acid sequence selected from SEQ ID NOs: 1-5.

Further, such "first arm specifically bound to PD-1" is, for example, at least 80% identical to any one amino acid sequence selected from SEQ ID NOs: 1-5, preferably at least. It has a VH consisting of an amino acid sequence that is 90% identical, more preferably at least 95% identical, even more preferably at least 98% identical, and even more preferably at least 99% identical, and the original first. It also includes those in which the difference from the amino acid sequence of VH of one arm does not substantially affect the binding activity to PD-1 (hereinafter, may be abbreviated as homologous first arm). As used herein, "% identity" as used in the comparison of identity with respect to an amino acid sequence refers to an amino acid sequence that aligns and references two sequences (where necessary to achieve the maximum percent identity). Is defined as a percentage of the amino acid sequence that is identical to the reference amino acid sequence after the introduction of the gap). Further, here, "the difference from the amino acid sequence of 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 It means that it is 95% or more, preferably 98% or more, and more preferably 99% or more of the binding activity of the original first arm.

In still another embodiment, the "first arm specifically bound to PD-1" in the present invention includes (1) the VH or SEQ ID NO: shown in any of the above (1b) to (5b). To PD-1 of the first arm having VH consisting of any one amino acid sequence selected from 1 to 5 and VL of the common light chain herein (preferably VL consisting of the amino acid sequence of SEQ ID NO: 25). (2) The variable region of the anti-PD-1 antibody that cross-competites with the binding of the variable region of the monoclonal antibody that specifically binds to PD-1 consisting of the VH and VL to PD-1 (here, the relevant). The variable region includes those having VH and VL that compose it), and the binding to PD-1 is (3) any of the above (1b) to (5b). Specific to VH represented by or the first arm having VL of the common light chain and VH consisting of any one amino acid sequence selected from SEQ ID NOs: 1 to 5 or (4) PD-1 consisting of VH and VL. Also included are those having a variable region of an anti-PD-1 antibody that is cross-competitive by the variable region of a monoclonal antibody that binds specifically. Here, "cross-competition for binding to PD-1" means PD- of the first arm by binding to an epitope that is the same as or partially overlaps with that of the first arm exemplified herein. Binding to PD-1 by an antibody that inhibits binding to 1 to any extent, or binds to an epitope that is the same as or partially overlaps with the exemplified first arm is the illustrated first arm. It means that it is inhibited regardless of the degree of cross-competition, and whether or not it cross-competites can be evaluated by a competitive binding assay. For example, it can be determined using viacore analysis, ELISA assay, flow cytometry, enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer measurement (FRET) or fluorescence trace measurement technology (FMAT®). ..

For example, it is selected from, for example, (1b) to (4b) above, as a cross-competition for binding to PD-1 by the VH shown in (5b) above and the first arm having the VL of the common light chain. VH represented by any of the above and VL of the common light chain (preferably 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 consisting of the amino acid sequence of SEQ ID NO: 28. -The first arm with CDR3), as well as VH consisting of the amino acid sequence selected from SEQ ID NOs: 1-4 and VL of the common light chain (preferably VL consisting of the amino acid sequence of SEQ ID NO: 25). First arm having.

Further, for example, by a first arm having VH represented by any of the above (1b) to (4b) or VH consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 4 and VL of the common light chain. As cross-competitors for binding to PD-1, for example, VH shown in (5b) above and VL of the common light chain (preferably VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27). A first arm having VL-CDR2 consisting of the amino acid sequence and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28), and VH consisting of the amino acid sequence of SEQ ID NO: 5 and VL of the common light chain (preferably). Includes a first arm having a VL) consisting of the amino acid sequence of SEQ ID NO: 25.

Here, the "first arm that specifically binds to PD-1" in the present invention preferably includes the first arm having VH shown in (1b) to (5b) above, and further, this preferred first arm. In one arm, as described above, in each CDR of the VH, any 1-5 amino acid residues thereof is replaced with another amino acid (preferably the conservative amino acid thereof) and the amino acid substitution thereof. Including those that do not substantially affect the binding activity to PD-1, and further, as described above, the amino acid sequence of the VH framework is the germline type V gene IGHV7-4-1 or the same. Also included are those having the J gene JH6c or the VH encoded by the somatically mutated gene thereof. And more preferably, the first arm having VH consisting of any one amino acid sequence selected from SEQ ID NOs: 1 to 5 can be mentioned.

Further, the "first arm specifically bound to PD-1" in the present invention preferably contains the VL of the common light chain in the present specification, and such a common light chain is preferably used, for example, IGVK1-. It is a 39 / JK1 common light chain, and more preferably, for example, 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. It is a light chain having a VL containing, and more preferably, for example, a 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, a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29 is preferable.

In addition, the "first arm that specifically binds to PD-1" allows interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both. The one is more preferable. Here, "allowing the interaction with PD-1 and PD-L1, the interaction with PD-1 and PD-L2, or both of them" means the PD-1 / CD19 double of the present invention. Even in the situation where the specific antibody is present in an excess of 20 times the concentration of soluble PD-L1 or PD-L2, the interaction between PD-L1 and PD-1, the PD-L2 and PD-1 The interaction, or both of them, is maintained by 50% or more, preferably 70% or more, and more, as compared with the interaction in the absence of the PD-1 / CD19 bispecific antibody of the present invention. It means that it is preferably maintained at 80% or more. In addition, the definition of "accepting interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both of them" is defined as "interaction with PD-1 and PD-L1". It may be used synonymously with the meaning of "does not substantially inhibit interactions, interactions with PD-1 and PD-L2, or both interactions."

FIG. 6 shows 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, their VH amino acid sequences and their SEQ ID NOs. , 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.

Second arm that specifically binds to CD19 In the present specification, "second arm that specifically binds to CD19" (hereinafter, may be abbreviated as "second arm") means an antibody or an antibody fragment. Contains, at least, the VH of an antibody that specifically binds to CD19, and specifically binds to CD19, whether or not it is part of, or not part of, and exists as a single entity. It means an antibody moiety that can, for example, such a second arm consists of the VH of the anti-CD19 antibody and the VL of the common light chain constituting the anti-CD19 antibody, and further the second arm is the VH. And the Fab part of the antibody containing VL is also included. Here, "specifically binds to CD19" has at least an affinity (dissociation constant (Kd value)) higher than 1x10 ^-5 M, preferably 1x10 ^-7 M, more preferably 1x10 ^-9 M. It can bind directly to CD19 with its binding activity and is used as a feature that does not substantially bind to other proteins. Further, the "antibody" in the "antibody that specifically binds to CD19" or "anti-CD19 antibody" is a full-length antibody, that is, a full-length antibody consisting of two heavy chains linked by a disulfide bond and two light chains. It means an antibody, but is preferably a monoclonal antibody thereof.

Here, as the "second arm that specifically binds to CD19", for example, (a) the amino acid sequence represented by SYWIJ ⁴ [in the sequence, J ⁴ represents G (glycine) or A (alanine). The other alphabets each represent a one-letter abbreviation for an amino acid. ] VH-CDR1, (b) IIU ⁴ PGDSDTRYSPSFQG amino acid sequence [ ^In the sequence, U4 represents W (tryptophan) or Y (tyrosine), and the other alphabets have the same meanings as above. .. ] VH-CDR2, and (c) X ⁴ TIVZ ⁴ J ⁵ U ⁵ X ⁵ Z ⁵ AJ ⁶ DU ⁶ amino acid sequence [in the sequence, X ⁴ is K (glycine), Q (glutamine), H (histidine) or R (arginine) is represented, Z ⁴ is represented by G (glycine) or A (alanine), J ⁵ is represented by T (threonine) or V (valine), and U ⁵ is represented by V (valine). Valin), I (isoleucine) or T (threonine), X ⁵ represents M (methionine), Y (tyrosine), G (glycine) or H (histidine), Z ⁵ represents T (threonine), N (asparagin), L (leucine) or W (tryptophan), J ⁶ represents F (phenylalanine) or S (serine), U ⁶ represents I (isoleucine), F (phenylalanine) or Y (tyrosine). ), And the other alphabets have the same meaning as above. ] With VH having VH-CDR3.

Further, as another aspect of the "second arm specifically bound to CD19", for example,
(1d) 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 containing VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 37,
(2d) 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 containing VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 40,
(3d) 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 containing VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 43,
(4d) 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, VH containing VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (5d) VH consisting of the amino acid sequence of SEQ ID NO: 47. Examples thereof include those having any one VH selected from VH-CDR1 consisting of an amino acid sequence, 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.

Further, the "second arm specifically bound to CD19" in the present invention includes any one of the above (1d) to (5d) in each VH-CDR in any one of the VHs. Substantially equivalent to the binding activity to CD19 by the original second arm in which up to 5 amino acid residues have been replaced with another amino acid (preferably its conservative amino acid) and not replaced with that amino acid. Those having binding activity are also included. For example, in the case of VH-CDR1, one amino acid residue is replaced with another amino acid (preferably its conservative amino acid), and in the case of VH-CDR2 or VH-CDR3, 1 to 5 each. Amino acid residues are substituted with other amino acids (preferably the conservative amino acids thereof). Also, as shown in FIG. 5, in each CDR of the anti-CD19 antibody clone corresponding to the second arm that specifically binds to CD19, each amino acid that differs between the clones or any combination thereof is present. , Mutually replaceable between the clones. Here, "having substantially the same binding activity to CD19 by the original second arm not substituted with the amino acid" means the binding activity of the second arm substituted with the amino acid to CD19. However, it means that the binding activity of the original second arm not substituted with the amino acid is 95% or more, preferably 98% or more, and more preferably 99% or more. The "substitution with conservative amino acids" in each VH-CDR of the second arm includes, for example, the above-mentioned example of amino acid substitution in the first arm.

Further, in the present invention, the "second arm specifically bound to CD19" includes each CDR having the above-mentioned specific amino acid sequence in the VH, and the FR amino acid sequence of the VH is specified. Also included are those with a germline gene or a VH encoded by the gene that has undergone a somatic mutation thereof. For example, any one of the VHs selected from (1d) to (5d) above is encoded by the VDJ recombinant gene whose germline V gene is IGHV5-51 or the gene subjected to somatic mutation thereof. Can be done. 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).

The framework of the second arm VH that specifically binds to CD19 of the present invention may be encoded by the germline VDJ recombinant gene that has undergone somatic mutation. For example, FR1 and FR3 of VH shown in any of the above (1d) to (5d), wherein the germline V gene is IGHV5-51, are at the amino acid positions shown in FIG. 5 IGHV5-51. Because it differs from the amino acid sequence encoded by the gene, it has undergone somatic mutation at each position. For example, for FR1, glutamic acid at position 1 in the amino acid sequence of SEQ ID NO: 22 is replaced with glutamine, proline at position 14 is replaced with serine, tyrosine at position 27 is replaced with phenylalanine, or threonine at position 30 is replaced with isoleucine. Alternatively, it may be replaced with any combination of them. For FR3, isoleucine at position 76 in the amino acid sequence of SEQ ID NO: 22 is phenylalanine, serine at position 77 is threonine or asparagine, threonine at position 78 is valine, serine at position 84 is asparagine, and methionine at position 93. Isoleucine or leucine, or alanine at position 97 may be replaced with valine, respectively, or with any combination of plurals. FR1 and FR3, respectively, having any of the above amino acid substitution combinations also have no substantial effect on the function of the second arm that specifically binds to CD19 and can be used as a framework.

Furthermore, the "second arm specifically bound to CD19" in the present invention contains each CDR having the above-mentioned specific amino acid sequence, and the FR amino acid sequence of the VH is a specific germline type. It also includes those having a gene or a VH encoded by the gene that has undergone a somatic mutation thereof. For example, such a second arm may have a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30-34.

Further, such a "second arm specifically bound to CD19" is, for example, at least 80% identical to, preferably at least 90%, the amino acid sequence of any one selected from SEQ ID NOs: 30-34. It has a VH consisting of an amino acid sequence that is identical, more preferably at least 95% identical, even more preferably at least 98% identical, and even more preferably at least 99% identical, and the original second arm. Also included are those in which the difference from the amino acid sequence of VH does not substantially affect the binding activity to CD19 (hereinafter, may be abbreviated as homologous second arm). Here, "the difference from the amino acid sequence of 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 the original second arm. It means that the binding activity is 95% or more, preferably 98% or more, and more preferably 99% or more.

In yet another embodiment, the "second arm specifically bound to CD19" in the present invention comprises (1) VH or SEQ ID NO: 30-represented by any of the above (1d)-(5d). Binding or (preferably VL of the amino acid sequence of SEQ ID NO: 25) to CD19 of the second arm having VH consisting of any one amino acid sequence selected from 34 and VL of the shared light chain herein. 2) The variable region of the anti-CD19 antibody that cross-competites with the binding of the variable region of the monoclonal antibody that specifically binds to CD19 consisting of the VH and VL to CD19 (where, the variable region is the VH and the VH constituting the variable region). Includes VL), and the binding to CD19 is selected from (3) VH or SEQ ID NOs: 30-34 shown in any of the above (1d)-(5d). Cross-competition by the variable region of a monoclonal antibody that specifically binds to VH consisting of any one of the amino acid sequences and the VL of the shared light chain or (4) CD19 consisting of the VH and VL. Those having a variable region of anti-CD19 antibody are also included. Here, "cross-competition in binding to CD19" means binding of the second arm to CD19 by binding to an epitope that is the same as or partially overlaps with the second arm exemplified herein. Binding to CD19 by an antibody that inhibits to any extent, or binds to an epitope that is the same as or partially overlaps with the exemplified second arm, regardless of its degree by the exemplified second arm. It means that it is hindered. Here, whether or not there is cross-competition can be similarly measured according to the method described in the description of "the first arm specifically bound to PD-1".

Here, the "second arm specifically bound to CD19" in the present invention preferably includes the second arm having VH shown in (1d) to (5d) above, and further, this preferred second arm. In each CDR of the VH, any 1-5 amino acid residues are replaced with another amino acid (preferably its conservative amino acid) and the amino acid substitution is CD19. Including those that do not substantially affect the binding activity to, and further, as mentioned above, the amino acid sequences of the VH framework are germline V gene IGHV5-51 or their somatic mutations. Those having VH encoded by the received gene are also included. And more preferably, the second arm having VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30 to 34 can be mentioned.

Furthermore, in another embodiment, the 114th amino acid in each amino acid sequence of SEQ ID NOs: 30 to 34 is substituted or substituted with arginine, preferably as the "second arm specifically bound to CD19" in the present invention. A second arm having a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30 to 34 may be mentioned, more preferably the 114th glutamine in each amino acid sequence of SEQ ID NOs: 30 to 34. Is a second arm having a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30-34, substituted with arginine, each of which is any one amino acid selected from SEQ ID NOs: 62-66. Corresponds to the second arm having a VH consisting of an array. These amino acid substitutions can increase the isoelectric point (pI value) of the complex consisting of heavy and light chains having the second arm (hereinafter referred to as the second arm heavy chain / light chain complex). This facilitates the separation of the PD-1 / CD19 bispecific antibody of the present invention, especially from the homodimer of the second arm heavy chain / light chain complex. 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. The isoelectric point of the complex consisting of a heavy chain having a first arm and a light chain (hereinafter referred to as a first arm heavy chain / light chain complex) is preferably about 7.4 to about 7.7, and more preferably. It is about 7.5 to about 7.6.

Further, the "second arm specifically bound to CD19" in the present invention preferably contains the VL of the common light chain in the present specification, and such a common light chain is preferably used, for example, IGVK1-39 /. It is a JK1 common light chain, and more preferably contains, for example, 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 light chain having a VL, more preferably, for example, a 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, a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29 is preferable.

The correspondence between each clone of the anti-CD19 antibody for constructing the PD-1 / CD19 bispecific antibody of the present invention and their VH amino acid sequences and their SEQ ID NOs is shown in FIG. 8, and the anti-CD19 antibody. The correspondence between the amino acid sequence of each CDR in VH of each clone of the above and its SEQ ID NO is shown in FIG.

A preferred embodiment of the PD-1 / CD19 bispecific antibody of the present invention is, for example, a first arm that specifically binds to PD-1.
(A) In any one or more of VH-CDR1, VH-CDR2 and VH-CDR3 in VH indicated by any of the above (1b) to (5b), any one thereof. VH in which up to 5 amino acid residues may be replaced with another amino acid (preferably its conservative amino acid), and (B) VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, amino acid of SEQ ID NO: 27. It has a VL containing VL-CDR2 consisting of the sequence and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28, and
The second arm that specifically binds to CD19
(C) In any one or more CDRs selected from VH-CDR1, VH-CDR2 and VH-CDR3 in the VH indicated by any of the above (1d) to (5d), respectively. VH in which any 1-5 amino acid residues may be substituted with other amino acids (preferably conservative amino acids), and (D) VL-CDR1, SEQ ID NO: 27 consisting of the amino acid sequence of SEQ ID NO: 26. Included are PD-1 / CD19 bispecific antibodies characterized by having a VL comprising VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 28 and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28.

More preferably, for example, the first arm that specifically binds to PD-1
(A) VH represented by any of the above (1b) to (5b), (B) 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 It has a VL comprising VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28, as well as
The second arm that specifically binds to CD19
(C) VH represented by any of the above (1d) to (5d), (D) 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 Included are PD-1 / CD19 bispecific antibodies characterized by having a VL comprising VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 28.

Further, as another preferred embodiment of the PD-1 / CD19 bispecific antibody of the present invention, for example, a first arm that specifically binds to PD-1 may be used.
(A) VH consisting of any one amino acid sequence selected from SEQ ID NOs: 1 to 5, VH consisting of an amino acid sequence that is at least 80% identical to the amino acid sequence of the VH, and (B) amino acid of SEQ ID NO: 25. Has a VL consisting of sequences, as well as
The second arm that specifically binds to CD19
(C) VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30-34 and 62-66, or VH consisting of an amino acid sequence that is at least 80% identical to the amino acid sequence of the VH, and (D) sequence. Included are PD-1 / CD19 bispecific antibodies characterized by having a VL consisting of the amino acid sequence of number 25.

More preferably, for example, a first arm that specifically binds to PD-1
It has (A) a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 1-5, and (B) a VL consisting of the amino acid sequence of SEQ ID NO: 25, and
The second arm that specifically binds to CD19
PD-1 comprising (C) a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 30-34 and 62-66, and (D) a VL consisting of the amino acid sequence of SEQ ID NO: 25. / CD19 Bispecific antibody is mentioned.

Furthermore, as another aspect of this, more preferably, for example, the first arm that specifically binds to PD-1
It has (A) a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 1-5, and (B) a VL consisting of the amino acid sequence of SEQ ID NO: 25, and
The second arm that specifically binds to CD19
PD-1 / CD19 dual, characterized in that (C) a VH consisting of any one amino acid sequence selected from SEQ ID NOs: 62-66, and (D) a VL consisting of the amino acid sequence of SEQ ID NO: 25. Specific antibodies can be mentioned.

The preferred isotype of 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, leucine at position 235 is replaced with glycine and / or glycine at position 236 is replaced with arginine by the EU numbering system on each of the two heavy chain constant regions or hinge regions. The obtained IgG ₁ antibody is preferable. Further, antibodies lacking the amino acid at the C-terminal heavy chain of these bispecific antibodies, eg, lysine at position 447 according to the EU numbering system, are more preferred. When the PD- ₁ / CD19 bispecific antibody is an IgG4 antibody, an antibody in which serine at position 228 by the EU numbering system, which is located in the hinge region thereof, is replaced with proline is preferable.

Furthermore, when these PD-1 / CD19 bispecific antibodies are IgG ₁ antibodies, a preferred embodiment is the EU in a constant region in a heavy chain with a VH of the first arm that specifically binds to PD-1. The 351st leucine by the numbering system was replaced with lysine, and the 366th threonin was replaced with lysine, and the 351st leucine in the constant region in the heavy chain with VH of the second arm specifically bound to CD19 was Examples thereof include IgG ₁ antibody in which aspartic acid is substituted and leucine at position 368 is substituted with glutamate. In addition, leucine at position 351 and leucine at position 368 by the EU numbering system in the constant region in the heavy chain having VH of the first arm specifically bound to PD-1 was replaced with aspartic acid, and leucine at position 368 was replaced with glutamic acid. Also preferred is an IgG ₁ antibody in which leucine at position 351 and threonine at position 366 in the constant region of a heavy chain with a second arm VH that specifically binds to CD19 is replaced with lysine. ..

A preferred embodiment of the PD-1 / CD19 bispecific IgG ₁ antibody incorporating all of the above amino acid substitutions in the heavy chain constant region is, for example, a first arm VH that specifically binds to PD-1. A heavy chain having a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23 and having a VH of the second arm specifically bound to CD19 is selected from SEQ ID NO: 24 and SEQ ID NOs: 71-75. Examples thereof include an antibody having a heavy chain constant region consisting of any one of the amino acid sequences. The amino acid sequences of some of them are illustrated in FIG.

Here, the heavy chain having the VH of the second arm that specifically binds to CD19 has Gly (glycine) and Gly-Lys-Lys at its C-terminal via an amide bond with its C-terminal amino acid. -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). May have. Addition of these amino acids or peptides can increase the isoelectric point (pI value) of the second arm heavy chain / light chain complex, thereby providing the above-mentioned case of amino acid substitution in VH of the second arm. Similarly, in particular, the separation of the bispecific antibody from the homodimer of the second arm heavy / light chain complex is facilitated. The second arm heavy chain / light chain complex to which the amino acid or peptide is added to the C-terminal is also preferably about 8.3 to about 8.9, more preferably about 8.4 to, as its isoelectric point. It is about 8.8.

The most preferable embodiment of the PD-1 / CD19 bispecific antibody of the present invention is clone CD19-1 (Bi), clone CD19-2 (Bi), and clone CD19-produced in Example 12 of the present specification. Examples thereof include cloned CD19-4 (Bi) and cloned CD19-5 (Bi) as well as cloned CD19-6 (Bi) prepared in Example 13.

Preferred features of the PD-1 / CD19 bispecific antibody of the present invention include interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both. To do. Here, "tolerate interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both of them" means "specifically binds to PD-1." It has the same meaning as the definition given in the description of "first arm".

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

Further, a preferred feature of the PD-1 / CD19 bispecific antibody of the present invention is that it has an inhibitory effect on the activation of T cells (for example, memory T cells). The inhibitory effect can be evaluated as a cytokine (eg, IL-2) production inhibitory effect from peripheral blood mononuclear cells including T cells.

Methods for Producing and Purifying PD-1 / CD19 Bispecific Antibodies The PD-1 / CD19 bispecific antibodies of the invention and antibody fragments thereof are described in WO2014 / 051433, WO2013 / 157953 or WO2013 / 157954. But it can be manufactured.

Specifically, (1) a polynucleotide encoding a heavy chain having a VH of the first arm that specifically binds to PD-1, and (2) a weight having a VH of the second arm that specifically binds to CD19. An expression vector in which a polynucleotide encoding a chain and (3) a polynucleotide encoding a common light chain are inserted is introduced into mammalian cells and transformed to express both heavy chains and shared light chains. It can also be produced by secreting it.

Here, the host cell expressing the PD-1 / CD19 bispecific antibody of the present invention may be any host cell capable of introducing an expression vector into a gene and expressing the introduced expression vector. Preferred are 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, primates such as COS and Vero cells. Examples include mammalian cells such as similar cells, MDCK cells, BRL 3A cells, hybridomas, tumor cells, immortalized primary cells, embryonic retinal cells such as W138, HepG2, HeLa, HEK293, HT1080 or PER.C6. In selecting the expression system, an expression vector of mammalian cells and a host may be used so that the antibody is appropriately glycosylated. Human cell lines, preferably PER.C6, are advantageously used to obtain antibodies consistent with the glycosylation pattern in humans.

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

Here, the culture conditions of the host cell can be optimized by a known method, and the amount of protein produced can be optimized. The culture can be carried out, for example, in a culture dish, a roller bottle or a reaction tank by batch culture, fed-batch culture, continuous culture, or culture with hollow fibers. In order to carry out large-scale and continuous production of recombinant proteins by cell culture, it is preferable to grow the cells in a suspension. In addition, it is preferable to culture the cells under the condition that there is no animal or human-derived serum or components of animal or human-derived serum.

Antibodies expressed in host cells and recovered from the cells or cell media 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 and the like. In addition, protein A or protein G affinity chromatography may be preferably used (see, eg, US4801687 and US5151504).

Anti-CD19 Monoclonal Antibody The present invention shall be abbreviated as "monoclonal antibody that specifically binds to CD19" (hereinafter, abbreviated as "anti-CD19 monoclonal antibody" for constructing the PD-1 / CD19 bispecific antibody of the present invention. ) And their antibody fragments.

One aspect of the anti-CD19 monoclonal antibody of the present invention is a monoclonal antibody capable of specifically binding to CD19 by association of VH and VL of the common light chain in the present invention. Here, "specifically binds to CD19" has at least an affinity (dissociation constant (Kd value)) higher than 1x10 ^-5 M, preferably 1x10 ^-7 M, more preferably 1x10 ^-9 M. It can bind directly to CD19 with its binding activity 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 full-length antibody consisting of two heavy chains and two light chains linked by a disulfide bond. .. Further, the "fragment of a monoclonal antibody that specifically binds to CD19" is an antibody that is a part of a full-length antibody and contains at least an antigen-binding portion, and is, for example, Fab, Fab', Fv, scFv and F ( ab') ₂ and the like.

As the anti-CD19 monoclonal antibody of the present invention, for example, any one VH selected from the above (1d) to (5d) constituting the VH of the "second arm specifically binding to CD19" or SEQ ID NO: 30. Included are VHs consisting of any one amino acid sequence selected from ~ 34 and those having a variable region of the common light chain herein (preferably VL consisting of the amino acid sequence of SEQ ID NO: 25).

Further, in the anti-CD19 monoclonal antibody of the present invention, any 1 to 5 amino acid residues thereof are other amino acids in each CDR in any one VH selected from the above (1d) to (5d). Those having a VH having substantially the same binding activity to CD19 by an anti-CD19 monoclonal antibody having the original VH substituted with (preferably a conservative amino acid) and not substituted with the amino acid. Is also included. For example, in the case of CDR1, one amino acid residue is replaced with another amino acid (preferably a conservative amino acid), and in the case of CDR2 or CDR3, 1 to 5 amino acid residues are each other. Amino acids (preferably conservative amino acids) have been substituted. Also, as shown in FIG. 5, in each CDR of an anti-CD19 monoclonal antibody clone, each amino acid that differs between each clone or any combination thereof is interchangeable between the clones. Here, "having substantially the same binding activity to CD19 by an anti-CD19 monoclonal antibody having the original VH not substituted with the amino acid" means anti-PD-1 substituted with the amino acid. The binding activity of the monoclonal antibody 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. It means that it is more than%.

Further, the anti-CD19 monoclonal antibody of the present invention contains each CDR having the above-mentioned specific amino acid sequence in the VH, and the amino acid sequence of the framework of the VH is a specific germline gene or its somatic cell. Some have a VH encoded by the mutated gene, eg, the specific germline gene mentioned above in the description of "the second arm that specifically binds to CD19" or Those with a specific VH encoded by the gene that has undergone the somatic mutation.

Furthermore, among the anti-CD19 monoclonal antibodies of the present invention, each CDR in any one VH selected from the above (1d) to (5d) is contained, and the FR amino acid sequence of the VH is a specific germ line. Examples of those having a VH encoded by the type gene or the gene encoded by the somatic mutation thereof include those having a VH consisting of an amino acid sequence selected from SEQ ID NOs: 30 to 34. Furthermore, such anti-CD19 monoclonal antibodies are, for example, at least 80% identical, preferably at least 90% identical, and more preferably at least at least one amino acid sequence selected from SEQ ID NOs: 30-34. To CD19 by an anti-CD19 monoclonal antibody having a VH consisting of an amino acid sequence that is 95% identical, more preferably at least 98% identical, and even more preferably at least 99% identical, and having the original VH. Those having substantially the same binding activity as the binding activity are also included. Here, "having substantially the same binding activity to CD19 by the anti-CD19 monoclonal antibody having the original VH" means that the anti-CD19 monoclonal antibody having VH consisting of any one of the amino acid sequences is used. It means that the binding activity to CD19 is 95% or more, preferably 98% or more, and more preferably 99% or more.

In yet another embodiment, the anti-CD19 monoclonal antibody of the invention comprises (1) any one VH selected from (1d)-(5d) above or an amino acid sequence selected from SEQ ID NOs: 30-34. An anti-PD-1 monoclonal antibody that cross-competites for binding to CD19 of an anti-CD19 monoclonal antibody having VH and the VL of the common light chain herein (preferably VL consisting of the amino acid sequence of SEQ ID NO: 25), and (2). ) VH consisting of any one VH whose binding to CD19 is selected from (1d) to (5d) above or any one amino acid sequence selected from SEQ ID NOs: 30 to 34 and VL of the common light chain. Also included are anti-CD19 monoclonal antibodies that are cross-competitive with their anti-CD19 monoclonal antibodies.

In still another embodiment, the anti-CD19 monoclonal antibody of the present invention has the 114th amino acid in any one amino acid sequence selected from SEQ ID NOs: 30 to 34 substituted or substituted with arginine. Examples thereof include VHs having a good amino acid sequence and those having a variable region of a common light chain (preferably VL consisting of the amino acid sequence of SEQ ID NO: 25) in the present specification, and more preferably selected from SEQ ID NOs: 30 to 34. The 114th glutamine in any one of the amino acid sequences is having a VH consisting of an amino acid sequence substituted with arginine and a VL of the common light chain, which are selected from SEQ ID NOs: 62 to 66, respectively. Corresponds to an anti-CD19 monoclonal antibody having a VH consisting of one amino acid sequence and a VL of the common light chain.

Polynucleotide encoding PD-1 / CD19 bispecific antibody
The polynucleotide encoding the PD-1 / CD19 bispecific antibody is specific to (1) a polynucleotide encoding a heavy chain having a VH of the first arm that specifically binds to PD-1, and (2) CD19. It is composed of a polynucleotide encoding a heavy chain having a VH of a second arm to which it binds, and (3) a polynucleotide encoding a common light chain. Here, the polynucleotide encoding the heavy chain having the VH of the first arm specifically bound to PD-1 is the polynucleotide encoding the VH of the first arm specifically bound to PD-1 and the VH. A polynucleotide encoding a heavy chain having a second arm VH that specifically binds to CD19 is composed of a polynucleotide encoding a constant region of the heavy chain having a CD19. It is composed of a polynucleotide encoding a two-arm VH and a polynucleotide encoding a constant region of a heavy chain having the VH.

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

Pharmaceutical Use The PD-1 / CD19 bispecific antibodies of the present invention are useful for the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of autoimmune diseases or graft-versus-host disease (GVHD).

Examples of autoimmune diseases that can be prevented, suppressed and / or treated by the PD-1 / CD19 bispecific antibody of the present invention include Bechet's disease, systemic erythematosus, chronic discoid erythematosus, and multiple scleroderma. Diseases (systemic scleroderma, progressive systemic scleroderma), scleroderma, polymyositis, dermatomyitis, nodular periarteritis (nodular polyarteritis, microscopic polyangiitis), aortitis syndrome ( (Hyoan arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Sjogren's syndrome, adult Still disease, vasculitis, allergic granulomatous vasculitis, irritable vasculitis, Rheumatoid vasculitis, macroangiitis, ANCA-related vasculitis (eg, polyangiitis granulomatosis and eosinophilia polyangiitis granulomatosis), Cogan syndrome, RS3PE syndrome, temporal arteritis, rheumatic polymorphism Scleroderma, fibromyopathy, antiphospholipid antibody syndrome, eosinophilia, IgG ₄ -related diseases (eg, primary scleroderma, autoimmune pancreatitis, etc.), Gillan Valley syndrome, severe Myasthenia, chronic atrophic gastric inflammation, autoimmune hepatitis, non-alcoholic steatosis, primary biliary cirrhosis, Good Pasture syndrome, rapidly progressive glomerulonephritis, giant erythroblastic anemia, autoimmune hemolytic anemia , Malignant anemia, autoimmune neutrophilia, idiopathic thrombocytopenic purpura, Basedou disease (Graves disease (scleroderma)), Hashimoto disease, autoimmune adrenal dysfunction, primary hypothyroidism , Azison's disease (chronic hypocortical dysfunction), idiopathic Azison's disease, type I diabetes, slowly progressive type I diabetes (adult latent autoimmune diabetes), localized scleroderma, psoriasis, psoriatic arthritis, blisters Sexual scleroderma, scleroderma, scleroderma, gestational scleroderma, linear IgA bullous dermatosis, acquired epidermal scleroderma, alopecia, white spots, white spots vulgaris, optic neuromyelitis, chronic inflammatory demyelinating Polyneuritis, polyfocal movement neuropathy, sarcoidosis, giant cell arteritis, muscle atrophic scleroderma, Harada's disease, autoimmune optic neuropathy, idiopathic asthenia, habitual abortion, inflammatory bowel disease ( For example, ulcerative colitis, Crohn's disease), Celiac disease, scleroderma, severe asthma, chronic urticaria, transplant immunity, familial Mediterranean fever, autoimmune sinusitis, dilated myocardial disease, systemic obesity Examples include cytosis and inclusion myopathy.

In the present invention, "treatment" means, for example, curing or ameliorating a certain disease or symptom thereof, and "prevention" means preventing or delaying the onset of a certain disease or symptom for a certain period of time. That is, "suppression of symptom progression" means suppressing the progression or worsening of the symptom and stopping the progression of the pathological condition. The meaning of "prevention" also includes suppression of recurrence. "Relapse suppression" means preventing or reducing the likelihood of recurrence of a disease or symptom.

In another aspect, the PD-1 / CD19 bispecific antibody and the like of the present invention are useful for prevention, suppression of symptom progression, suppression of recurrence and / or treatment of autoreactive B cell-mediated diseases. Autoimmune B-cell-mediated diseases include systemic lupus erythematosus, Graves' disease, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary bile duct sclerosis and Pernicious anemia and the like. In the said pharmaceutical use, the PD-1 / CD19 bispecific antibody of the present invention and the like mediate an inhibitory action 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 and the like of the present invention have an inhibitory effect on memory T cell activation. Here, the inhibitory effect on memory T cell activation includes a cytokine production inhibitory effect.

The PD-1 / CD19 bispecific antibodies and the like of the present invention are usually administered systemically or locally in a parenteral form. Specific examples of such an administration method include injection administration, nasal administration, pulmonary administration, and transdermal administration. Examples of the injection administration include intravenous injection, intramuscular injection, intraperitoneal injection and the like, and in the case of intravenous injection, administration by infusion is preferable. The dose varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but is usually in the range of 0.1 μg / kg to 300 mg / kg per adult, particularly preferably. , 0.1 mg / kg to 10 mg / kg, given parenterally once to several times daily, or given intravenously continuously for 30 minutes to 24 hours daily. Of course, as described above, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or administration beyond the range may be necessary.

Preparation When the PD-1 / CD19 bispecific antibody or the like of the present invention is formulated and used as an infusion solution for injection or infusion, the injection or infusion solution is an aqueous solution, suspension or emulsion. It may be in any form of the above, and it is formulated as a solid agent together with a pharmaceutically acceptable carrier so that it can be dissolved, suspended or emulsionized by adding a solvent at the time of use. May be good. Solvents used in infusions for injections or infusions include, for example, distilled water for injection, saline, glucose solutions and isotonic solutions (eg, sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, etc. A solution of sodium chloride, propylene glycol, etc.) can be used.

Here, examples of the pharmaceutically acceptable carrier include stabilizers, lysis aids, suspending agents, emulsifiers, soothing agents, buffers, preservatives, preservatives, pH regulators, antioxidants and the like. Can be mentioned. Stabilizers include, for example, 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, etc. Dibutyl hydroxytoluene and the like can be used. Examples of the solubilizing agent include alcohols (eg, ethanol, etc.), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate 20 (registered trademark), polysorbate 80 (registered trademark)). ), HCO-50, etc.) can be used. As the suspending agent, for example, glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the emulsifier, for example, gum arabic, sodium alginate, tragant and the like can be used. As the pain-relieving agent, for example, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the buffer, for example, a phosphate buffer solution, an acetate buffer solution, a borate buffer solution, a carbon dioxide buffer solution, a citrate buffer solution, a Tris buffer solution, a glutamate buffer solution, an epsilon aminocaproic acid buffer solution and the like can be used. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borophyllate. Sand or the like can be used. As the preservative, for example, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used. As the pH adjuster, for example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid and the like can be used. As antioxidants, for example, (1) water-soluble antioxidants such as (1) ascorbic acid, cysteine hydrochloride, sodium bicarbonate, sodium metabisulfate, sodium sulfite, etc., (2) ascorbic palmitate, butylated hydroxyanisol, etc. Use oil-soluble antioxidants such as butylated hydroxytoluene, lecithin, propyl gallate, α-tocopherol and (3) metal chelating agents such as citric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid, phosphoric acid and the like. Can be done.

Infusions or infusions for infusions can be produced by sterilization in the final step or by aseptic technique, eg, filtering through a filter or the like to sterilize and then filling in a sterile container. Infusions for injections or infusions are vacuum-dried and freeze-dried sterile powders (which may contain pharmaceutically acceptable carrier powders) that are dissolved in a suitable solvent before use. You can also do it.

Combinations or Combinations In addition, the PD-1 / CD19 bispecific antibodies of the present invention may be used in combination with other agents used for prevention, suppression of symptom progression, suppression of recurrence and / or treatment of autoimmune diseases. May be. In the present invention, when used in combination with other drugs (combination), the dosage form may be a combination drug in which both components are mixed in one formulation, or may be administered as separate formulations. There may be. By the combined use, the preventive effect of other drugs, suppression of symptom progression, suppression of recurrence and / or therapeutic effect can be complemented, and the dose or frequency of administration can be maintained or reduced. When the PD-1 / CD19 bispecific antibody or the like of the present invention and another drug are separately administered, they are co-administered for a certain period of time, and then only the PD-1 / CD19 bispecific antibody or the like or another drug is administered. Only the drug may be administered. Further, the PD-1 / CD19 bispecific antibody or the like of the present invention may be administered first, and another drug may be administered after the administration is completed, or the other drug may be administered first, and after the administration is completed. The PD-1 / CD19 bispecific antibody or the like of the present invention may be administered later, and the respective administration methods may be the same or different. It can also be provided as a kit of a preparation containing the PD-1 / CD19 bispecific antibody of the present invention and other agents. Here, the dose of the other drug can be appropriately selected based on the clinically used dose. In addition, other drugs may be administered in combination of any two or more at an appropriate ratio. In addition, the other drugs include not only those found so far but also those found in the future.

For example, when the PD-1 / CD19 bispecific antibody of the present invention is applied to the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of type I diabetes, insulin preparations (eg, human insulin, insulin glargine, insulin) Lispro, insulin detemil and insulin aspart, etc.), sulfonylurea agents (eg, glybenclamid, glycladide, glymepyrid, etc.), fast-acting insulin secretagogues (eg, nateglunide, etc.), biguanide preparations (eg, metformin, etc.), improving insulin resistance Drugs (eg, pioglycazone, etc.), α-glucosidase inhibitors (eg, acarbose and boglibose, etc.), therapeutic agents for diabetic neuropathy (eg, epalrestat, mexiretin, imidazole, etc.), GLP-1 analog preparations (eg, liraglutide, excentide, etc.) , Lixisenatide and semaglutide, etc.) and DPP-4 inhibitors (eg, sitagliptin, vildagliptin, allogliptin, etc.) and may be used in combination with any one or more agents selected.

In addition, for example, when the PD-1 / CD19 bispecific antibody of the present invention is applied to the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of multiple sclerosis, steroid drugs (eg, cortisone acetate, hydrocortisone) , Hydrocortisone sodium phosphate, hydrocortisone sodium succinate, fludrocortisone acetate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisolone butyl acetate, prednisolone sodium phosphate, halopredone acetate, methylprednisolone, methylprednisolone acetate, methylprednisolone succinate , Triamsinolone, Triamsinolone acetate, Triamsinolone acetonide, Dexamethasone, Dexamethasone acetate, Dexamethasone phosphate sodium, Dexamethasone palmitate, Prednisolone acetate and Betamethasone, etc.) It may be used in combination with any one or more agents selected from cyclophosphamide, cyclosporin, methotrexate, cladribine, corticostimulatory hormone (ACTH), corticotropin, misolibin, tachlorimus, fingolimod and alemtuzumab.

Further, for example, when the PD-1 / CD19 bispecific antibody of the present invention is applied to the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of systemic lupus erythematosus, a steroid drug (for example, the steroid drug described above) is used. ), Immunosuppressants (eg, cyclosporine, tacrolimus and fingolimod, etc.) and belimumab may be used in combination with any one or more agents.

For example, when the PD-1 / CD19 bispecific antibody or the like of the present invention is applied to the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of rheumatoid arthritis, a steroid drug (for example, the steroid drug described above), anti. Any one selected from rheumatoid drugs (eg, methotrexate, sulfasalazine, bushilamine, reflunomid, misolivin and tacrolimus, etc.) or anti-cytogenic drugs (eg, infliximab, adalimumab, tosirizumab, etanelcept, golimumab and sertrizumab, etc.) and abatacept. It may be used in combination with a drug.

When applied to 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 and any one of the other agents described above. It may be used in combination with the above.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited thereto. Various modifications and modifications can be made by those skilled in the art based on the description of the present invention, and these modifications and modifications are also included in the present invention.

Example 1: Immunization of MeMo® Mice Using Recombinant Human PD-1-Fc Fusion Protein As a method for obtaining a first arm that specifically binds to PD-1 of the present invention, MeMo (Registration) A method of immunizing recombinant human PD-1 protein in mice (see WO2009 / 157771) was selected. MeMo® mice are gene fragments containing the non-recombinant human heavy chain V gene region, D gene region and J gene region and recombinant human κ light chain IgVκ1-39 * 01 / IGJκ1 * 01 germ cell lineage gene. Is a mouse genetically modified to be linked to a mouse constant region gene, and by directly immunizing the target protein of the antibody, an antibody consisting of a heavy chain having diversity and a common light chain can be produced. ..

Recombinant human PD-1-Fc fusion protein (R & D Systems, model number) emulsified with 12 to 16 week old MeMo (registered trademark) mice using Gerbu adjuvant MM (Gerbu Biotechnik, model number # 3001). 1086-PD) were each immunized at 14-day intervals. The recombinant human PD-1-Fc fusion protein was subcutaneously administered on the 0th, 14th, and 28th days of immunization, and at the subsequent timing, the recombinant human PD-1-Fc fusion protein dissolved in PBS was administered. It was administered subcutaneously. Serum antibody titers were evaluated by flow cytometry using human PD-1 forced expression HEK293T cell lines on days 21, 35, 56, 77 and 98 of immunization. When the human PD-1 forced expression HEK293T cell line was stained with 1000-fold diluted serum, the lymphoid tissue of the mouse whose MFI value was increased by 3 times or more as compared with the control human PD-1 non-expressing HEK293T cell line was obtained. It was used to construct a phage display library. Mice that met the criteria for library construction were boost-immunized with the recombinant PD-1-Fc fusion protein for 3 days from the date of antibody titer evaluation to recover the spleen and spleen lymph nodes. Spleen and spleen lymph nodes were also recovered from mice with serum antibody titers of 1/100 or higher against human PD-1 and cynomolgus monkey PD-1, and whose antibody titers did not increase due to booster immunization. After RNA was extracted from those lymphoid tissues, cDNA synthesis was performed.

Example 2: Construction of a phage display library for obtaining anti-PD-1 antibody (protein immunity)
Using the DNA prepared in Example 1, a PCR reaction was carried out using primers specific for the immunoglobulin heavy chain variable region family. The PCR product was cleaved with restriction enzymes SfiI and XhoI, and then cleaved with the restriction enzyme. Phagemid vector [Gene encoding common light chain (human κ light chain IgVκ1-39 * 01 / IGJκ1 * 01 germline type Includes gene)] to construct the same library.

Example 3: Screening for anti-PD-1 antibody Human PD-1-Fc fusion protein, human PD-1-His tag fusion protein, crab monkey PD-1-His tag fusion protein or mouse PD-1-His tag fusion protein A coated plate was used to perform phage selection based on binding to PD-1. When the human PD-1-Fc fusion protein was used, Fc-reactive clones were absorbed by adding human IgG (SIGMA, model number I4506) during incubation with phage. Binding phage that bind to human PD-1, cynomolgus monkey PD-1 and mouse PD-1 were enriched. Forced expression of cynomolgus monkey PD-1 Selection on the HEK293T cell line enriched phage that bind to cynomolgus monkey PD-1. A clone of Escherichia coli strain TG1 transformed with the phage obtained by the selection was obtained, and a master plate was prepared.

Furthermore, phage selection was performed from the periplasmic extracts of clones obtained from the above selection based on the binding to PD-1 on the plate on which the human PD-1-Fc fusion protein was adsorbed. As a criterion for selection, a positive clone was defined as one in which a signal of 3 times or more was obtained with respect to the signal (OD ₄₅₀ value) obtained in the negative control well (PBS).

Example 4: DNA sequence of candidate clones of anti-PD-1 antibody DNA sequence of heavy chain variable region gene of positive clone obtained by screening of Example 3 was performed. The analyzed DNA sequences are supercluster (a group in which the heavy chain CDR3 has the same length and the amino acid sequence of the same CDR3 is 70% or more homologous) and a cluster (a group in which the amino acid sequence of the heavy chain CDR3 is the same). It was classified into. Hundreds of clones were obtained and they were classified into superclusters and clusters.

Example 5: Screening by evaluation of binding to PD-1 expressing cells From each classified supercluster, anti-PD-1 monoclonal antibody clones satisfying the following conditions were selected and isolated.
(1) Somatic mutations are frequently introduced into the CDR regions,
It has (2) a germline gene for VH that is frequently used, and (3) a high signal was obtained in the binding screening for the human PD-1-Fc fusion protein.

Using the Fab fragment contained in these periplasmic extracts, the binding to the human PD-1 forced expression CHO-S cell line and the crab monkey PD-1 forced expression CHO-S cell line should be detected with an anti-mouse IgG polyclonal antibody. Evaluated by. Of the 117 clones evaluated (105 clusters), 22 clones including anti-PD-1 monoclonal antibody clones PD1-1, PD1-2, PD1-3 and PD1-4 expressed human PD-1 expression CHO. -Binding to S cell lines was observed.

Example 6: Preparation of Amino Acid Substitutes for Anti-PD-1 Monoclonal Antibodies Clone PD1-1 and PD1-4 contain a deamidation motif (Asn-Gly) in framework region 4 of their heavy chain variable regions. For the purpose of obtaining a PD-1 arm with a reduced risk of deamidation, a mutant in which this deamidation motif was converted was prepared. Clone PD1-5 was prepared and isolated so that asparagine (Asn) at position 119 by the EU numbering system of clone PD1-4 was modified to be glutamine by a known site-specific mutation method. The clone also had the same binding ability to human PD-1 forced expression CHO-S cells as clone PD1-4.

Example 7: Immunization of MeMo® Mice Using CD19 Expression plasmid Vector As a method for obtaining a second arm that specifically binds to CD19 of the present invention, see MeMo® mice (WO2009 / 157771). ) Was immunized with a human CD19 expression plasmid vector and a crab monkey CD19 expression plasmid vector.

MeMo® mice are gene fragments containing the non-recombinant human heavy chain V gene region, D gene region and J gene region and recombinant human κ light chain IgVκ1-39 * 01 / IGJκ1 * 01 germ cell lineage gene. Is a mouse genetically modified to be linked to a mouse constant region gene, and is common to heavy chains having diversity by immunizing MeMo® mice with a plasmid vector expressing the target protein of the antibody. Antibodies consisting of light chains can be produced.

Twelve MeMo® mice aged 12-16 weeks were immunized with the human CD19 expression plasmid vector and / or the cynomolgus monkey CD19 expression plasmid vector individually or alternately. Expression plasmid vectors are available on days 0, 3, 6, 14, 17, 28, 31, 42, 49, 63 and / or 70 of immunization. Administered to the eyes. Serum antibody titers were evaluated by flow cytometry using human CD19-expressing cell lines. When a human CD19-expressing cell line was stained with 100-fold diluted sera, a phage display library was constructed of mouse lymphoid tissue in which the MFI value was increased by 3 times or more as compared with the control human CD19-non-expressing cell line. Used for. Mice that met the criteria for library construction recovered the spleen and spleen lymph nodes after boost immunization. After RNA was extracted from those lymphoid tissues, cDNA synthesis was performed by a reverse transcription reaction using an IgG constant region-specific primer.

Example 8: Construction of Phage Display Library for Obtaining Anti-CD19 Antibody A PCR reaction was carried out from the DNA prepared in Example 7 using primers specific for the immunoglobulin heavy chain variable region family. After cleaving the PCR product with a restriction enzyme, a phagemid vector cleaved with the restriction enzyme [gene encoding a common light chain (human κ light chain IgVκ1-39 * 01 / IGJκ1 * 01 germline gene) Included] to build the same library.

Example 9: Screening for anti-CD19 antibody Human CD19-Fc fusion protein (R & D systems, model number 9269-CD), cynomolgus monkey CD19-Fc fusion protein (NovoPro Bioscience, model number 504385), human B cell line Raji or cynomolgus monkey CD19 forced expression HEK293T Cell lines were used to perform phage selection based on binding to CD19. A clone of Escherichia coli strain TG1 transformed with the phage obtained by the selection was obtained, and a master plate was prepared. As a selection criterion, a positive clone was defined as a signal obtained by 3 times or more the signal obtained by the negative control (OD ₄₅₀ value or MFI).

Example 10: DNA sequence of candidate clones of anti-CD19 antibody DNA sequence of heavy chain variable region gene of positive clone obtained by screening of Example 9 was performed. The analyzed DNA sequences were superclusters (a group in which heavy chain CDR3s have the same length and the amino acid sequences of the heavy chain variable regions are 70% or more homologous to each other) and clusters (heavy chain CDR3s and heavy chain variable regions). Classified into a group) in which the amino acid sequences of the above are the same as each other.

In the initial screening, hundreds of clones were obtained and they were classified into superclusters, clusters and 4 germline lines. In the second screening, hundreds of clones were obtained and classified into superclusters, clusters and 8 germline lines. Among them, 19 super clusters were different from the initial screening.

Example 11: Screening by evaluation of binding to CD19 From each classified supercluster, anti-CD19 monoclonal antibody clones satisfying the following conditions were selected and isolated.
(1) Somatic mutations are frequently introduced into the CDR regions,
(2) It has a germline gene for VH, which is frequently used, and (3) a high signal was obtained in the binding screening for CD19.

The Fab fragments contained in those periplasmic extracts were used to assess binding to CD19.

Among the clones evaluated, a plurality of clones including the anti-CD19 monoclonal antibody clones CD19-1, CD19-2, CD19-3, CD19-4 and CD19-5 were found to have binding properties to human CD19-expressing cell lines.

Example 12: Preparation of PD-1 / CD19 bispecific antibody
Expression vectors expressing each heavy chain of the first arm that specifically binds to PD-1 are the anti-PD-1 monoclonal antibody clones PD1-1 to PD1-5 selected in Examples 5 and 6. The DNA encoding each heavy chain variable region was ligated to the DNA encoding the IgG ₁ heavy chain constant region. On the other hand, the expression vector expressing the heavy chain of the second arm that specifically binds to CD19 is the DNA encoding the heavy chain variable region of the anti-CD19 monoclonal antibody clones CD19-1 to CD19-5 selected in Example 11. Was ligated to DNA encoding the IgG ₁ heavy chain constant region. Here, the genes expressing those heavy chain constant regions are those expressing the Fc region having the L351D / L368E mutation (DE mutation) in the case of the first arm that specifically binds to PD-1. In the case of the second arm that specifically binds to CD19, the one expressing the Fc region having the L351K / T366K mutation (KK mutation) was used. These expression vectors were constructed to contain the gene encoding them so that they would also express the IGVK1-39 / JK1 common light chain. Furthermore, in each of the genes expressing these heavy chain constant regions, the 235th leucine in the heavy chain constant region is replaced with glycine and the 236th glycine is replaced with arginine in order to eliminate the Fc effector activity. It was modified and further modified to delete the 447th lysine at the C-terminal of the heavy chain constant region was used to avoid post-translational processing. Both of these expression vectors were gene-introduced into Free Style 293F cells to produce antibodies in the culture supernatant. Clone CD19-1 (Bi) and clone CD19-2 (Bi), which are PD-1 / CD19 bispecific monoclonal antibodies of the present invention, were collected by collecting the culture supernatant and treating with protein A affinity chromatography. ), Clone CD19-3 (Bi), clone CD19-4 (Bi) and clone CD19-5 (Bi), respectively.

These PD-1 / CD19 bispecific antibody clones are derived from the anti-CD19 monoclonal antibody clones CD19-1, CD19-2, CD19-3, CD19-4 and CD19-5 used in the preparation thereof. Each of these anti-CD19 monoclonal antibody clones corresponds in that it has a second arm that specifically binds to CD19. In addition, all of these PD-1 / CD19 bispecific antibody clones have a first arm derived from PD1-5 that specifically binds to PD-1.

Example 13: Preparation of variants of anti-CD19 monoclonal antibody and corresponding PD-1 / CD19 bispecific antibody The PD-1 / CD19 bispecific antibody clone prepared in Example 12 was used for pharmaceutical production. In the scaled-up production process for this purpose, separation and purification from the first arm heavy chain / light chain complex, the second arm heavy chain / light chain complex and / or their respective homodimer by-products are fully achieved. It may not be possible. Therefore, an amino acid variant of an anti-CD19 monoclonal antibody with an increased isoelectric point was prepared for the purpose of improving the separation of the bispecific antibody from the by-product during purification by cation exchange chromatography.

A mutant was prepared in which the 114th glutamine in SEQ ID NO: 30, which represents the amino acid sequence of VH of clone CD19-5, was replaced with arginine by a known site-specific mutation method. In the present invention, the mutant was named "CD19-6". The isoelectric point of each antibody was calculated using gene and amino acid sequence analysis software Geneticx (Genetics Co., Ltd.).

Furthermore, each variation in the form in which glycine and the peptides represented by SEQ ID NOs: 67 to 70 are added to the C-terminal of the heavy chain having VH of CD19-6 (provided that it has the C-terminal amino acid lysine). A body (hereinafter collectively referred to as "CD19-6 / C-terminal peptide adduct") was prepared by a known gene modification method. In the present invention, the variant in which the peptide represented by SEQ ID NO: 67 is added to the C-terminal of CD19-6 was named "CD19-7".

According to the same method as in Example 12, each expression vector described in the same example in which each DNA encoding the variant of these anti-CD19 monoclonal antibodies and the DNA encoding the clone PD1-5 were inserted was used in Free Style 293F cells. The gene was introduced into the culture supernatant to produce an antibody. The culture supernatant was collected and treated by protein A affinity chromatography to purify the bispecific antibody of the present invention derived from each variant prepared in this example. Of these, the same bispecific antibody derived from CD19-6 is referred to as "CD19-6 (Bi)" and the clone of the same bispecific antibody derived from CD19-7 is referred to as "CD19-7 (Bi)". I named it.

Example 14: Examination of purification separation of PD-1 / CD19 bispecific antibody and by-product The PD-1 / CD19 bispecific antibody prepared in Examples 12 and 13, respectively, was produced in their production. We examined the success or failure of purification separation from by-products of concern.

The culture supernatant containing the bispecific antibody recovered in Examples 12 and 13, respectively, was separately treated with protein A affinity chromatography and size exclusion chromatography to obtain the bispecific antibody and by-products. Corresponding anti-PD-1 and anti-CD19 antibodies were purified respectively.

The solvent containing each of these purified antibodies was buffer-replaced by an ultrafiltration method so as to have a pH of 6.0. Each purified antibody after buffer substitution was applied to a cation exchange column TSKgel SP-STAT Column (Tosoh, model number 0021964) equilibrated using buffer A (pH 7.0). Each purified antibody bound to the column was eluted with a salt gradient using buffer B (pH 7.0) containing 1 mol / L sodium chloride. The flow rate of the mobile phase is 0.5 mL / min, elution is a linear gradient from buffer A to buffer B, buffer B is 0% for 0 to 10 minutes after the start of application of each purified antibody, and buffer B is applied for 10 to 40 minutes. Was linearly increased from 0 to 100%, and buffer B was set to 100% for 40 to 50 minutes.

In the cation exchange columns 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). The retention times (minutes) were 15.167, 15.185, 15.749 and 17.521, respectively, and the retention times were extended for clones CD19-6 and CD19-7. On the other hand, clones PD1-3 (isoelectric point: 7.67) and PD1-5 (isoelectric point: 7.52) were eluted without binding to the cation exchange column.

On the other hand, PD-1 / CD19 bispecific antibody clones CD19-2 (Bi) and CD19-5 (Bi) were eluted without binding to the cation exchange column, but clones CD19-6 (Bi) and CD19-7. (Bi) were their retention times (minutes) 13.715 and 14.955.

As described above, the isoelectric point is increased by amino acid substitution in the second arm or addition of a specific peptide to the C terminal of the heavy chain thereof, which is the object of the present invention at the time of cation exchange chromatography purification. The separation between the heavy specific antibody and the by-products anti-PD-1 antibody and anti-CD19 antibody was improved, and it became possible to provide the bispecific antibody of the present invention in which the contamination of the by-product was extremely reduced.

Example 15: Evaluation of binding of PD-1 / CD19 bispecific antibody By Biacore measurement using human IgG1-Fc fusion human PD-1 extracellular region recombinant protein (R & D systems, model number 1086-PD). The binding affinity of the PD-1 / CD19 bispecific monoclonal antibody obtained in Examples 12 and 13 to the PD-1 recombinant protein of the first arm was evaluated. A Series S Sensor Chip CM5 sensor chip (GE Healthcare, model number 29-1049-88) was used to immobilize the recombinant protein.

Similarly, the CD19 binding affinity of the second arm of the antibody was evaluated by Biacore measurement using a human IgG1-Fc fusion human CD19 extracellular space recombinant protein (R & D systems, model 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. It was confirmed that the binding affinity of CD19-6 (Bi) for PD-1 and CD19 was improved as compared with the activity of CD19-5 (Bi) having the structure not subjected to the amino acid substitution of Example 13. rice field.

Example 16: Confirmation of binding of PD-1 / CD19 bispecific antibody The PD-1 / CD19 bispecific antibody obtained in Examples 12 and 13 is human PD-1, crab monkey PD-1, human CD19 and It was confirmed that each specifically binds to crab monkey CD19. Human PD-1 forced expression CHO-S cell line, Crab monkey PD-1 forced expression CHO-S cell line, CHO-S cell line, Human CD19 forced expression CHO-K1 cell line, Crab monkey CD19 forced expression CHO-K1 cell line and Clone CD19-1 (Bi) to CD19-6 (Bi) were added to each of the CHO-K1 cell lines and incubated on ice for 20 minutes. After washing the cells, 100 μL of PE-labeled goat anti-human IgG-Fc F (ab') ₂ fragment antibody (Thermo Fisher, model number H10104) was added and incubated on ice for 20 minutes. After washing the cells, the PD-1 binding property of the first arm and the CD19 binding property of the second arm of the antibody were evaluated by flow cytometry. The results of the assay are shown in FIGS. 12-16.

Both clones bound to human PD-1, cynomolgus monkey PD-1, human CD19 and cynomolgus monkey CD19. No non-specific binding was detected in this experimental system.

It was confirmed that the PD-1 / CD19 bispecific monoclonal antibody obtained in Example 12 simultaneously specifically binds to PD-1 and CD19, respectively. First, clones CD19-1 (Bi) to CD19-5 (Bi) were added to the human CD19 forced expression 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 6 × His tag fusion human PD-1 extracellular space recombinant protein (R & D systems, model number 8986-PD) was added and incubated on ice for 20 minutes. After washing the cells, 100 μL of Alexa Fluor 488-labeled mouse anti-His tag antibody (MBL, model number D291-A48) was added and incubated on ice for 20 minutes. After washing the cells, the amount of binding of human PD-1 extracellular space recombinant protein was evaluated by flow cytometry. The results of the assay are shown in FIG.

Both clones bound to PD-1 and CD19 simultaneously. No non-specific binding was detected in this experimental system.

Example 17: Evaluation of binding characteristics of the first arm of PD-1 / CD19 bispecific antibody PD-1 / PD-L1 of the first arm of PD-1 / CD19 bispecific antibody obtained in Example 12 To evaluate the effect on binding, a competitive binding assay was performed for the binding of the bispecific antibody clone and the soluble PD-L1 recombinant protein to PD-1. First, clones CD19-1 (Bi) to CD19-5 (Bi), nivolumab and anti-human PD-1 antibody J105 (Immunology Letters, 2002, Vol.83,) against human PD-1 forced expression CHO-S cell lines. Issue 3, p.215-220) were added on ice respectively. Furthermore, a soluble PD-L1 recombinant protein (R & D systems, model number 156-B7) biotinylated using a biotin labeling kit (Dojin Kagaku Kenkyusho, model number LK03) was added on ice. After washing the cells, APC-labeled streptavidin (BioLegend, model number 405207) was added on ice. After washing the cells, the amount of soluble PD-L1 recombinant protein bound was evaluated by flow cytometry. The result is shown in FIG.

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

In addition, the bispecific antibody using the anti-PD-1 monoclonal antibody clones PD1-1 to PD1-4 obtained in Example 5 as the first arm was also evaluated as soluble PD-L1 for PD-1. It was confirmed that the binding of recombinant protein was allowed.

Example 18: Activation of PD-1 / CD19 bispecific antibody In vitro inhibitory effect on B cells From healthy human-derived peripheral blood mononuclear cells (LONZA, model number CC-2702), B Cell Isolation Kit II, human (Miltenyi) B cells isolated by Biotec, model number 130-091-151) were used to evaluate their inhibitory effect on human IgM production. Human B cells are seeded on a cell culture plate, and anti-human CD79B antibody (LifeSpan Biosciences, model number LS-C134648), human CD40L recombinant protein (Enzo Life Sciences, model number ALX-522-110), human IL-21 recombinant protein. (R & D systems, model number 8879-IL) was added to perform activation treatment. The clones CD19-1 (Bi) to CD19-5 (Bi) or the control antibody were treated, and IgM contained in the culture supernatant after the activation treatment was quantified by the ELISA method (Thermo Fisher, model number BMS2098). The result is shown in FIG. The clones CD19-1 (Bi) to CD19-5 (Bi) all suppressed IgM production. The IgM production amount (ng / mL) in the figure is shown by the mean value ± standard error (N = 4).

Example 19: Activation of PD-1 / CD19 bispecific antibody In vivo inhibitory effect on B cells NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ mice transplanted with peripheral blood mononuclear cells (LONZA, model number CC-2702) derived from healthy humans (Hereinafter, abbreviated as NSG mouse) was used to evaluate the inhibitory effect on human IgG ₂ production. 1 × 10 ⁷ healthy human-derived peripheral blood mononuclear cells were transplanted per NSG mouse. Clone CD19-1 (Bi) -CD19-4 (Bi) or control antibody at doses of 3 mg / kg daily on days 3, 7, 10, 14, and 17 of transplantation. Each dose was administered intraperitoneally. On the 21st day of transplantation, blood was collected from the tail vein to prepare serum. On the other hand, clones CD19-5 (Bi) and CD19-6 (Bi) were intraperitoneally administered at the same dosages on the 3rd, 7th and 10th days of transplantation, respectively. At this time, the control antibody was intraperitoneally administered in parallel. On the 14th day of transplantation, blood was collected from the tail vein to prepare serum. Human IgG ₂ contained in serum was quantified by the ELISA method (Thermo Fisher, model number BMS2093). The results are shown in FIGS. 20 and 21. The clones CD19-1 (Bi) to CD19-6 (Bi) all suppressed IgG ₂ production. The amount of IgG ₂ produced (μg / mL) in the figure is shown by the average value ± standard error (N = 4 to 8).

Example 20: Assessment of cross-competitiveness of PD-1 / CD19 bispecific antibodies to PD-1 binding
Binding of bispecific antibodies to PD-1 using PD1-1 to PD1-5 clones of anti-PD-1 monoclonal antibodies used to prepare PD-1 / CD19 bispecific antibodies as the first arm. Competitive binding assays were performed to assess cross-competitiveness against.

First, a bispecific antibody using clone PD1-5 as the first arm was added to a human PD-1-expressing CHO-S cell line on ice. Furthermore, bispecific antibodies each using biotin-labeled clones PD1-1 to PD1-5 as the first arm were added 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 amount of biotin-labeled antibody bound to the antibody was evaluated by flow cytometry.

Bispecific antibodies with clone PD1-5 as the first arm inhibit the binding of the same antibody with clones PD1-1 to PD1-4 as the first arm to PD-1, and they are PD-1. Cross-conflict was confirmed for binding to.

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 PD-1 / CD19 bispecific antibody, human peripheral blood mononuclear of the bispecific antibody clone of the present invention and mouse anti-human CD3 antibody OKT3 (BioLegend, model number 317304). Experiments on addition to cells (hereinafter referred to as human PBMC) were carried out.

Clone CD19-6 (Bi) or OKT3 was added to human PBMC (LONZA, model number CC-2702) and cultured. IL-2 contained in the culture supernatant was quantified by flow cytometry using a cytometric bead array (BD Biosciences, model number 551809). The result is shown in FIG. The IL-2 production amount (pg / mL) in the figure is shown by the mean ± standard error (N = 3).

Although OKT3 markedly induced IL-2 production, no IL-2 production by CD19-6 (Bi) was observed.

Example 22: Evaluation of physicochemical stability of PD-1 / CD19 bispecific antibody The PD-1 / CD19 bispecific antibody of the present invention is evaluated for structural stability by differential scanning calorimetry (DSC) and has a diffusion coefficient. Colloidal stability assessment by rate of change (DLS) measurement and chemical stability assessment under stress conditions (eg, pH 3-4 / 5 ° C or pH 7/5 ° C / 5 freeze-thaw) (eg, protein concentration changes, It was confirmed that good performance was exhibited in any of the physical and chemical stability evaluations (molecular structure change, presence / absence of association / aggregation, presence / absence of charge variant generation, structural change and binding to CD19).

PD-1 / CD19 bispecific antibodies or antibody fragments thereof of the present invention are useful for the prevention, suppression of progression of symptoms, suppression of recurrence and / or treatment of autoimmune diseases or graft-versus-host disease (GVHD).

Claims (41)

A bispecific antibody or antibody fragment thereof that has a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19 and which specifically binds to PD-1 and CD19, respectively, is effective. The first arm, which is a pharmaceutical composition contained as an ingredient and specifically binds to PD-1,
(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) VH- consisting of the amino acid sequence of SEQ ID NO: 18. CDR1,
It has any one VH selected from (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.
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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
It has any one VH selected from (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, wherein PD- Remaining any 1-5 amino acids in any one or more CDRs selected from VH-CDR1, VH-CDR2 and VH-CDR3 in VH of the first arm specifically bound to 1. The group may be substituted with a conservative amino acid and / or any one selected from VH-CDR1, VH-CDR2 and VH-CDR3 in VH of the second arm that specifically binds to CD19. Alternatively, a pharmaceutical composition comprising the bispecific antibody or an antibody fragment thereof as an active ingredient, wherein any 1 to 5 amino acid residues thereof may be replaced with conservative amino acids in a plurality of CDRs. The first arm that specifically binds to PD-1
(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) VH- consisting of the amino acid sequence of SEQ ID NO: 18 CDR1,
It has any one VH selected from (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.
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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
The pharmaceutical according to claim 1, which has any one VH selected from (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. Composition. (I) The VH of the first arm that specifically binds to PD-1
(A) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 6.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 7 and (c) 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) 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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
The invention according to claim 1 or 2, wherein (b) any one is selected from VH-CDR2 having 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. Pharmaceutical composition. (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: 9.
It has (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
(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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
The invention according to claim 1 or 2, wherein (b) any one is selected from VH-CDR2 having 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. Pharmaceutical composition. (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: 12.
It has (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.
(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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
The invention according to claim 1 or 2, wherein (b) any one is selected from VH-CDR2 having 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. Pharmaceutical composition. (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.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and (c) VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 17.
(Ii) The VH of the second arm that specifically binds to CD19
(A) (a) VH-CDR1, consisting of the amino acid sequence of SEQ ID NO: 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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
The invention according to claim 1 or 2, wherein (b) any one is selected from VH-CDR2 having 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. Pharmaceutical composition. (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.
It has (b) VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and (c) 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, (c) VH having VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and (E) VH- consisting of the amino acid sequence of SEQ ID NO: 47. CDR1,
The invention according to claim 1 or 2, wherein (b) any one is selected from VH-CDR2 having 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. Pharmaceutical composition. The first arm VHs FR1, FR2 and FR3 that specifically bind to PD-1 are encoded by such genes that may be somatically mutated in the germline V gene IGHV7-4-1. An amino acid sequence encoded by the gene corresponding to each amino acid sequence and in which FR4 may be subjected to a somatic mutation of the germline J gene JH6c (excluding the amino acid sequence contained in the VH-CDR3 region). .) The pharmaceutical composition according to any one of claims 1 to 7. The second arm VHs FR1, FR2 and FR3 that specifically bind to CD19 may be somatically mutated in the germline V gene IGHV5-51, respectively, to the amino acid sequence encoded by that gene. The corresponding pharmaceutical composition according to any one of claims 1 to 8. The VH of the first arm that specifically binds to PD-1 is 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 the VH of the said VH. The pharmaceutical composition according to any one of claims 1 to 9, which comprises an amino acid sequence that is at least 80% identical to the amino acid sequence. The VHs of the second arm that specifically bind to CD19 are 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 The pharmaceutical composition according to any one of claims 1 to 10, comprising any one amino acid sequence selected from SEQ ID NO: 66, or an amino acid sequence that is at least 80% identical to the amino acid sequence of the VH. The VH of the first arm that specifically binds to PD-1 consists of any one of the amino acid sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and is assigned to CD19. The VHs of the second arm specifically bound are 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: The pharmaceutical composition according to claim 1 or 2, which comprises any one amino acid sequence selected from 66. 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 is SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. , Any one of claims 1 to 3, consisting of any one of the amino acid sequences selected from 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 pharmaceutical composition according to the section. 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 is SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and any one of the amino acid sequences selected from SEQ ID NO: 66, according to claim 1, 2 or 4. Pharmaceutical composition. 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 is SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and any one of the amino acid sequences selected from SEQ ID NO: 66, according to claim 1, 2 or 5. Pharmaceutical composition. 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 is SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and any one of the amino acid sequences selected from SEQ ID NO: 66, according to claim 1, 2 or 6. Pharmaceutical composition. 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 is SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. 33, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and any one of the amino acid sequences selected from SEQ ID NO: 66, according to claim 1, 2 or 7. Pharmaceutical composition. A first arm that specifically binds to PD-1 and / or a second arm that specifically binds to CD19, respectively.
(A) VL-CDR1, consisting of the amino acid sequence of SEQ ID NO: 26,
The pharmaceutical composition according to any one of claims 1 to 17, having (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. thing. Any one of claims 1-17, wherein the first arm specifically bound to PD-1 and / or the second arm specifically bound to CD19 each has a VL consisting of the amino acid sequence of SEQ ID NO: 25. The pharmaceutical composition according to the item. A bispecific antibody or antibody fragment thereof that has a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19 and which specifically binds to PD-1 and CD19, respectively, is effective. A pharmaceutical composition contained as an ingredient, which is a pharmaceutical composition.
(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 as the first arm specifically bound to PD-1. The second arm having the VL consisting of the amino acid sequence of SEQ ID NO: 25 and (B) specifically binding to CD19 is SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, The pharmaceutical composition comprising VH consisting of any one of the amino acid sequences selected from 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. A bispecific antibody or antibody fragment thereof that has 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, is effective. The first arm of the pharmaceutical composition contained as an ingredient that specifically binds to PD-1 is selected from (1) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. Binding to PD-1 of the first arm that specifically binds to PD-1, having VH consisting of any one of the amino acid sequences and VL consisting of the amino acid sequence of SEQ ID NO: 25, or (2) the VH and VL. The pharmaceutical composition comprising, cross-competing with the binding of a variable region of a monoclonal antibody specifically binding to PD-1 to PD-1. A bispecific antibody or antibody fragment thereof that has 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, is effective. In the pharmaceutical composition contained as an ingredient, the binding to PD-1 by the first arm that specifically binds to the PD-1 is as follows: (1) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 And a first arm specifically bound to PD-1 or (2) the VH and VL having a VH consisting of any one amino acid sequence selected from SEQ ID NO: 5 and a VL consisting of the amino acid sequence of SEQ ID NO: 25. The pharmaceutical composition comprising, cross-competing by the variable region of a monoclonal antibody that specifically binds to PD-1. Further, the second arm specifically bound to the CD19 is (1) 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, Binding to CD19 of the second arm that specifically binds to CD19, having VH consisting of any one of the amino acid sequences selected from SEQ ID NO: 65 and SEQ ID NO: 66 and VL consisting of the amino acid sequence of SEQ ID NO: 25 ( 2) The pharmaceutical composition according to claim 21 or 22, which cross-competites with the binding of the variable region of the monoclonal antibody specifically binding to CD19 consisting of the VH and VL to CD19. The pharmaceutical composition according to any one of claims 1 to 23, wherein the first arm specifically bound to PD-1 allows an interaction between PD-1 and PD-L1. The pharmaceutical composition according to any one of claims 1 to 24, wherein the bispecific antibody is an IgG antibody. The pharmaceutical composition according to claim 25, wherein the IgG antibody is an IgG ₁ antibody or an IgG ₄ antibody. The pharmaceutical composition according to claim 25, wherein the IgG antibody is an IgG ₁ antibody. The pharmaceutical composition according to claim 27, wherein the binding to the Fc receptor is abolished or attenuated. 28. Claim 28, wherein leucine at position 235 and / or glycine at position 236 in the EU numbering system in the two heavy chain constant regions of the bispecific antibody were each replaced with glycine. Pharmaceutical composition. Leucine at position 351 was replaced with lysine and threonine at position 366 was replaced with lysine by the EU numbering system in the constant region in the heavy chain with VH of the first arm specifically bound to PD-1 to CD19. Any of claims 27-29, wherein the leucine at position 351 in the constant region in the heavy chain having the VH of the second arm specifically bound was replaced with aspartic acid, and the leucine at position 368 was replaced with glutamic acid. The pharmaceutical composition according to paragraph 1. The 351st leucine by the EU numbering system in the constant region in the heavy chain with the VH of the first arm specifically bound to PD-1 was replaced with aspartic acid, and the 368th leucine was replaced with glutamic acid, CD19. Any one of claims 27-29 in which the 351st leucine in the constant region in the heavy chain having the VH of the second arm specifically bound to lysine was replaced with lysine and the 366th threonine was replaced with lysine. The pharmaceutical composition according to the section. The pharmaceutical composition according to any one of claims 27 to 31, each lacking lysine at position 447 according to the EU numbering system in the two heavy chain constant regions of the bispecific antibody. The pharmaceutical composition according to any one of claims 1 to 28, wherein the heavy chain having VH of the first arm specifically bound to PD-1 contains a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23. .. The heavy chain having the VH of the second arm specifically bound to CD19 is any one amino acid 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 pharmaceutical composition according to any one of claims 1 to 29, 31 and 33, which comprises a heavy chain constant region consisting of a sequence. A light chain having a VL of the first arm that specifically binds to PD-1 and / or a light chain having a VL of the second arm that specifically binds to CD19 is a light chain constant consisting of the amino acid sequence of SEQ ID NO: 29. The pharmaceutical composition according to any one of claims 1 to 34, which comprises an area. A bispecific antibody or antibody fragment thereof that has a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD19 and which specifically binds to PD-1 and CD19, respectively, is effective. A pharmaceutical composition contained as an ingredient, which is a pharmaceutical composition.
(A) Any one of the heavy chains having the VH of the first arm that specifically binds to PD-1 is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. It contains VH consisting of the amino acid sequence and a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 23.
(B) A light chain having a VL of the first arm that specifically binds to PD-1 comprises a VL consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 29.
(C) The heavy chain having the VH of the second arm specifically bound to CD19 is 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: It contains a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 24 and VH consisting of any one of the amino acid sequences selected from No. 64, SEQ ID NO: 65 and SEQ ID NO: 66, and specifically binds to (D) CD19. The pharmaceutical composition comprising a light chain having a VL of the second arm comprising 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. The pharmaceutical composition according to any one of claims 1 to 36, further comprising at least one pharmaceutically acceptable carrier. Use of the pharmaceutical composition according to any one of claims 1 to 37 for the prevention, suppression of symptom progression, suppression and / or treatment of autoimmune diseases. Autoimmune diseases include Bechet's disease, systemic erythematosus, chronic discoid erythematosus, polysclerosis, scleroderma, polymyositis, dermatomyitis, nodular periarteritis, aortitis syndrome, malignant rheumatoid arthritis, rheumatoid arthritis. , Juvenile idiopathic arteritis, spondyloarthritis, mixed connective tissue disease, Schegren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, macroangiitis, ANCA-related blood vessels Flame, Cogan syndrome, RS3PE syndrome, temporal arteritis, rheumatic polymyopathy, fibromyalgia, antiphospholipid antibody syndrome, autoimmune myocarditis, IgG ₄ -related disease, Gillan Valley syndrome, severe Myasthenia, chronic atrophic vasculitis, autoimmune hepatitis, non-alcoholic steatosis, primary biliary cirrhosis, Good Pasture syndrome, rapidly progressive glomerulonephritis, giant cell arteritis, autoimmune hemolytic anemia , Malignant anemia, autoimmune neutrophilia, idiopathic thrombocytopenic purpura, Basedow's disease, Hashimoto's disease, autoimmune adrenal dysfunction, primary hypothyroidism, Azison's disease, idiopathic Azison's disease, I Type diabetes, slowly progressive type I diabetes, localized strong skin disease, psoriasis, psoriatic arteritis, vasculitis vasculitis, vasculitis, vasculitis, gestational herpes, linear IgA vesicular dermatosis, acquired epidermis Bulles, round alopecia, leukoplakia, leukoplakia vulgaris, optic neuromyelitis, chronic inflammatory demyelinating polyneuritis, polyfocal motor neuropathy, sarcoidosis, giant cell arteritis, muscle atrophic lateral sclerosis, Harada Diseases, autoimmune optic neuropathy, idiopathic asperemia, habitual abortion, inflammatory bowel disease, ceriac disease, tonic spondylitis, severe asthma, chronic urticaria, transplant immunity, familial Mediterranean fever, eosinophilia Use of the pharmaceutical composition according to claim 38, which is sinusitis, dilated myocardial disease, systemic obesity cell disease or encapsulated myopathy. Insulin preparations, sulfonylurea agents, haste-type insulin secretagogues, biguanide preparations, insulin resistance improving agents, α-glucosidase inhibitors, diabetic neuropathy therapeutic agents, GLP-1 analog preparations, DPP-4 inhibitors, steroids, Interferon β-1a, Interferon β-1b, glatiramer acetate, mitoxanthrone, azathiopurine, cyclophosphamide, cyclosporin, methotrexate, cladribine, adrenocorticotropic hormone (ACTH), corticotropin, misolibin, tacrolimus, fingolimod, alemtuzumab, immunosuppressive The use of the pharmaceutical composition according to claim 38 or 39, characterized in that it is administered with any one or more agents selected from the agent, belimumab, anti-rheumatic agent, anti-cytogenic agent and avatacept. Use of the pharmaceutical composition according to any one of claims 1-37 for the prevention, suppression of symptom progression, suppression and / or treatment of graft-versus-host disease (GVHD).

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