RU2796019C9 - Bispecific antibody - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to a bispecific antibody against PD-1/CD3. Also a pharmaceutical composition for the prevention, suppression of symptoms of progression or recurrence of autoimmune diseases, using the specified antibody is disclosed.

EFFECT: invention allows to carry out effective prophylaxis, suppress symptoms of progression or recurrence of autoimmune diseases.

36 cl, 19 dwg, 15 ex

Description

The field of technology to which the invention belongs

[0001] The present invention relates to a bispecific antibody capable of specifically binding to PD-1 and CD3, respectively (herein referred to as "anti-PD-1/CD3 bispecific antibody"), and a pharmaceutical composition containing said antibody as an active ingredient, as well as to the use of said antibody in therapy.

Prior Art

[0002] PD-1 is an immunosuppressive receptor belonging to the immunoglobulin family and is a molecule having the function of suppressing immune activation signals of T cells activated by stimulation through the antigen receptor. Analysis of PD-1 knockout mice, and the like, has shown that PD-1 signaling pathways play an important role in the suppression of autoimmune diseases such as autoimmune dilated cardiomyopathy, lupus-like syndrome, autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease, type I diabetes, and rheumatoid arthritis. Accordingly, it has been stated that the agent for enhancing the PD-1 signaling pathway can be a prophylactic or therapeutic agent for autoimmune diseases.

[0003] Bispecific antibodies recognizing PD-1 have been previously known as a means of enhancing the PD-1 signaling pathway (Patent Refs 1-3). Bispecific antibodies consist of an antigen recognition site of an antibody recognizing CD3, which is an element of the T-cell receptor complex, and an antigen recognition site of an antibody recognizing PD-1, linked together by genetic engineering techniques. The bispecific antibodies have an activity that enhances the inhibitory signal of PD-1 against the T-cell receptor complex by increasing the frequency of bringing PD-1 into close proximity to the T-cell receptor complex. In addition, patent documents also state that anti-PD-1 bispecific antibodies can be used to prevent or treat autoimmune diseases.

[0004] Where, a side effect in the case of a protein composition is an infusion reaction or a cytokine release syndrome that occurs immediately after administration of the composition. There is a need for formulations in which such reactions are reduced or suppressed.

[0005] For the PD-1/CD3 bispecific antibody of the present invention, post-administration stimulation of cytokine production, which is believed to be the cause of the aforementioned adverse reactions, is significantly reduced. Therefore, the anti-PD-1/CD3 bispecific antibody is expected to be a pharmaceutical formulation that suppresses this undesirable side effect.

[0006] So far, no bispecific antibodies having these characteristics have been reported.

List of cited literature

Patent links

[0007] Patent Reference 1: International Publication No. WO2003/011911

Patent citation 2: International Publication No. WO2004/072286

Patent citation 3: International Publication No. WO2013/022091

The essence of the invention

Technical task

[0008] The purpose of the present invention is to provide a new pharmaceutical agent for the prevention, suppression of symptoms of progression or recurrence of autoimmune diseases, and the like, or their treatment.

The solution of the problem

[0009] The present inventors have carefully studied and focused their studies on the anti-PD-1/CD3 bispecific antibody of the present invention as an agent capable of solving the above problem. The inventors of the present invention also confirmed that the anti-PD-1/CD3 bispecific antibody can be an agent that reduces the manifestation of reactions called infusion reaction or cytokine release syndrome, and came to the present invention.

[0010] In addition, the present inventors found that the anti-PD-1/CD3 bispecific antibody has characteristics that allow interaction between PD-1 and PD-L1 as their ligand, and found that such characteristics contribute to enhancing or maintaining the action of the anti-PD-1/CD3 bispecific antibody.

[0011] That is, the present invention relates to the following objects.

[1] An anti-PD-1/CD3 bispecific antibody or antibody fragment having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3,

where the first arm specifically binding to PD-1 contains any one VH selected from:

(A) a heavy chain variable region (herein, "heavy chain variable region" may be abbreviated as "VH") having

(a) heavy chain variable complementarity determining region 1 (herein, "heavy chain variable complementarity determining region 1" may be abbreviated as "VH-CDR1") comprising the amino acid sequence of SEQ ID NO:6;

(b) heavy chain variable complementarity determining region 2 (herein, "heavy chain variable complementarity determining region 2" may be abbreviated as "VH-CDR2") comprising the amino acid sequence of SEQ ID NO:7; And

(c) heavy chain variable complementarity determining region 3 (herein, "heavy chain variable complementarity determining region 3" may be abbreviated as "VH-CDR3") comprising the amino acid sequence of SEQ ID NO:8;

(B) VH having

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:9;

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:10; And

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:11;

(C) VH having

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:12;

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:13, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:14;

(D) VH having

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:15;

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:16; And

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:17; And

(E) VH having

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:18;

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:19; And

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:20; And

where the second arm specifically binding to CD3 contains a VH having

(a) VH-CDR1 containing the amino acid sequence in SEQ ID NO:37;

(b) VH-CDR2 containing the amino acid sequence in SEQ ID NO:38; And

(c) VH-CDR3 containing the amino acid sequence in SEQ ID NO:39.

[2] The bispecific anti-PD-1/CD3 antibody or antibody fragment of [1], wherein one to five arbitrary amino acid residues can be respectively substituted for another amino acid(s) (preferably a conserved amino acid(s)) in any one or more VH-CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3 in the first arm specifically binding to PD-1; and/or an arbitrary amino acid residue (one to five residues) can be respectively substituted for another amino acid(s) (preferably a conservative amino acid(s)) in any one or more VH-CDRs selected from VH-CDR1, VH-CDR2 and VH-CDR3 in the second arm specifically binding to CD3.

[3] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to the previous paragraph [1] or [2], wherein

(i) The VH of the first arm specifically binding to PD-1 has:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:6,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:7, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:8; And

(ii) VH of the second arm specifically binding to CD3 has:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:37,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:38, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:39.

[4] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to the previous paragraph [1] or [2], wherein

(i) The VH of the first arm specifically binding to PD-1 has:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:9,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:10, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:11; And

(ii) VH of the second arm specifically binding to CD3 has:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:37,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:38, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:39.

[5] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to the previous paragraph [1] or [2], wherein

(i) The VH of the first arm specifically binding to PD-1 has

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:12,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:13, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:14, and

(ii) VH of the second arm specifically binding to CD3 has:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:37,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:38, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:39.

[6] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to the previous paragraph [1] or [2], wherein

(i) The VH of the first arm specifically binding to PD-1 has:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:15,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:16, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:17; And

(ii) VH of the second arm specifically binding to CD3 has:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:37,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:38, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:39.

[7] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to the previous paragraph [1] or [2], wherein

(i) The VH of the first arm specifically binding to PD-1 has:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:18,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:19, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:20; And

(ii) VH of the second arm specifically binding to CD3 has:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:37,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:38, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:39.

[8] An anti-PD-1/CD3 bispecific antibody or antibody fragment having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3,

where the first arm specifically binding to PD-1 contains a VH having:

(a) VH-CDR1 containing the amino acid sequence HYJ ¹ LH [wherein J ¹ represents G (glycine) or (alanine), and the letter designations represented by J ¹ or otherwise represent the single letter abbreviation of the amino acid, respectively]

(b) VH-CDR2 containing the amino acid sequence WJ²NTNTU²NPTX²AQGFTG [where J² represents L (leucine) or I (isoleucine), U² represents E (glutamic acid) or G (glycine), X² represents F (phenylalanine) or Y (tyrosine), and letter designations represented by J², U² or X², or otherwise, represent, respectively, the same as above] and

(c) VH-CDR3 containing the amino acid sequence of GDJ³VVPTTIWNYYU³X³MZ³V [where J³ represents M (methionine) or L (leucine), U³ represents H (histidine) or Y (tyrosine), X³ represents F (phenylalanine) or Y (tyrosine), Z³ represents D (aspartic acid) or E (glutamic acid), and letter designations represented by J³, U³, X³ or Z³, or other letter designations represent, respectively, the same as above] and;

where the second arm specifically binding to CD3 contains a VH having:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:37,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:38, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:39.

[9] The bispecific antibody or antibody fragment against PD-1/CD3 according to the previous paragraph [8], where

(a) J ¹ is G (glycine), J ² is L (leucine), U ² is E (glutamic acid), X ² is F (phenylalanine), J ³ is M (methionine), U ³ is H (histidine), X ³ is F (phenylalanine), and Z ³ is D (aspartic acid);

(b) J ¹ is G (glycine), J ² is I (isoleucine), U ² is G (glycine), X ² is Y (tyrosine), J ³ is L (leucine), U ³ is H (histidine), X ³ is Y (tyrosine), and Z ³ is E (glutamic acid);

(c) J ¹ is A (alanine), J ² is L (leucine), U ² is E (glutamic acid), X ² is Y (tyrosine), J ³ is M (methionine), U ³ is Y (tyrosine), X ³ is Y (tyrosine), and Z ³ is D (aspartic acid); or

(d) J ¹ is A (alanine), J ² is L (leucine), U ² is E (glutamic acid), X ² is F (phenylalanine), J ³ is M (methionine), U ³ is H (histidine), X ³ is F (phenylalanine), and Z ³ is D (aspartic acid).

[10] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1]-[9], wherein framework 1 (herein, may be abbreviated as "FR1"), framework 2 (herein, may be abbreviated as "FR2"), and framework 3 (herein, may be abbreviated as "FR3") in a framework (herein, "framework" may be abbreviated as "FR "") in the VH of the first arm specifically binding to PD-1 correspond to the amino acid sequences encoded by the IGHV7-4-1 germline V gene or its gene with somatic mutation(s), respectively.

[11] The bispecific anti-PD-1/CD3 antibody or antibody fragment according to the previous item [10], wherein the framework 4 (herein, "framework 4" may be abbreviated as "FR4") region in the VH of the first arm specifically binding to PD-1 includes an amino acid sequence (excluding the amino acid sequence included in VH-CDR3) encoded by the germline JH6c gene or its gene with co matic mutation(s).

[12] The bispecific anti-PD-1/CD3 antibody or antibody fragment according to the previous paragraph [10] or [11], wherein the FR region in the VH of the first arm specifically binding to PD-1 is encoded by the IGHV7-4-1 germline V gene with somatic mutation(s), and wherein the FR region contains an FR1 region in which, in the amino acid sequence of SEQ ID NO:21, due to somatic mutation, whether the zine at position 13 is or may be replaced by glutamine, the alanine at position 16 is or may be replaced by valine, or the lysine at position 19 is or may be replaced by methionine, respectively, or they are or may be implemented in an arbitrary variety of combinations thereof.

[13] The bispecific anti-PD-1/CD3 antibody or antibody fragment according to any one of the preceding [10]-[12], wherein the FR region of the VH in the first arm specifically binding to PD-1 is encoded by the germline V gene IGHV7-4-1 with somatic mutation(s), and wherein the FR region contains a FR2 region in which, in the amino acid sequence of SEQ ID NO:21, valine at position 37 is or can be replaced by leucine due to somatic mutation.

[14] The bispecific anti-PD-1/CD3 antibody or antibody fragment according to any one of the preceding [10]-[13], wherein the FR region of the VH in the first arm specifically binding to PD-1 is encoded by the germline V gene IGHV7-4-1 with somatic mutation(s), and wherein the FR region contains an FR3 region in which, in the amino acid sequence of SEQ ID NO:21, due to somatic mutation the serine at position 77 is or can be replaced by threonine, or the cysteine at position 84 is or can be replaced by serine or asparagine, respectively, or they are or can be implemented in an arbitrary variety of combinations thereof.

[15] The bispecific anti-PD-1/CD3 antibody or antibody fragment according to any one of [10]-[14], wherein the FR4 region of the first arm VH specifically binding to PD-1 is encoded by the JH6c germline J gene with somatic mutation(s) (excluding the gene region encoding VH-CDR3), and where in the amino acid sequence (Trp-Gly-Lys-Gly- Thr-Thr*-Val-Thr-Val-Ser-Ser) (SEQ ID NO:41) FR4 regions, lysine (Lys) is or can be replaced by glutamine or asparagine, and/or threonine (Thr) marked with an asterisk is or can be replaced by leucine.

[16] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1]-[15], wherein the VH of the first arm specifically binding to PD-1 comprises any amino acid sequence from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, or an amino acid sequence that is at least 80%, 90%, 95% identical, 98% or 99% by amino acid sequence.

[17] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1] and [3]-[7], wherein the VH of the first arm specifically binding to PD-1 comprises any amino acid sequence from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[18] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1]-[17], wherein the VH of the second arm specifically binding to CD3 comprises the amino acid sequence of SEQ ID NO:36, or an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% amino acid sequence identical.

[19] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1] and [3]-[18], wherein the CD3-specific second-arm VH contains the amino acid sequence of SEQ ID NO:36.

[20] The bispecific antibody or antibody fragment against PD-1/CD3 according to the previous paragraph [1], where the VH of the first arm specifically binding to PD-1 contains any amino acid sequence from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; and the VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36.

[21] An anti-PD-1/CD3 bispecific antibody or antibody fragment having a first arm specifically binding to PD-1 and a second arm specifically binding to CD3, wherein the VH of the first arm specifically binding to PD-1 comprises any amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, or an amino acid sequence that is at least 8 0%, 90%, 95%, 98%, or 99% identical in amino acid sequence; and wherein the VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36, or an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% amino acid sequence identical.

[22] Bispecific antibody or antibody fragment against PD-1/CD3 according to the previous paragraph [1] or [3], where the VH of the first arm, specifically binding to PD-1, contains the amino acid sequence of SEQ ID NO:1; and where the VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36.

[23] The bispecific antibody or antibody fragment against PD-1/CD3 according to the previous paragraph [1] or [4], where the VH of the first arm that specifically binds to PD-1 contains the amino acid sequence of SEQ ID NO:2; and where the VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36.

[24] The bispecific antibody or antibody fragment against PD-1/CD3 according to the previous paragraph [1] or [5], where the VH of the first arm specifically binding to PD-1 contains the amino acid sequence of SEQ ID NO:3; and where the VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36.

[25] The bispecific antibody or antibody fragment against PD-1/CD3 according to the previous paragraph [1] or [6], where the VH of the first arm that specifically binds to PD-1 contains the amino acid sequence of SEQ ID NO:4; and where the VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36.

[26] The bispecific antibody or antibody fragment against PD-1/CD3 according to the previous paragraph [1] or [7], where the VH of the first arm specifically binding to PD-1 contains the amino acid sequence of SEQ ID NO:5; and where the VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36.

[27] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1]-[26], wherein the first arm specifically binding to PD-1 and/or the second arm specifically binding to CD3 has a light chain variable region (herein, "light chain variable region" may be abbreviated as "VL") having

(a) light chain variable complementarity determining region 1 (herein, "light chain variable complementarity determining region 1" may be abbreviated as "VL-CDR1") comprising the amino acid sequence of SEQ ID NO:26;

(b) a light chain variable complementarity determining region 2 (herein, "light chain variable complementarity determining region 2" may be abbreviated as "VL-CDR2") comprising the amino acid sequence of SEQ ID NO:27; And

(c) Light chain variable complementarity determining region 3 (herein, "light chain variable complementarity determining region 3" may be abbreviated as "VL-CDR3") comprising the amino acid sequence of SEQ ID NO:28, respectively.

[28] The bispecific anti-PD-1/CD3 antibody or antibody fragment according to any one of the previous paragraphs [1]-[27], wherein the first arm specifically binding to PD-1 and/or the second arm specifically binding to CD3 have a VL containing the amino acid sequence of SEQ ID NO:25, respectively.

[29] An anti-PD-1/CD3 bispecific antibody or antibody fragment having a first arm specifically binding to PD-1 and a second arm specifically binding to CD3, wherein (A) the first arm specifically binding to PD-1 has a VH containing any amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, and a VL containing the general amino acid sequence of SEQ ID NO:25; And

(B) the second arm specifically binding to CD3 has a VH containing the amino acid sequence of SEQ ID NO:36; and VL containing the amino acid sequence of SEQ ID NO:25.

[30] An anti-PD-1/CD3 bispecific antibody or antibody fragment having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3,

where the first arm specifically binding to PD-1 cross-competes for (1) binding to PD-1 with the first arm specifically binding to PD-1 having a VH containing any amino acid sequence from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; and a VL containing the amino acid sequence of SEQ ID NO:25, or for (2) binding to PD-1 with the variable region of a monoclonal antibody that specifically binds to PD-1, having the same VH and VL.

[31] An anti-PD-1/CD3 bispecific antibody or antibody fragment having a first arm specifically binding to PD-1 and a second arm specifically binding to CD3, wherein the binding to PD-1 of the first arm specifically binding to PD-1 is cross-competed (1) by the first arm specifically binding to PD-1 having a VH containing any of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 , SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; and VL containing the amino acid SEQ ID NO:25, or (2) from the variable region of a monoclonal antibody that specifically binds to PD-1, having the same VH and VL.

[32] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to [30] or [31], wherein the CD3-specific second arm furthermore cross-competes for (1) CD3 binding with the CD3-specific second arm having a VH containing the amino acid sequence of SEQ ID NO:36; and a VL comprising the amino acid sequence of SEQ ID NO:25, or for (2) binding to CD3 with the variable region of a CD3-specific monoclonal antibody having the same VH and VL.

[33] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to [30] or [31], wherein the CD3-specific second arm contains a VH having:

(a) VH-CDR1 containing the amino acid sequence in SEQ ID NO:37;

(b) VH-CDR2 containing the amino acid sequence in SEQ ID NO:38; And

(c) VH-CDR3 containing the amino acid sequence in SEQ ID NO:39.

[34] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [30], [31] and [33], wherein the VH in the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36, or an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% amino acid sequence identical.

[35] The bispecific anti-PD-1/CD3 antibody or antibody fragment of [30] or [31], wherein the VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36.

[36] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [30], [31] and [33]-[35], wherein the CD3-specific second arm has a VL having:

(a) VL-CDR1 containing the amino acid sequence of SEQ ID NO:26;

(b) VL-CDR2 containing the amino acid sequence of SEQ ID NO:27; And

(c) VL-CDR3 containing the amino acid sequence of SEQ ID NO:28.

[37] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [30], [31] and [33]-[35], wherein the CD3-specific second arm has a VL containing the amino acid sequence of SEQ ID NO:25.

[38] The anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1] to [37], wherein the PD-1/CD3 bispecific antibody is an IgG antibody.

[39] The bispecific anti-PD-1/CD3 antibody or antibody fragment of [38], wherein the IgG antibody of [38] is an IgG ₁ or IgG ₄ antibody.

[40] The bispecific antibody or antibody fragment against PD-1/CD3 according to the previous paragraph [38], wherein the IgG antibody according to the previous paragraph [38] is an IgG ₁ antibody.

[41] The bispecific antibody or antibody fragment against PD-1/CD3 according to the previous paragraph [38], wherein the IgG antibody according to the previous paragraph [38] is an IgG ₄ antibody.

[42] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to the previous paragraph [40], wherein in the two heavy chain constant regions of the IgG ₁ antibody according to the previous paragraph [40], the leucines at position 235 according to the EU numbering system are substituted with glycine, and/or the glycines at position 236 according to the EU numbering system are substituted with arginine, respectively.

[43] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to [40] or [42], wherein in the heavy chain constant region having the first arm VH specifically binding to PD-1, both the leucine at position 351 according to the EU numbering system and the threonine at position 366 according to the EU numbering system are replaced by lysine, and in the heavy chain constant region having the second arm VH specifically binding to CD3, the leucine at position 351 according to the EU numbering system is replaced by aspartic acid, and the leucine at position 368 according to the EU numbering system is replaced by glutamic acid.

[44] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to [40] or [42], wherein in the heavy chain constant region having the first arm VH specifically binding to PD-1, the leucine at position 351 according to the EU numbering system is replaced by aspartic acid, and the leucine at position 368 according to the EU numbering system is replaced by glutamic acid, and in the heavy chain constant region having the VH of the second arm specifically binding to CD3, both the leucine at position 351 according to the EU numbering system and the threonine at position 366 according to the EU numbering system are replaced by lysine.

[45] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any of [40] and [42]-[44], wherein in the two heavy chain constant regions of the IgG ₁ antibody according to any of the previous [40] and [42]-[44] Each lysine at position 447 according to the EU numbering system is deleted.

[46] The bispecific anti-PD-1/CD3 antibody or antibody fragment of the previous item [41], wherein in the two heavy chain constant regions of the IgG ₄ antibody of the previous item [41] serines at position 228 according to the EU numbering system are substituted with proline, respectively.

[47] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1]-[38], [40], [42] and [44], wherein the heavy chain having a first arm VH specifically binding to PD-1 has a heavy chain constant region containing the amino acid sequence of SEQ ID NO:23.

[48] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of the preceding paragraphs [1]-[38], [40], [42] [44] and [47], wherein the heavy chain having a second arm VH specifically binding to CD3 has a heavy chain constant region containing the amino acid sequence of SEQ ID NO:24.

[49] The bispecific antibody or antibody fragment against PD-1/CD3 according to any one of the preceding paragraphs [1]-[48], wherein the light chain having the VL of the first arm specifically binding to PD-1 and/or the light chain having the VL of the second arm specifically binding to CD3 has/have a light chain constant region containing the amino acid sequence of SEQ ID NO:29.

[50] An anti-PD-1/CD3 bispecific antibody or antibody fragment having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3, wherein:

(A) a heavy chain having a first arm VH specifically binding to PD-1 has a VH containing any of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and a heavy chain constant region containing the amino acid sequence of SEQ ID NO:23;

(B) a light chain having a first arm VL specifically binding to PD-1 has a VL containing the amino acid sequence of SEQ ID NO:25 and a light chain constant region containing the amino acid sequence of SEQ ID NO:29;

(C) a heavy chain having a second arm VH specifically binding to CD3 has a VH containing the amino acid sequence of SEQ ID NO:36 and a heavy chain constant region containing the amino acid sequence of SEQ ID NO:24; And

(D) A light chain having a second arm VL specifically binding to CD3 has a VL containing the amino acid sequence of SEQ ID NO:25 and a light chain constant region containing the amino acid sequence of SEQ ID NO:29.

[51] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1]-[50], wherein the first arm specifically binding to PD-1 provides interaction between PD-1 and PD-L1.

[52] The bispecific anti-PD-1/CD3 antibody or antibody fragment according to any one of [1] to [51], wherein cytokine production is sufficiently reduced.

[53] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1]-[50], wherein the first arm specifically binding to PD-1 allows interaction between PD-1 and PD-L1, and wherein cytokine production is sufficiently reduced.

[54] The bispecific anti-PD-1/CD3 antibody or antibody fragment according to the previous item [52] or [53], wherein the cytokine comprises at least IL-2, IFN-γ or TNF-α.

[55] An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1] to [54], wherein PD-1 is human PD-1 and CD3 is human CD3, respectively.

[56] The bispecific anti-PD-1/CD3 antibody or antibody fragment according to any one of [1]-[55], wherein CD3 is CD3ε.

[57] The anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1] to [56], wherein the anti-PD-1/CD3 bispecific antibody is a monoclonal antibody.

[58] The anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1] to [57], wherein the anti-PD-1/CD3 bispecific antibody is an isolated antibody.

[1-1] A pharmaceutical composition containing a bispecific antibody or antibody fragment against PD-1/CD3 according to any one of the previous paragraphs [1]-[58] and a pharmaceutically acceptable carrier.

[2-1] A pharmaceutical agent for the prevention, suppression of symptoms of progression or recurrence and/or treatment of an autoimmune disease, comprising an anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1] to [58] as an active ingredient.

[2-2] The pharmaceutical agent according to the previous item [2-1], wherein the autoimmune disease is Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis (systemic scleroderma and progressive systemic sclerosis), scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa and microscopic polyangiitis rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, large vessel vasculitis, ANCA-associated vasculitis (eg, granulomatous matosis with polyangiitis and eosinophilic granulomatosis with polyangiitis), Cogan's syndrome, RS3PE, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related disease (eg, primary sclerosing cholangitis, autoimmune insulitis), Guillain-Barré syndrome, myasthenia gravis , chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic steatohepatitis, primary biliary cirrhosis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease (disease Graves (hyperthyroidism)), Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, Addison's disease (chronic hypoadrenocorticism), Addison's disease of unknown origin, type I diabetes mellitus, slowly progressive type I diabetes mellitus (latent autoimmune diabetes mellitus in adults), focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pe mfigus, pemphigoid, gestational herpes, linear IgA-dependent bullous dermatosis, acquired epidermosis bullosa, alopecia areata, vitiligo, vitiligo vulgaris, optic neuromyelitis, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral mic sclerosis, Harad's disease, autoimmune optic neuropathy, azoospermia of unknown origin, recurrent abortion, inflammatory bowel disease (eg, ulcerative colitis, and Crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, transplantation immunity, familial Mediterranean fever, eosinophilic chronic rhinosinusitis, dilated cardiomyopa tiya, systemic mastocytosis or myositis with body inclusions.

[2-3] A pharmaceutical agent for the prevention, suppression of symptoms of progression or recurrence, and/or treatment of graft-versus-host disease (GVHD), containing a bispecific antibody or antibody fragment against PD-1/CD3 according to any one of the previous [1]-[58] as an active ingredient.

[2-4] A pharmaceutical agent for the prevention, suppression of symptoms of progression or relapse, and/or treatment of type I diabetes mellitus, comprising a bispecific antibody or antibody fragment against PD-1/CD3 according to any one of the preceding paragraphs [1]-[58] as an active ingredient and administered together with any one or more agents selected from an insulin preparation (e.g., human insulin, insulin glargine, insulin lispro, insulin detemir, insulin aspart, etc. the like), a sulfonylurea drug (eg, glibenclamide, gliclazide, or glimepiride), a fast-acting insulin secretion promoter (eg, nateglinide), a biguanide drug (eg, metformin), an agent that affects insulin resistance (eg, pioglitazone), an α-glucosidase inhibitor (eg, acarbose, voglibose, and the like), an agent for treating diabetic neuropathy (eg, epal restat, mexiletine, imidapril, and the like), a GLP-1 analog drug (eg, liraglutide, exenatide, lixisenatide, and the like), and a DPP-4 inhibitor (eg, sitagliptin, vildagliptin, alogliptin, and the like).

[2-5] A pharmaceutical agent for the prevention, suppression of symptoms of progression or recurrence and/or treatment of multiple sclerosis, comprising a bispecific antibody or antibody fragment against PD-1/CD3 according to any of the previous paragraphs [1]-[58] as an active ingredient and administered together with any one or more agents selected from a steroid agent (for example, 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 sodium succinate, triamcinolone, triamcinolone acetate, triamcinolone acetonide, dexam etasone, dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone palmitate, paramethasone acetate or betamethasone and the like), interferon β-1a, interferon β-1b, glatiramer acetate, mitoxantrone, azathioprine, cyclophosphamide, cyclosporine, methotrexate, cladribine, adrenocor ticotropic hormone (ACTH), corticotropin, mizoribine, tacrolimus, fingolimod, and alemtuzumab.

[2-6] A pharmaceutical agent for the prevention, suppression of symptoms of progression or recurrence and/or treatment of systemic lupus erythematosus, containing a bispecific antibody or antibody fragment against PD-1/CD3 according to any of the previous items [1]-[58] as an active ingredient and administered together with any one or more selected from a steroid agent (for example, steroid agents mentioned in the previous item [2-5]), an immunosuppressive agent (for example, cyclo sporin, tacrolimus or fingolimod) and belimumab.

[2-7] A pharmaceutical agent for the prevention, suppression of symptoms of progression or recurrence and/or treatment of rheumatoid arthritis, comprising a bispecific antibody or antibody fragment against PD-1/CD3 according to any of the previous items [1]-[58] as an active ingredient and administered together with any one or more selected from a steroid agent (for example, the steroid agents mentioned in the previous item [2-5]), an antirheumatic agent (for example, a method trexate, sulfasalazine, bucillamine, leflunomide, mizoribine, tacrolimus, and the like), an anti-cytokine agent (eg, infliximab, adalimumab, tocilizumab, etanercept, golimumab, sertolizumab), and abatacept.

[2-8] A pharmaceutical agent for the prevention, suppression of symptoms of progression or relapse and/or treatment of an autoimmune disease, comprising a bispecific antibody or antibody fragment against PD-1/CD3 according to any of the previous items [1]-[58] as an active ingredient and administered together with any one or more of the agents described in the previous items [2-4]-[2-7].

[2-9] A pharmaceutical agent for the prevention, suppression of symptoms of progression or relapse and/or treatment of the diseases mentioned in the previous paragraphs [2-4]-[2-8], administered to a patient who is administered any one or more of the drugs mentioned in the previous paragraphs [2-4]-[2-7].

[2-10] A pharmaceutical agent for the prevention, suppression of symptoms of progression or relapse and/or treatment of the diseases mentioned in the previous paragraphs [2-4]-[2-8], administered after the administration of any one or more drugs mentioned in the previous paragraphs [2-4]-[2-7].

[2-11] A pharmaceutical agent for the prevention, suppression of symptoms of progression or relapse and/or treatment of the diseases mentioned in the previous paragraphs [2-4]-[2-8], administered before the administration of any one or more drugs mentioned in the previous paragraphs [2-4]-[2-7].

[3-1] A composition for intravenous injection, comprising a bispecific antibody or antibody fragment against PD-1/CD3 according to any one of the previous paragraphs [1]-[58] and a pharmaceutically acceptable carrier.

[3-2] Intravenous injection composition according to [3-1] For use in prophylaxis, suppression of progression of symptoms, suppression of recurrence, and/or treatment of an autoimmune disease.

[3-3] Composition for intravenous injection according to the previous paragraph [3-1] or [3-2] For use in drip infusion.

[4-1] An isolated polynucleotide or fragment thereof encoding a heavy chain having a first arm VH specifically binding to PD-1 constituting any one of the PD-1/CD3 bispecific antibodies selected from the previous paragraphs [1]-[58].

[4-2] An isolated polynucleotide or fragment thereof encoding a first arm VH specifically binding to PD-1 constituting any one of the PD-1/CD3 bispecific antibodies selected from the previous paragraphs [1]-[58].

[4-3] The isolated polynucleotide or fragment thereof in the previous paragraphs [4-1] or [4-2], wherein the VH of the first arm specifically binding to PD-1 is encoded by a polynucleotide containing the sequence in any of SEQ ID NOs: 30-34.

[4-4] An isolated polynucleotide or fragment thereof, including a polynucleotide containing the sequence in any of SEQ ID NOs: 30-34.

[5-1] An expression vector having a polynucleotide according to any one of the previous [4-1]-[4-4].

[6-1] An animal cell into which the expression vector of the previous [5-1] is transfected or which is transformed with the transfection vector.

[7-1] A method for preventing, suppressing symptoms of progression or recurrence, and/or treating an autoimmune disease, comprising administering to a patient an effective amount of an anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of [1]-[58].

[8-1] An anti-PD-1/CD3 bispecific antibody or antibody fragment selected from the previous [1] to [58] used for prevention, suppression of symptoms of progression or relapse, and/or treatment of an autoimmune disease.

[9-1] Use of an anti-PD-1/CD3 bispecific antibody or antibody fragment selected from the previous [1] to [58] For the preparation of a pharmaceutical agent for preventing, suppressing symptoms of progression or relapse, and/or treating an autoimmune disease.

[10-1] An isolated anti-PD-1 monoclonal antibody or antibody fragment that cross-competes for binding to PD-1 with an antibody specifically binding to PD-1 having a VH containing any of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and a VL containing the amino acid sequence of SEQ ID NO:25.

[10-2] An isolated anti-PD-1 monoclonal antibody or antibody fragment, wherein binding to PD-1 of the isolated anti-PD-1 monoclonal antibody or antibody fragment is cross-competed by an antibody that specifically binds to PD-1, having a VH containing any amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, and VL, containing the amino acid sequence of SEQ ID NO:25.

[10-3] An isolated anti-PD-1 monoclonal antibody or antibody fragment having any one VH selected from:

(A) VH having:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:6,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:7, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:8,

(B) VH having:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:9,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:10, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:11,

(C) VH having:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:12,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:13, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:14,

(D) VH having:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:15,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:16, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:17, and

(E) VH having:

(a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:18,

(b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:19, and

(c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:20 and VL having:

(a) VL-CDR1 containing the amino acid sequence of SEQ ID NO:26,

(b) VL-CDR2 containing the amino acid sequence of SEQ ID NO:27, and

(c) VL-CDR3 containing the amino acid sequence of SEQ ID NO:28.

[10-4] The isolated anti-PD-1 monoclonal antibody or antibody fragment according to the previous item [10-3], wherein an arbitrary amino acid residue (one to five residues) in any one or more CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3 can be replaced with another amino acid(s) (preferably a conservative amino acid(s)).

[10-5] The isolated monoclonal antibody or anti-PD-1 antibody fragment according to the previous paragraph [10-3] or [10-4] having a VH containing any amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, or a VH containing an amino acid sequence having at least 80%, 90%, 95%, 98% or 99% identity of its amino acid sequence, and VL containing the amino acid sequence of SEQ ID NO:25.

[10-6] The isolated anti-PD-1 monoclonal antibody or antibody fragment according to any one of [10-1] to [10-5], wherein PD-1 is human PD-1.

[10-7] The isolated anti-PD-1 monoclonal antibody or antibody fragment according to any one of [10-1] to [10-6], wherein the anti-PD-1 antibody is an IgG antibody.

[10-8] The isolated anti-PD-1 monoclonal antibody or antibody fragment of the previous item [10-7], wherein the IgG antibody described in the previous item [10-7] is an IgG ₁ antibody or an IgG ₄ antibody.

[10-9] The isolated anti-PD-1 monoclonal antibody or antibody fragment according to the previous item [10-7], wherein the IgG antibody described in the previous item [10-7] is an IgG ₁ antibody.

[10-10] The isolated anti-PD-1 monoclonal antibody or antibody fragment according to the previous item [10-7], wherein the IgG antibody described in the previous item [10-7] is an IgG ₄ antibody.

Results of the invention

[0012] Since the inducibility of PD-1/CD3 cytokine production by the bispecific antibody of the present invention is reduced, the occurrence of an infusion reaction or cytokine release syndrome after administration is reduced. In addition, the PD-1/CD3 bispecific antibody provides an interaction between PD-1 and PD-L1 and is expected to enhance or maintain an effect on preventing, suppressing symptoms of progression or recurrence of an autoimmune disease and/or treating it.

Brief Description of Drawings

[0013] Figure 1 shows each amino acid sequence of the VL and constant region of a conventional light chain.

Figure 2 shows the amino acid sequence of each CDR in the VL of a conventional light chain.

Figure 3 shows the amino acid sequences encoded by the genes: IGHV7-4-1 and IGHV3-33 germline V, respectively.

Figure 4 shows the sequence alignment among the VH of each clone of antibodies specifically binding to PD-1 (which may be abbreviated herein as "anti-PD-1 antibody") and the germline IGHV7-4-1 and JH6c genes. In the drawing, in the amino acid sequence of each clone, "-" represents the same amino acid as the amino acid of the corresponding germline IGHV7-4-1 or JH6c gene, and the simplified character portion of each amino acid represents an amino acid different from the amino acid of the germline gene.

Figure 5 shows the VH amino acid sequence of each anti-PD-1 antibody clone.

6 shows the amino acid sequence of each CDR in the VH of each anti-PD-1 antibody clone.

Figure 7 shows the amino acid sequence of the VH clone CD3-2 as an antibody specifically binding to CD3 (which may be abbreviated herein as "anti-CD3 antibody").

Figure 8 shows the amino acid sequence of each CDR in the VH of clone CD3-2 as an anti-CD3 antibody.

Figure 9 shows the amino acid sequence of the constant region in each heavy chain bispecific anti-PD-1/CD3 monoclonal antibody.

Figure 10 shows the amino acid sequence of the VH clone 15C3 of the anti-CD3 antibody described in WO2005/118635. Here, it should be noted that the underlined amino acid represents the 55th glycine, which is converted to alanine when clone CD3-1 is obtained.

FIG. 11 shows Biacore measurement results showing PD-1 and CD3 binding activity of each clone of the anti-PD-1/CD3 bispecific monoclonal antibody.

FIG. 12 shows flow cytometry demonstrating the property of simultaneous binding to PD-1 and CD3 of each clone of the anti-PD-1/CD3 bispecific monoclonal antibody.

Figure 13 shows flow cytometry showing the effect on interaction with PD-1/PD-L1 of each clone of a bispecific anti-PD-1/CD3 monoclonal antibody.

Figure 14 shows the effect on IFN-γ production in activated human T cells of each clone of a bispecific anti-PD-1/CD3 monoclonal antibody. It should be noted here that in the drawing "Ctrl" represents the control group.

In FIG. 15 shows the therapeutic effect of anti-PD-1/CD3 bispecific monoclonal antibody clones (PD1-1(Bi) and PD1-2(Bi)) in an experimental mouse model of allergic encephalomyelitis (EAE models).

In FIG. 16 shows the therapeutic effect of anti-PD-1/CD3 bispecific monoclonal antibody clones (PD1-3(Bi) and PD1-4(Bi)) in an experimental mouse model of allergic encephalomyelitis.

In FIG. 17 shows the therapeutic effect of anti-PD-1/CD3 bispecific monoclonal antibody clones (PD1-5(Bi) and PD1-6(Bi)) in an experimental mouse model of allergic encephalomyelitis.

In FIG. 18 shows the effect on cytokine production in human peripheral blood mononuclear cell of each clone of anti-PD-1/CD3 bispecific monoclonal antibody.

In FIG. 19 shows PD1-5(Bi) cross-competitive activity against PD-1 binding of each anti-PD-1/CD3 bispecific monoclonal antibody clone.

Description of Embodiments

[0014] Human PD-1 (programmed cell death protein-1) is a membrane-type protein consisting of the amino acid sequence represented by GenBank accession number NP_005009. In the present description, the term "PD-1", unless specifically indicated otherwise, can be used in the sense that includes all its isoforms, and its variants, which retain the epitopes for the "first arm that specifically binds to PD-1" in the present invention. In the present invention, PD-1 is preferably human PD-1.

[0015] CD3 is a membrane-type protein that forms a T-cell receptor complex by binding to a T-cell receptor. In the present description, the term "CD3", unless specifically indicated otherwise, can be used in the sense that includes the subtype (subtype ε, δ, γ and ζ) and variants thereof, which retains the epitopes for the "second arm specifically binding to CD3", according to the present invention. In the present invention, CD3 is preferably CD3ε and human CD3, and more preferably human CD3ε.

[0016] As used herein, the term "isolated" means that a single, substantially pure component is obtained by identifying, separating, and/or purifying impurities, including a plurality or a large number of components recovered from a host cell.

[0017] In the present description, the term "monoclonal antibody" means an antibody derived from a group of essentially homogeneous antibodies that bind to the same specific antigen.

[0018] In this specification, the term "bispecific antibody" means an antibody having binding specificity for two different antigen molecules or epitopes in the molecule. In addition, the term "bispecific monoclonal antibody" means a bispecific antibody derived from a group of substantially homogeneous antibodies.

[0019] The present invention relates to a bispecific antibody capable of specifically binding to PD-1 and CD3 (may be abbreviated herein as "anti-PD-1/CD3 bispecific antibody"). In the present invention, the anti-PD-1/CD3 bispecific antibody is preferably an anti-PD-1/CD3 bispecific monoclonal antibody, and more preferably an isolated anti-PD-1/CD3 monoclonal bispecific antibody and even more preferably an isolated anti-human PD-1/human CD3 monoclonal antibody. As used herein, "isolated anti-human PD-1/human CD3 bispecific monoclonal antibody" means an isolated bispecific monoclonal antibody for human PD-1 and human CD3.

[0020] Exemplary embodiments of an anti-PD-1 bispecific antibody include, for example, diabodies, sc(Fv) ₂ bispecifics, mini-antibodies, F(ab') 2 bispecifics, bispecific fusion antibodies, covalent diabodies (DART bispecifics), ( _{FvCys) 2 bispecifics, F(ab'-zipper) 2 bispecifics, bispecific (Fv-zipper) 2 , bispecific three} -chain antibodies and bispecific mAb ² and the like.

[0021] Diatelo is a dimer of single-stranded peptides in which VH and VL, recognizing different antigens, are linked to each other by a peptide linker (Proc. Natl. Acad. Sci. USA, 1993, Vol. 90, No. 14, pp. 6444-6448).

[0022] A bispecific sc(Fv) ₂ is a small molecule antibody modified such that two VH/VL pairs in two antibodies recognizing different antigens are linked to each other by a peptide linker in a continuous single chain form (see J. Biological Chemistry, 1994, 269: pp. 199-206).

[0023] A bispecific F(ab') ₂ is a small molecule antibody in which Fab' fragments of antibodies recognizing two different antigens are covalently linked, for example, via a disulfide bond.

[0024] A bespecifically mini-antibody is a small molecule antibody in which small molecular weight antibody fragments modified such that the antibody constant region CH3 domains are linked to scFvs that recognize different antigens are covalently linked, for example, by disulfide bonds on the CH3 domains (see Biochemistry, 1992, Vo. 31, No. 6, pp. 1579-1584).

[0025] A bispecific hybrid antibody is an intact antibody in which heavy chain/light chain complexes recognizing two different antigens are covalently linked to each other, eg, by a disulfide bond.

[0026] In the present invention, examples of a form of a preferred bespecifically antibody include a bespecifically hybrid antibody.

[0027] A bispecific hybrid antibody can be obtained, for example, from a hybridoma using the interspecific hybridoma method (see US4474893). Alternatively, the bispecific hybrid antibody can be produced by secretion from a mammalian cell co-expressing four types of cDNA, respectively, encoding heavy chain and light chain of antibodies recognizing different antigens.

[0028] Monoclonal antibodies used in the present invention can be obtained using the hybridoma method (see, for example, Kohler and Milstein et al. Natur (1975), Vol. 256, p. (Cold Spring Harbor Laboratory Press (1988), Vol. 2) and Hammerling et al. Monoclonal Antibodies and T-Cell Hybridomas, pp.563-681 (Elsevier, N.Y. 1981)), recombinant DNA method (see e.g. US4816567), phage display method (e.g. Ladner et al. US5223409, US5403 484 and US5571698, Dower et al. 93081).

[0029] An antibody or monoclonal antibody, when administered to a human, can be formulated as a chimeric antibody, a humanized antibody, or a complete human antibody to reduce or eliminate its antigenicity.

[0030] The term "chimeric antibody" means an antibody whose constant region sequence and variable region sequence are derived from another mammal. Examples of the chimeric antibody include an antibody whose variable region sequence is derived from a mouse antibody and whose constant region sequence is derived from a human antibody. A chimeric antibody can be obtained by linking a gene encoding the variable region of an antibody isolated from the aforementioned antibody-producing hybridoma isolated by hybridoma, recombinant DNA or phage display using well-known techniques to a gene encoding a constant region of an antibody of human origin using a well-known method (see, for example, Cabilly et al. US4816567).

[0031] The term "humanized antibody" means an antibody obtained, for example, by transplanting a complementarity determining region (CDR) sequence of an antibody derived from the germline of a non-human mammal, such as a mouse, into human human antibody framework sequences. Also, a humanized antibody can be obtained by linking genes encoding the CDR region of an antibody isolated from the aforementioned antibody-producing hybridoma isolated by the aforementioned method to a gene encoding a framework region of an antibody of human origin using a well-known method (see, for example, Winter, US5225539 and US5530101; Queen et al. US5585089 and US6180370.

[0032] The term "human antibody" or "total human antibody" means an antibody in which both the variable region, consisting of framework regions and CDR regions, and the constant region are derived from human germline immunoglobulin sequences. a human antibody to be used in the present invention can be obtained using a method using a mouse transformed to produce human antibodies, for example, a Humab mouse (see, for example, Lonberg and Kay et al. 14318, US5874299 and US5770429), KM mice (see e.g. Ishida et al. WO2002/43478), Xeno mice (see e.g. US5939598, US6075181, US6114598, US6150584 and US6162963) or Tc mice (see e.g. Tomizuka et al. Proc Natl Acad Sci USA (2000), pp.722-727). human antibody can also be generated using SCID mice that have been transplanted with human immune cells such that a human antibody response occurs upon immunization (see, for example, US5476996 and US5698767 to Wilson et al). In addition, the human antibody used in the present invention can also be obtained using the above-mentioned phage display method.

[0033] As used herein, the term "antibody fragment" of an anti-PD-1/CD3 bispecific antibody is part of a full-length antibody, and is an antibody having an anti-PD-1 antigen-binding portion and an anti-CD3 antigen-binding portion. His examples include F(ab′) ₂ and the like. As used herein, antigen-binding portion means the smallest unit of an antibody that can bind to its antigen and, for example, it consists of three CDRs in each of VH and VL and framework regions to align the CDRs such that the target antigen can be recognized by a combination of these CDRs.

[0034] As used herein, the term "total light chain" means a light chain that can be linked to two or more different heavy chains and that can exhibit the ability to bind to each antigen (De Wildt RM, J. Mol. Biol. (1999), Vol. 285, pp. 895-901, De Kruif et al. J. Mol. Biol. (2009), Vol. 387, pp. 548 -58, WO2004/009618, WO2009/157771 and WO2014/051433). Preferred examples of such a conventional light chain include the light chain encoded by the κ IgVκ1-39*01/IGJκ1*01 human light chain germline gene (IMGT database nomenclature) (herein, abbreviated "IGVK1-39/JK1 common light chain"). More preferred examples include a light chain containing a VL having a CDR1 containing the amino acid sequence of SEQ ID NO:26, a CDR2 containing the amino acid sequence of SEQ ID NO:27, and a CDR3 containing the amino acid sequence of SEQ ID NO:28, and even more preferred examples include a light chain containing a VL containing the amino acid sequence of SEQ ID NO:25. In addition, preferred examples of a conventional light chain constant region include a light chain constant region comprising the amino acid sequence of SEQ ID NO:29. Each amino acid sequence of the VL and conventional light chain constant region used in the present invention is shown in FIG. 1 is shown, and the amino acid sequence of each variable region CDR is shown in FIG. 2.

[0035] As used herein, the term "isotype" refers to the class of antibody (eg, IgM or IgG) that is encoded by heavy chain constant region genes. Preferred isotype examples for the PD-1/CD3 bispecific antibody of the present invention include IgG, and more preferably IgG ₁ or IgG ₄ . Preferred is the IgG ₁ used herein, the binding of which to the Fc receptor is eliminated or reduced. Specifically, an IgG ₁ antibody whose binding to an Fc receptor is eliminated or reduced can be obtained by substitution, deletion or insertion of an arbitrary amino acid of the heavy chain constant region. Examples thereof include an antibody in which the leucine at position 235 according to the EU numbering system is replaced by glycine and/or the glycine at position 236 according to the EU numbering system is replaced by arginine in each of the two heavy chain constant regions or the hinge region. In addition, in order to reduce antibody heterogeneity, an antibody in which an amino acid at the C-terminus has been removed, such as lysine at position 447 according to the EU numbering system, is preferred. In addition, when the bispecific antibody is IgG ₄ , in order to prevent rearrangement in the antibody molecule, it is preferable to replace, delete or insert an arbitrary amino acid in the constant region of its heavy chain. For example, an antibody is preferred in which the serine, located in the hinge region and at position 228 according to the EC numbering system, is replaced by a proline. It should be noted here that in the description, the positions of amino acids destined for CDRs and scaffolds in the variable region of an antibody are indicated according to Kabat numbering (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987 and 1991)). In addition, amino acids in the constant region are represented according to the EC numbering system based on Kabat amino acid positions (see Sequences of proteins of immunological interest, NIH Publication No. 91-3242).

[0036] In the Fc regions of the anti-PD-1/CD3 bispecific antibody of the present invention, arbitrary amino acids can be changed therein so that two different heavy chains easily bind to each other. Examples of preferred embodiments include an anti-PD-1/CD3 bispecific antibody in which, in the heavy chain constant region having a first arm VH specifically binding to PD-1, the leucine at position 351 according to the EU numbering system is replaced by lysine and the threonine at position 366 according to the EU numbering system is replaced by lysine; and in which, in the heavy chain constant region having a second arm VH specifically binding to CD3, the leucine at position 351 according to the EU numbering system is replaced by aspartic acid, and the leucine at position 368 according to the EU numbering system is replaced by glutamic acid. Examples also include an anti-PD-1/CD3 bispecific antibody in which, in a constant region in the heavy chain having a first arm VH specifically binding to PD-1, the leucine at position 351 according to the EU numbering system is replaced with aspartic acid, and the leucine at position 368 is replaced with glutamic acid; and in which, in the constant region in the heavy chain having the VH of the second arm specifically binding to CD3, the leucine at position 351 according to the EU numbering system is replaced by lysine, and the threonine at position 366 according to the EU numbering system is replaced by lysine.

[0037] First arm specifically binding to PD-1

As used herein, "PD-1 specific binding first arm" (which may be abbreviated herein as "first arm") means an antibody portion containing at least the VH of an antibody that specifically binds to PD-1 (which may be abbreviated herein as "anti-PD-1 antibody"), whether contained in an antibody portion or an antibody fragment, or exists not as a part but as a separate body. For example, such a first arm consists of the VH of an anti-PD-1 antibody and the VL of a conventional light chain constituting the same anti-PD-1 antibody. In addition, examples of the first arm also include the Fab portion of the antibody containing VH and VL. In this document, the term "specifically binding to PD-1" is used as an indication that it can bind directly to PD-1 with a binding activity of at least 1x10 ^-5 M, preferably 1x10 ^-7 M, and more preferably more than 1x10 ^-9 M (dissociation constant (Kd value)), and essentially does not bind to other receptor elements belonging to the so-called receptor of the CD28 family, for example, at least CD28, CTLA-4 and ICOS. In addition, "antibody" in "antibody that specifically binds to PD-1" or "anti-PD-1 antibody" means a full-length antibody consisting of two heavy chains and two light chains linked by disulfide bonds, and preferably a monoclonal antibody.

[0038] As used herein, examples of the "first arm specifically binding to PD-1" include:

(a) VH-CDR1 containing the amino acid sequence HYJ ¹ LH [wherein J ¹ represents G (glycine) or (alanine), and herein, the letter designations represented by J ¹ or otherwise represent the single letter abbreviation of the amino acid, respectively];

(b) VH-CDR2 containing the amino acid sequence WJ²NTNTU²NPTX²AQGFTG [where J² represents L (leucine) or I (isoleucine), U² represents E (glutamic acid) or G (glycine), X² represents F (phenylalanine) or Y (tyrosine), and herein the letter designators represented by J², U² or X² or otherwise, represent, respectively, the same as above]; And

(c) VH-CDR3 containing the amino acid sequence of GDJ³VVPTTIWNYYU³X³MZ³V [where J³ represents M (methionine) or L (leucine), U³ represents H (histidine) or Y (tyrosine), X³ represents F (phenylalanine) or Y (tyrosine), Z³ represents D (aspartic acid) or E (glutamic acid), and herein the letter designators represented by J³, U³, X³ or Z³ or other letter designations represent, respectively, the same as above].

[0039] As used herein, preferred embodiments of the "first arm specifically binding to PD-1" include those comprising:

(1a) a VH having each VH-CDR wherein J ¹ in the sequence HYJ ¹ LH as VH-CDR1 is G (glycine); in WJ ² NTNTU ² NPTX ² AQGFTG as VH-CDR2, J ² is L (leucine), U ² is E (glutamic acid), and X ² is F (phenylalanine), respectively; in the sequence GDJ ³ VVPTTIWNYYU ³ X ³ MZ ³ V as VH-CDR3, J ³ is M (methionine), U ³ is H (histidine), X ³ is F (phenylalanine), and Z ³ is D (aspartic acid), respectively;

(2a) a VH having each VH-CDR wherein J ¹ in the sequence HYJ ¹ LH as VH-CDR1 is G (glycine); in WJ ² NTNTU ² NPTX ² AQGFTG as VH-CDR2, J ² is I (isoleucine), U ² is G (glycine), and X ² is Y (tyrosine), respectively; in the sequence GDJ ³ VVPTTIWNYYU ³ X ³ MZ ³ V as VH-CDR3, J ³ is L (leucine), U ³ is H (histidine), X ³ is Y (tyrosine), and Z ³ is E (glutamic acid), respectively;

(3a) a VH having each VH-CDR wherein J ¹ in the sequence HYJ ¹ LH as VH-CDR1 is A (alanine); in WJ ² NTNTU ² NPTX ² AQGFTG as VH-CDR2, J ² is L (leucine), U ² is E (glutamic acid), and X ² is Y (tyrosine), respectively; in the sequence GDJ ³ VVPTTIWNYYU ³ X ³ MZ ³ V as VH-CDR3, J ³ is M (methionine), U ³ is Y (tyrosine), X ³ is Y (tyrosine), and Z ³ is D (aspartic acid), respectively; And

(4a) a VH having each VH-CDR wherein J ¹ in the sequence HYJ ¹ LH as VH-CDR1 is A (alanine); in WJ ² NTNTU ² NPTX ² AQGFTG as VH-CDR2, J ² is L (leucine), U ² is E (glutamic acid), and X ² is F (phenylalanine), respectively; J ³ is M (methionine), U ³ is H (histidine), in the sequence GDJ ³ VVPTTIWNYYU ³ X ³ MZ ³ V as VH-CDR3, X ³ is F (phenylalanine) and Z ³ is D (aspartic acid), respectively.

[0040] In addition, as preferred examples of another embodiment, the "PD-1 specific binding first arm" comprises any one VH selected from:

(1b) a VH having a VH-CDR1 containing the amino acid sequence of SEQ ID NO:6, a VH-CDR2 containing the amino acid sequence of SEQ ID NO:7, and a VH-CDR3 containing the amino acid sequence of SEQ ID NO:8;

(2b) a VH having a VH-CDR1 containing the amino acid sequence of SEQ ID NO:9, a VH-CDR2 containing the amino acid sequence of SEQ ID NO:10, and a VH-CDR3 containing the amino acid sequence of SEQ ID NO:11;

(3b) a VH having a VH-CDR1 containing the amino acid sequence of SEQ ID NO:12, a VH-CDR2 containing the amino acid sequence of SEQ ID NO:13, and a VH-CDR3 containing the amino acid sequence of SEQ ID NO:14;

(4b) a VH having a VH-CDR1 containing the amino acid sequence of SEQ ID NO:15, a VH-CDR2 containing the amino acid sequence of SEQ ID NO:16, and a VH-CDR3 containing the amino acid sequence of SEQ ID NO:17; And

(5b) a VH having a VH-CDR1 containing the amino acid sequence of SEQ ID NO:18, a VH-CDR2 containing the amino acid sequence of SEQ ID NO:19, and a VH-CDR3 containing the amino acid sequence of SEQ ID NO:20.

[0041] In addition, the "first arm specifically binding to PD-1" of the present invention includes those in which 1-5 arbitrary amino acid residue(s) is/are replaced by another amino acid(s) (preferably, its conserved amino acid(s) in each VH-CDR of any one of the VHs selected from the above (1a)-(4a) or (1b)-(5b), and which have both essentially the same PD-1 binding activity as the original first arm without any amino acid(s) substitutions.For example, for VH-CDR1, one amino acid residue is replaced by its conservative amino acid.For VH-CDR2 or VH-CDR3, one or two amino acid residues are/are replaced by its conservative amino acid(s), respectively.As used herein, a substitution by a conservative amino acid means the ability to be substituted for a residue having a similar side chain.For example, a group of amino acids having aliphatic side chain, includes glycine, alanine, valine, leucine and isoleucine; a group of amino acids having an aliphatic hydroxyl side chain includes serine and threonine; a group of amino acids having an amide-containing side chain includes asparagine and glutamine; a group of amino acids having an aromatic side chain includes phenylalanine, tyrosine and tryptophan; a group of amino acids having a basic side chain includes lysine, arginine and histidine; and the group of amino acids having a sulfur-containing side chain includes cysteine and methionine. Preferred examples of a conservative amino acid substitution include a substitution between valine, leucine and isoleucine, a substitution between phenylalanine and tyrosine, a substitution between lysine and arginine, and a substitution between alanine and valine, and a substitution between asparagine and glutamine. In addition, herein, the expression "which have substantially the same PD-1 binding activity as the original first arm without any amino acid(s) substitutions" mentioned above means that the PD-1 binding activity of the first arm with the same amino acid(s) substitution is 95% or more, preferably 98% or more, and more preferably 99% or more, compared to the activity of the original first arm without any same amino acid substitutions. oh (amino acids).

[0042] In addition, the "first arm specifically binding to PD-1" of the present invention includes an arm that contains a VH having each VH-CDR having the aforementioned specific amino acid sequence, and in which the VH backbone is encoded by a specific germline gene or a gene with somatic mutation(s). For example, a VH represented by any one selected from the above (1a)-(4a) or (1b)-(5b) may be encoded by a recombinant VDJ gene, wherein the V germline gene is IGHV7-4-1 and the J germline gene is JH6c or a gene with somatic mutation(s) thereof. As used herein, the amino acid sequence encoded by the V germline IGHV7-4-1 gene corresponds to the amino acid sequence of SEQ ID NO:21 (FIG. 3).

[0043] The first arm VH frameworks specifically binding to PD-1 of the present invention may be encoded by a recombinant VDJ germline gene with somatic mutation(s). For example, since the FR1, FR2 and FR3 of the VH represented by any one selected from the above (1a)-(4a) or (1b)-(5b) in which the V germline genome is IGHV7-4-1 differs from the amino acid sequence is encoded by the IGHV7-4-1 gene, at the amino acid position shown in FIG. 4, they undergo somatic mutation in each of the positions. For example, with respect to the FR1 region, in the amino acid sequence of SEQ ID NO:21, lysine at position 13 can be replaced by glutamine, alanine at position 16 can be replaced by valine, or lysine at position 19 can be replaced by methionine, respectively, or they can be replaced in an arbitrary variety of combinations thereof. With respect to the FR2 region, the valine at position 37 in the amino acid sequence of SEQ ID NO:21 has been replaced by leucine. With regard to the FR3 region, in the amino acid sequence of SEQ ID NO:21, the serine at position 77 can be replaced by threonine, or the cysteine at position 84 can be replaced by serine or asparagine, respectively, or they can be replaced in an arbitrary variety of combinations thereof. In addition, with regard to the FR4 VH region represented by any one selected from the above (1a)-(4a) or (1b)-(5b), in the amino acid sequence (Trp-Gly-Lys-Gly-Thr-Thr*-Val-Thr-Val-Ser-Ser) (SEQ ID NO:41) of the FR4 region derived from the J gene of JH6c, lysine (Lys) can be replaced by glutamine or asparagine and/or threonine (Thr) marked with an asterisk can be substituted for leucine. Each of FR1, FR2, FR3 and FR4 containing a combination of any of the amino acid substitution(s) mentioned above has no significant effect on the functions of the first arm specifically binding to PD-1 and can be used as scaffolds.

[0044] In addition, the "first arm specifically binding to PD-1" of the present invention includes variants where each CDR having the above amino acid sequence is present, and in which the FR amino acid sequences in the VH are encoded by a specific germline gene or a gene with somatic mutation(s). Examples of such a first arm include includes variants that have a VH containing any amino acid sequence from SEQ ID NOs: 1-5.

[0045] In addition, examples of such a "PD-1 specific binding first arm" include variants that have a VH containing an amino acid sequence having at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, even more preferably at least 98% identity, and even more preferably at least 99% identity with the amino acid sequence shown in any one, selected from SEQ ID NOs: 1-5, and in which a difference from the VH amino acid sequence in the original first arm does not significantly affect PD-1 binding activity (herein, may be abbreviated as "homologous first arm"). As used herein, the term "% identity" as used when comparing amino acid sequence identity is defined as the percentage of amino acid sequence identical to a reference amino acid sequence (herein, a reference amino acid sequence in which a gap has been introduced to maximize sequence identity) when two sequences are aligned. In addition, as used herein, "the difference from the VH amino acid sequence in the original first arm does not significantly affect the PD-1 binding activity" means that the PD-1 binding activity of the homologous first arm is 95% or more, preferably 98% or more, and more preferably 99% or more of the binding activity of the original first arm.

[0046] In another embodiment, the "first arm specifically binding to PD-1" of the present invention also includes an arm that has a variable region (herein, the variable region includes its constituent VH and VL) of anti-PD-1 antibodies and cross-competes (1) for binding to PD-1 with the first arm having a VH of any one selected from the above (1a)-(4a) or (1b)-( 5b), or a VH containing any amino acid sequence from SEQ ID NOs: 1-5 and a conventional light chain VL, or (2) for binding to PD-1 with the variable region of a monoclonal antibody that specifically binds to PD-1 having the same VH and VL, and further includes variants when having an anti-PD-1 antibody variable region, where binding to PD-1 is cross-competed (3) from the first arm having VH of any one selected from the above (1a)-(4a) or (1b)-(5b), or VH containing any amino acid sequence from SEQ ID NO:1-5, and VL of a conventional light chain, or (4) from the variable region side of a monoclonal antibody that specifically binds to PD-1, having the same VH and VL. As used herein, the expression "cross-competition for binding to PD-1" means that it inhibits binding of the first arm to PD-1, regardless of its extent, by binding to an epitope that is the same as or overlaps with the epitope of the first arm exemplified in this specification, or that binding to PD-1 of an antibody that binds to an epitope that is the same as or partially overlaps the epitope of the exemplary first arm is suppressed by the exemplary first arm. om, regardless of its degree. Cross-competition can be assessed using competitive binding analysis. For example, it can be determined using Biacore assay, ELISA assay, flow cytometry, enzyme immunoassay (ELISA), fluorescence energy transfer (FRET) method, and microvolume fluorometric assay (FMAT (registered trademark)) technology.

[0047] Examples of a first arm cross-competing for binding to PD-1 with a first arm having a VH in the above (5b) and a conventional light chain VL include a first arm having a VH of any one selected from the above (1b)-(4b) and a conventional light chain VL (preferably a VL having a VL-CDR1 containing the amino acid sequence of SEQ ID NO:26, VL-CDR2 , containing the amino acid sequence of SEQ ID NO: 27, and VL-CDR3, containing the amino acid sequence of SEQ ID NO: 28) and in addition, the first arm, having a VH containing any amino acid sequence from SEQ ID NO: 1-4, and a VL of a conventional light chain (preferably, a VL containing the amino acid sequence of SEQ ID NO: 25).

[0048] In addition, examples of the first arm cross-competing for binding to PD-1 with the first arm having a VH of any one selected from the above (1b)-(4b), or a VH containing any amino acid sequence from SEQ ID NO: 1-4, and a VL of a conventional light chain, include a first arm having a VH in the above (5b), and a VL of a conventional light chain (preferably a VL having a VL- CDR1 containing the amino acid sequence of SEQ ID NO: 26, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 27, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 28), and in addition the first arm having a VH containing the amino acid sequence in SEQ ID NO: 5, and a VL of a conventional light chain (preferably a VL containing the amino acid sequence of SEQ ID NO: 25).

[0049] Herein, preferred examples of the "first arm specifically binding to PD-1" of the present invention include a first arm having a VH of any one selected from the above (1b)-(5b). In addition, as mentioned above, the preferred first arm also includes arms in which 1-5 arbitrary amino acid residues are substituted with another amino acid(s) (preferably, its conserved amino acid(s) in each VH CDR, and the amino acid(s) substitution does not substantially affect PD-1 binding activity. Further, as mentioned above, the preferred first arm includes options when having a VH in which the amino acids framework sequences are encoded by the V germline IGHV7-4-1 gene, or the JH6c J gene, or gene(s) with somatic mutation(s) Further, more preferred examples of the first arm include variants that have a VH containing any amino acid sequence of SEQ ID NOs: 1-5.

[0050] In addition, the first arm specifically binding to PD-1 of the present invention preferably has a conventional light chain, and preferred examples of such a conventional light chain include a light chain coded as a conventional light chain IGVK1-39/JK1. More preferred examples include a light chain containing a VL having a CDR1 containing the amino acid sequence of SEQ ID NO:26, a CDR2 containing the amino acid sequence of SEQ ID NO:27, and a CDR3 containing the amino acid sequence of SEQ ID NO:28, and even more preferred examples include a light chain containing a VL containing the amino acid sequence of SEQ ID NO:25. In addition, preferred examples of a conventional light chain constant region include a light chain constant region comprising the amino acid sequence of SEQ ID NO:29.

[0051] In addition, preferred examples of the "first arm specifically binding to PD-1" include those that provide an interaction between PD-1 and PD-L1, an interaction between PD-1 and PD-L2, or both of these interactions. As used herein, the expression "provides an interaction between PD-1 and PD-L1, an interaction between PD-1 and PD-L2, or both" means that even when there is a bispecific anti-PD-1/CD3 antibody of the present invention with a 20-fold concentration of the soluble form of PD-L1 or PD-L2, the interaction between PD-L1 and PD-1, the interaction between PD-L2 and PD-1, or both of these interactions are maintained on 50% or more, preferably 70% or more, and more preferably 80% or more compared to when there is no anti-PD-1/CD3 bispecific antibody of the present invention. In addition, the definition "provides an interaction between PD-1 and PD-L1, an interaction between PD-1 and PD-L2, or both of these interactions" can have the same meaning as "which does not substantially inhibit the interaction between PD-1 and PD-L1, the interaction between PD-1 and PD-L2, or both of these interactions."

[0052] FIG. 5 shows the dependent relationship between each anti-PD-1 monoclonal antibody clone generated to construct the anti-PD-1/CD3 bispecific antibody of the present invention and its VH amino acid sequence and its SEQ ID numbers. In FIG. 6 shows the dependent relationship between the amino acid sequence of each CDR in the VH of each anti-PD-1 monoclonal antibody clone and its SEQ ID number.

[0053] Second arm specifically binding to CD3

As used herein, "CD3-specific-binding second arm" (which may be abbreviated herein as "second arm") means a portion of an antibody having at least the VH of an antibody that specifically binds to CD3 (which may herein be abbreviated as "anti-CD3 antibody"), whether it is contained in an antibody portion or an antibody fragment, or exists not as a part, but as a separate body. For example, such a second arm consists of a VH of an anti-CD3 antibody and a VL of a conventional light chain constituting the same anti-CD3 antibody, and further includes a Fab portion of the antibody containing VH and VL. As used herein, the expression "specifically binding to CD3" is used as an indication that an antibody can bind directly to CD3 with a binding activity with an affinity of at least 1×10 ^-5 M, preferably 1×10 ^-7 M, and more preferably greater than 1×10 ^-9 M (dissociation constant (Kd value)) and does not substantially bind to another protein. In addition, "antibody" in "CD3-specific binding antibody" or "anti-CD3 antibody" means a full-length antibody that consists of two heavy chains and two light chains linked by disulfide bonds, and preferably a monoclonal antibody thereof.

[0054] As used herein, examples of the "second arm specifically binding to CD3" include an arm that contains a VH having: (1c) a VH-CDR1 containing the amino acid sequence of SEQ ID NO:37, a VH-CDR2 containing the amino acid sequence of SEQ ID NO:38, and a VH-CDR3 containing the amino acid sequence of SEQ ID NO:39.

[0055] In addition, the "CD3-specific-binding second arm" of the present invention includes variants in which 1-5 arbitrary amino acid residues are replaced with another amino acid(s) (preferably their conserved amino acid(s) in each VH CDR in the above (1c), and which have essentially the same CD3-binding activity as the original second arm without any of the same amino acid(s) substitutions). For example, with respect to CDR1, one amino acid residue is replaced by its conservative amino acid. With respect to CDR2 or CDR3, one or two amino acid residues are/are replaced by its conservative amino acid(s), respectively. Herein, the expression "which have essentially the same CD3 binding activity as the original second arm without any substitutions for the same amino acid(s)", as mentioned above, means that the CD3 binding activity of the second arm with amino acid substitution is 95% or more, preferably 98% or more, and more preferably 99% or more of the activity of the original second arm without any substitutions for the same amino acid(s). It should be noted here that the examples of "substitution by conservative amino acid(s)" in each VH-CDR of the second arm include the above-mentioned examples of substitution of the amino acid in the first arm.

[0056] In addition, the "second arm specifically binding to CD3" in the present invention also includes variants that contain a VH having each CDR containing the specific amino acid sequence mentioned above, and in which the FR VH amino acid sequences are encoded by a specific germline gene or a gene with somatic mutation(s). For example, the VH of the above (1c) is encoded by the recombinant VDJ gene, where the germline V gene is IGHV3-33 or a gene with somatic mutation(s) thereof. Herein, the amino acid sequence encoded by the IGHV3-33 germline gene V (SEQ ID NO:22) is shown in FIG. 3.

[0057] In addition, the "second arm specifically binding to CD3" of the present invention also includes variants that contain each CDR having the aforementioned specific amino acid sequence, and in which the amino acid sequences of the FRs in the VH are encoded by a specific germline gene or a somatically mutated gene(s). Examples of such a second arm include includes variants that have a VH containing the amino acid sequence of SEQ ID NO:36.

[0058] In addition, examples of such a “CD3-specific-binding second arm” include those that have a VH containing an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with the amino acid sequence of SEQ ID NO:36, and in which the amino acid sequence of the VH of the original second arm does not significantly affect CD3 binding activity (herein, may be abbreviated as "homologous second arm"). As used herein, "the difference from the VH amino acid sequence of the original second arm does not significantly affect CD3 binding activity" means that the CD3 binding activity of the homologous second arm is 95% or more, preferably 98% or more, and more preferably 99% or more of the binding activity of the original second arm.

[0059] In yet another embodiment, the "second arm specifically binding to CD3" of the present invention includes includes variants that comprise the variable region of an anti-CD3 antibody (herein, the variable region includes its constituent VH and VL) cross-competing for (1) binding to CD3 with a second arm having a VH in the above (1c), or a VH containing the amino acid sequence of SEQ ID NO:36, and a normal VL light chain, or for (2) binding to CD3 with the variable region of a monoclonal antibody that specifically binds to CD3, having the same VH and VL. As used herein, "cross-competition for CD3 binding" means that it suppresses the binding of the second arm to CD3, regardless of its degree, by binding to an epitope that is the same as or partially overlaps the second arm exemplified herein. Herein, cross-competition can be similarly assessed using the same method as described for the "first arm specifically binding to PD-1".

[0060] Herein, preferred examples of the "second arm specifically binding to CD3" of the present invention include a second arm having a VH in the above (1c). In addition, as mentioned above, preferred examples of the second arm also include those in which 1-5 arbitrary amino acid residue(s) are/are substituted with another amino acid(s) (preferably their conserved amino acid(s) in each VH CDR, and the amino acid substitution does not substantially affect CD3 binding activity. Further, as mentioned above, preferred examples of the second arm include variants that have a VH in which the amino acid sequences scaffolds are encoded by the IGHV3-33 germline gene or its gene with somatic mutation(s) Also, more preferred examples of the second arm include variants that have a VH containing the amino acid sequence of SEQ ID NO:36.

[0061] Herein, FIG. 7 shows the dependent relationships between anti-CD3 antibody clones for constructing the bispecific anti-PD-1/CD3 antibodies of the present invention, their VH amino acid sequences, and their SEQ ID numbers, respectively. FIG. 8 shows the dependent relationships between the amino acid sequences of the CDRs in VH of anti-CD3 antibody clones and their SEQ ID numbers, respectively.

[0062] The "CD3 specific binding second arm" of the present invention preferably comprises conventional light chains, and preferred examples of such a conventional light chain include the IGVK1-39/JK1 conventional light chain. More preferred examples include a light chain containing a VL having a CDR1 containing the amino acid sequence of SEQ ID NO:26, a CDR2 containing the amino acid sequence of SEQ ID NO:27, and a CDR3 containing the amino acid sequence of SEQ ID NO:28, and even more preferred examples include a light chain containing a VL containing the amino acid sequence of SEQ ID NO:25. In addition, preferred examples of a conventional light chain constant region include a light chain constant region comprising the amino acid sequence of SEQ ID NO:29.

[0063] Preferred examples of the "second arm specifically binding to CD3" of the present invention include a second arm specifically binding to CD3ε.

[0064] Examples of a preferred isotype of the PD-1/CD3 bispecific antibody of the present invention include an IgG antibody, even more preferably an IgG ₁ antibody or an IgG ₄ antibody, and more preferably an IgG ₁ antibody.

[0065] On the other hand, a preferred embodiment of the anti-PD-1/CD3 bispecific antibody of the present invention is, for example, an anti-PD-1/CD3 bispecific antibody,

where the first arm specifically binding to PD-1 contains:

(A) a VH in which 1-5 arbitrary amino acid residue(s) can be replaced with another amino acid(s) (preferably conservative amino acid(s)) in any one or more CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3 in the VH represented by any one selected from (1a)-(4a) or (1b)-(5b) above; And

(B) a conventional light chain VL having a CDR1 containing the amino acid sequence of SEQ ID NO:26, a CDR2 containing the amino acid sequence of SEQ ID NO:27, and a CDR3 containing the amino acid sequence of SEQ ID NO:28; And

where the second arm specifically binding to CD3 contains:

(C) a VH in which 1-5 arbitrary amino acid residue(s) can be replaced with another amino acid(s) (preferably the conservative amino acid(s)) in any one or more CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3 in the VH shown in (1c) above; And

(D) A conventional light chain VL having a CDR1 containing the amino acid sequence of SEQ ID NO:26, a CDR2 containing the amino acid sequence of SEQ ID NO:27, and a CDR3 containing the amino acid sequence of SEQ ID NO:28.

[0066] A more preferred embodiment of the PD-1/CD3 bispecific antibody of the present invention is, for example, a PD-1/CD3 bispecific antibody,

where the first arm specifically binding to PD-1 contains:

(A) VH of any one selected from (1a)-(4a) or (1b)-(5b) above, and

(B) a conventional light chain VL having a CDR1 containing the amino acid sequence of SEQ ID NO:26, a CDR2 containing the amino acid sequence of SEQ ID NO:27, and a CDR3 containing the amino acid sequence of SEQ ID NO:28; And

where the second arm specifically binding to CD3 contains:

(C) VH in above (1c); And

(D) A conventional light chain VL having a CDR1 containing the amino acid sequence of SEQ ID NO:26, a CDR2 containing the amino acid sequence of SEQ ID NO:27, and a CDR3 containing the amino acid sequence of SEQ ID NO:28.

[0067] In addition, another preferred embodiment of the PD-1/CD3 bispecific antibody of the present invention is, for example, a PD-1/CD3 bispecific antibody,

in which the first arm specifically binding to PD-1 contains:

(A) a VH containing any amino acid sequence from SEQ ID NOs: 1-5, or a VH containing an amino acid sequence having at least 80% identity with the amino acid sequence of the same VH; And

(B) VL conventional light chain containing the amino acid sequence of SEQ ID NO:25; And

where the second arm specifically binding to CD3 contains,

(C) a VH containing the amino acid sequence of SEQ ID NO:36, or a VH containing an amino acid sequence that is at least 80% identical to the amino acid sequence of the same VH; And

(D) A conventional light chain VL containing the amino acid sequence of SEQ ID NO:25.

[0068] Other embodiments more preferably include, for example, an anti-PD-1/CD3 bispecific antibody,

in which the first arm specifically binding to PD-1 contains:

(A) VH containing any amino acid sequence from SEQ ID NO: 1-5; And

(B) VL conventional light chain containing the amino acid sequence of SEQ ID NO:25; And

in which the second arm specifically binding to CD3 contains:

(C) VH containing the amino acid sequence of SEQ ID NO:36; And

(D) A conventional light chain VL containing the amino acid sequence of SEQ ID NO:25.

[0069] In the PD-1/CD3 bispecific antibody of the present invention, when the antibody is an IgG ₁ antibody, an IgG ₁ antibody is preferred in which the leucine at position 235 according to the EU numbering system is replaced by glycine and/or the glycine at position 236 according to the EU numbering system is replaced by arginine in the two heavy chain constant regions or the hinge region. Also preferred is a bispecific antibody in which the C-terminal amino acid of the heavy chain has been removed, eg lysine at position 447 according to the EU numbering system. In addition, in the case where the anti-PD-1/CD3 bispecific antibody is an IgG ₄ antibody, an antibody in which the serine located in the hinge region and at position 228 according to the EU numbering system is replaced by a proline is preferred.

[0070] In addition, a preferred embodiment, in the case where the anti-PD-1/CD3 bispecific antibody is an IgG ₁ antibody, includes variants in which, in the heavy chain constant region having the first arm VH specifically binding to PD-1, the leucine at position 351 according to the EU numbering system is replaced by lysine, and the threonine at position 366 is replaced by lysine, and in the heavy chain constant region having the second arm VH and specifically binding to CD3, the leucine at position 351 is replaced by aspartic acid, and the leucine at position 368 is replaced by glutamic acid. Also preferred is an IgG ₁ antibody in which, in the heavy chain constant region having the first arm VH specifically binding to PD-1, the leucine at position 351 according to the EU numbering system is replaced by aspartic acid, and the leucine at position 368 is replaced by glutamic acid, and in the heavy chain constant region having the second arm VH specifically binding to CD3, the leucine at position 351 is replaced by lysine, and the threonine at position 366 is replaced by lysine.

[0071] Examples of a preferred embodiment of an anti-PD-1/CD3 IgG ₁ bispecific antibody in which all of the above amino acid substitutions have been made in the heavy chain constant region include those in which a heavy chain having a first arm VH specifically binding to PD-1 comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO:23 and a heavy chain having a second arm VH specifically binding to CD3 , contains a heavy chain constant region containing the amino acid sequence of SEQ ID NO:24. These amino acid sequences are shown in FIG. 9.

[0072] The most preferred embodiments of the PD-1/CD3 bispecific antibody of the present invention are PD1-1(Bi) clone, PD1-2(Bi) clone, PD1-3(Bi) clone, PD1-4(Bi) clone, and PD1-5(Bi) clone obtained using the method described in Example 8 of this specification.

[0073] Preferred features of the bispecific anti-PD-1/CD3 antibody of the present invention include: (1) providing an interaction between PD-1 and PD-L1, an interaction between PD-1 and PD-L2, or both interactions; and/or (2) sufficient reduction in cytokine production. In this document, the expression "providing an interaction between PD-1 and PD-L1, an interaction between PD-1 and PD-L2, or both interactions" means the same as described with respect to "the first arm specifically binding to PD-1". On the other hand, the expression "sufficient reduction in cytokine production" means that, for example, during intravenous administration or 24 hours after this administration by drip infusion of the anti-PD-1/CD3 bispecific antibody of the present invention, for example, the concentration of cytokine, including IL-2, IFN-γ and/or TNF-α in blood or tissue does not increase, or increases only to the extent that it can be suppressed by administration of steroids.

[0074] A method for preparing and purifying an anti-PD-1/CD3 bispecific antibody

The anti-PD-1/CD3 bispecific antibody and antibody fragment of the present invention can also be prepared using the method disclosed in WO2014/051433, WO2013/157953 or WO2013/157954.

[0075] Specifically, an anti-PD-1/CD3 bispecific antibody and an antibody fragment of the present invention can be produced using a transferable gene expression vector into which (1) a polynucleotide encoding a heavy chain containing a first arm VH specifically binding to PD-1, (2) a polynucleotide encoding a heavy chain containing a second arm VH specifically binding to CD3, and (3) a polynucleotide encoding an o conventional light chain, respectively, into mammalian animal cells for transformation, and then by expression and secretion of both the heavy chain and the conventional light chain.

[0076] As used herein, any host cell can be used to express the anti-PD-1/CD3 bispecific antibody of the present invention, as long as it can carry out gene transfer using an expression vector and allow expression of the transferred expression vector. The host cells are preferably insect cells such as SF-9 and SF-21, more preferably mammalian cells such as murine cells such as CHO cells, BHK cells, SP2/0 cells and NS-0 myeloma cells, primate cells such as COS and Vero cells, MDCK cells, BRL 3A cells, hybridoma, tumor cells, immortalized primary cells, W138, HepG2, HeLa, HEK293, HT1080 or embryonic retinal cells such as PER.C6. It should be noted here that when choosing an expression system, expression vectors and mammalian hosts can be used to ensure that the antibody is properly glycosylated. To obtain an antibody whose glycosylated structures correspond to human glycosylated structures, human cell lines are preferably used, preferably PER.C6.

[0077] Production of proteins in host cells transformed by gene transfer with expression vectors can be done with reference to, for example, Current Protocols in Protein Science (1995), Coligan JE, Dunn BM, Ploegh HL, Speicher DW, Wingfield PT, ISBN 0-471-11184-8, Bendig, 1988. In addition, general recommendations, techniques and practical ways to maximize productivity host cell cultures can be performed with reference to Mammalian Cell Biotechnology: a Practical Approach (M. Butler, ed. IRL Press, 1991). Expression of antibodies in host cells is described, for example, in publications such as EP0120694, EP0314161, EP0481790, EP0523949, US4816567, WO2000/63403 and the like.

[0078] Here, the culture conditions of the host cells can be optimized using a well-known method, and thus the production of proteins can be optimized. Examples of culture include batch culture, nutrient culture, continuous culture, and culture on hollow fibers in a petri dish, roller bottle, or reaction chamber. For cell culture production of the recombinant protein on a large scale and continuously, cell proliferation in a suspension liquid is preferred. In addition, it is more preferred to culture the cells under conditions without serum of animal or human origin or some components of serum of animal or human origin.

[0079] An antibody expressed in host cells and then recovered from cells or cell culture using well known methods can be purified using well known methods. Examples of the purification method include an immune precipitation method, a centrifugation method, 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 preferably be used (see, for example, US4801687 and US5151504).

[0080] Anti-PD-1 monoclonal antibody

the present invention includes a "PD-1 specific binding monoclonal antibody" (herein, may be abbreviated as "anti-PD-1 monoclonal antibody") for constructing an anti-PD-1/CD3 bispecific antibody of the present invention and an antibody fragment.

[0081] One embodiment of the anti-PD-1 monoclonal antibody of the present invention is a monoclonal antibody capable of specifically binding to PD-1 by binding the VH and VL of the conventional light chain of the present invention. Herein, the expression "specifically binding to PD-1" is used as an indication that it can bind directly to PD-1 with a binding activity (dissociation constant (Kd value)) of an affinity of at least 1×10 ^-5 M, preferably 1×10 ^-7 M, and more preferably more than 1×10 ^-9 M, and does not substantially bind to receptor elements belonging to the so-called receptor of the CD28 family, for example , CD28, CTLA-4 and ICOS. As used herein, "antibody" in "monoclonal antibody that specifically binds to PD-1" or "anti-PD-1 antibody" means a full-length antibody consisting of two heavy chains and two light chains linked by disulfide bonds. In addition, "PD-1 specific binding monoclonal antibody fragment" is a portion of a full-length antibody and an antibody containing at least an antigen-binding portion, and examples of which include Fab, Fab', Fv, scFv, F(ab') ₂ and the like.

[0082] Examples of an anti-PD-1 monoclonal antibody of the present invention include variants that have any one VH selected from (1a)-(4a) or (1b)-(5b) above, constituting a "PD-1 specific binding first arm" VH, or a VH containing any amino acid sequence from SEQ ID NOs: 1-5, and a conventional light chain VL as described herein.

[0083] In addition, the anti-PD-1 monoclonal antibody of the present invention includes variants in which 1-5 arbitrary amino acid residue(s) are/are substituted with another amino acid(s) (preferably their conserved amino acid(s) in each CDR of any one of the VHs selected from (1a)-(4a) or (1b)-(5b) above) and which have substantially the same binding activity to PD-1 as an anti-PD-1 monoclonal antibody having the original VH without any substitutions by the same amino acid(s).For example, as for CDR1, one amino acid residue is replaced by its conserved amino acid.As for CDR2 or CDR3, one or two amino acid residue(s) are substituted/replaced by its conservative amino acids, respectively.As used herein, the expression "which have substantially the same PD-1 binding activity as an anti-PD- monoclonal antibody 1 having the original VH without any substitutions of the same amino acid(s)"means that the PD-1 binding activity of an anti-PD-1 monoclonal antibody with an amino acid (amino acid) substitution is 95% or more, preferably 98% or more, and more preferably 99% or more, compared with the activity of an anti-PD-1 monoclonal antibody having the original VH without any substitutions of the same amino acid(s).

[0084] In addition, examples of the anti-PD-1 monoclonal antibody of the present invention include those that contain a VH having each CDR containing the specific amino acid sequence mentioned above, and in which the VH framework amino acid sequences are encoded by a specific germline gene or a gene with somatic mutation(s), and examples of which include a specific VH encoded by a specific germline gene or its genome with somatic mutation(s), which is described in relation to the aforementioned "first arm specifically binding to PD-1".

[0085] Further, in the anti-PD-1 monoclonal antibodies of the present invention, examples of those having a VH containing each CDR in any one of the VHs selected from (1a)-(4a) or (1b)-(5b) above, and in which the amino acid sequences of the FRs are encoded by a specific germline gene or a gene with somatic mutation(s) thereof, include variants that have a VH containing any amino acid sequence from SEQ ID NO: 1-5. In addition, examples of such an anti-PD-1 monoclonal antibody include variants that have a VH amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99% identity with the amino acid sequence shown in any one selected from SEQ ID NOs: 1-5, and have PD-1 binding activity, essentially the same as the anti-PD-1 monoclonal antibody having the original VH. As used herein, "having a PD-1 binding activity substantially the same as an anti-PD-1 monoclonal antibody having a parent VH" means that it has 95% or more, preferably 98% or more, and more preferably 99% or more, a PD-1 binding activity compared to the activity of an anti-PD-1 monoclonal antibody having a parent VH.

[0086] Further, other embodiments of the anti-PD-1 monoclonal antibody of the present invention also include an anti-PD-1 monoclonal antibody cross-competing for binding to PD-1 with an anti-PD-1 monoclonal antibody having any one VH selected from (1a)-(4a) or (1b)-(5b) above, or the VH amino acid sequence shown in any of SEQ ID NOs: 1-5, and an anti-PD-1 monoclonal antibody whose binding to PD-1 is cross-competed by an anti-PD-1 monoclonal antibody having any one VH selected from (1a)-(4a) or (1b)-(5b) above, or a VH containing any amino acid sequence from SEQ ID NOs: 1-5.

[0087] Preferred features of the anti-PD-1 monoclonal antibody of the present invention include providing an interaction between PD-1 and PD-L1, an interaction between PD-1 and PD-L2, or both interactions, and an anti-PD-1/CD3 bispecific antibody of the present invention.

[0088] A polynucleotide encoding an anti-PD-1/CD3 bispecific antibody

A polynucleotide encoding an anti-PD-1/CD3 bispecific antibody comprises: (1) a polynucleotide encoding a heavy chain having a first arm VH that specifically binds to PD-1, (2) a polynucleotide encoding a heavy chain having a second arm VH that specifically binds to CD3, and (3) polynucleotides encoding conventional light chains. As used herein, a polynucleotide encoding a heavy chain having a first arm VH specifically binding to PD-1 includes a polynucleotide encoding a first arm VH specifically binding to PD-1 and a polynucleotide encoding a heavy chain constant region having a VH. Similarly, a polynucleotide encoding a heavy chain having a second arm VH that specifically binds to CD3 includes a polynucleotide encoding a second arm VH that specifically binds to CD3 and a polynucleotide encoding a heavy chain constant region having a VH.

[0089] The polynucleotide encoding the anti-PD-1/CD3 bispecific antibody can be any polynucleotide, provided that they encode each of the parts that make up the anti-PD-1/CD3 bispecific antibody, and can be any genomic DNA, cDNA, synthesized DNA, RNA, and DNA-RNA hybrid. As a codon encoding one amino acid, one to six types of codons are known. For example, Phe is TTT or TTC, Leu is TTA, TTG, CTT, CTC, CTA, or CTG, Ile is ATT, ATC, or ATA, Met is ATG, Val is GTT, GTC, GTA, or GTG, Ser is TCT, TCC, TCA, or TCG, Pro is CCT, CCC, CCA, or CCG, Thr is ACT, ACC, ACA, or ACG, Ala is GCT, GCC, GCA, or GCG, Tyr matches TAT or TAC, His matches CAT or CAC, Gln matches CAA or CAG, Asn matches AAT or AAC, Lys matches AAA or AAG, Asp matches GAT or GAC, Glu matches GAA or GAG, Cys matches TGT or TGC, Trp matches TGG, Arg matches CGT, CGC, CGA or CGG, Ser matches AGT or AGC, Arg matches AGA or AGG, and Gly matches GGT, G GC, GGA or GGG, respectively. Accordingly, a polynucleotide encoding an anti-PD-1/CD3 bispecific antibody includes polynucleotides in which the codons corresponding to each amino acid are randomly combined. Preferred examples of a polynucleotide encoding a first arm VH specifically binding to PD-1 include a polynucleotide having the base sequence set forth in any of SEQ ID NOs: 30-34 encoding the VH of clones PD1-1 to PD1-5, respectively. Preferred examples of a polynucleotide encoding a second arm VH specifically binding to CD3 include a polynucleotide having the base sequence of SEQ ID NO:40. In addition, preferred examples of a polynucleotide encoding a conventional light chain variable region include a polypeptide having the base sequence of SEQ ID NO:35.

[0090] Pharmaceutical application

The PD-1/CD3 bispecific antibody of the present invention is useful for preventing, suppressing symptoms of progression or relapse, and/or treating autoimmune diseases or graft-versus-host disease (GVHD).

[0091] Examples of diseases that can be prevented, the progression or recurrence of symptoms of which can be suppressed, and/or that can be treated with the PD-1/CD3 bispecific antibody of the present invention are Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis (systemic scleroderma, and progressive systemic sclerosis), scleroderma, polymyositis, dermatomyosis Ozitis, periarteritis nodosa (polyarteritis nodosa, and microscopic polyangiitis), aortic syndrome (Takayasu's arteritis), rheumatoid arthritis with malignant disease, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Sjögren's syndrome, Still's disease in adults, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis culitis, large vessel vasculitis, ANCA-associated vasculitis (eg, granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis), Cogan's syndrome, RS3PE, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related disease (eg, primary sclerosing cholangitis, autoimmune disease). immune insulitis), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic steatohepatitis, primary biliary cirrhosis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune Neutropenia, idiopathic thrombocytopenic purpura, Graves' disease (Graves' disease (hyperthyroidism)), Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, Addison's disease (chronic hypoadrenocorticism), Addison's disease of unknown origin, type I diabetes, slowly progressive type I diabetes mellitus (latent autoimmune diabetes mellitus in adults), focal scleroderma ia, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, gestational herpes, linear IgA-dependent bullous dermatosis, acquired epidermosis bullosa, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis of the optic nerve, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harad's disease, autoimmune optic neuropathy, azoospermia of unknown origin, habitual abortion, inflammatory bowel disease (eg, ulcerative colitis, and Crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, transplantation immunity, familial Mediterranean high fever, eosinophilic chronic rhinosinusitis, dilated cardiomyopathy, systemic mastocytosis or inclusion body myositis.

[0092] In the present invention, the term "treatment" means the treatment or improvement of the disease or reduce the manifestation of its symptoms. The term "prevention" means preventing the onset of a disease or its symptoms, or delaying for a certain period. The term "suppressing the progression of symptoms" means suppressing the progression or worsening of symptoms to stop the progression of a disease state. The meaning of "prevention" includes the suppression of relapse. The term "relapse suppression" means preventing the recurrence of a disease or syndrome or reducing the possibility of relapse.

[0093] The PD-1/CD3 bispecific antibody of the present invention is typically administered systemically or topically via parenteral administration. Specific examples of such administration methods include injection administration, intranasal administration, transpulmonary administration, transdermal administration, and the like. Examples of injection administration include intravenous injection, intramuscular injection and intraperitoneal injection. For intravenous injection, intravenous drip infusion is preferred. Its dose varies depending on age, body weight, symptoms, therapeutic effect, administration route, treatment period, and the like. Its single dose for one adult patient is usually within the range of 0.1 μg/kg to 300 mg/kg, and particularly preferably within the range of 0.1 mg/kg to 10 mg/kg, once to several times a day by parenteral administration, or within the range of one hour to 24 hours per day by intravenous maintenance administration. Of course, as mentioned above, since the dose varies depending on different conditions, a sufficient dose may be lower than the above dose, or it may be necessary to administer more than indicated above.

[0094] Composition

In preparing the formulation of the anti-PD-1/CD3 bispecific antibody of the present invention to be used as an injection or infusion solution for drip infusion, the injection or infusion solution may be in any form of an aqueous solution, suspension or emulsion, or may be formulated as a solid together with a pharmaceutically acceptable carrier such that the agent is dissolved, suspended or emulsified in a solvent at the time of use. Examples of the solvent that is used in the injection or infusion solution for drip infusion include distilled water for injection, saline, glucose solutions, and isotonic solutions (for example, in which sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, and the like can be dissolved).

[0095] As used herein, examples of pharmaceutically acceptable carriers include stabilizers, solubilizers, suspending agents, emulsifiers, emollients, buffering agents, preservatives, disinfectants, pH adjusters, and antioxidants. Various amino acids, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, polyethylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, dibutylhydroxytoluene and the like can be used as stabilizers. As solubilizers, alcohols (for example, ethanol), polyols (for example, propylene glycol, polyethylene glycol, and the like), non-ionic surfactants (for example, Polysorbate 20 (registered trademark), Polysorbate 80 (registered trademark), HCO-50, and the like) and the like can be used. As suspending agents, glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate and the like can be used. As emulsifiers, gum arabic, sodium alginate, tragacanth and the like can be used. As emollients, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As buffering agents, phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamic acid buffer, epsilon aminocaproic acid buffer and the like can be used. As preservatives, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax and the like can be used. As disinfectants, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol and the like can be used. As pH adjusters, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid and the like can be used. The antioxidants that can be used are (1) aqueous antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite, (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, and α-tocopherol, and (3) metal chelating agents such as citric acid, ethylenediamine. traacetic acid, sorbitol, tartaric acid and phosphoric acid, and the like.

[0096] An injectable or infusion solution for drip infusion can be obtained by performing sterilization in the final process or decontamination treatment, for example, performing sterilization by filtration with a filter or the like, and then filling an aseptic container. An injectable or infusion solution for drip infusion may be used by dissolving a vacuum-dried or lyophilized aseptic powder (which may include a pharmaceutically acceptable carrier powder) in a suitable solvent at the time of use.

[0097] Combined use or combined formulation

In addition, the PD-1/CD3 bispecific antibody of the present invention can be used in combination with another agent that is used to prevent, suppress the progression of symptoms or recurrence of an autoimmune disease and/or treat it. In the present invention, examples of the form of administration in such combined use with other agents (combination use) may include the form of a combined formulation containing both ingredients in one formulation, or the form administered as separate formulations. Such combined use may be in addition to the prophylactic, symptomatic progression suppressing, relapse suppressing and/or therapeutic effect of the other agent, or may maintain or reduce the dose or frequency of administration of the other agent. In the present invention, in the case of separately administering the anti-PD-1/CD3 bispecific antibody and the other agent, both agents can be administered simultaneously for a period of time, and then only the anti-PD-1/CD3 bispecific antibody or the other agent can be administered. Alternatively, the PD-1/CD3 bispecific antibody of the present invention may be administered first, and the other agent may be administered after administration is complete. Another agent may be administered first, and then the anti-PD-1/CD3 bispecific antibody of the present invention may be administered. The route of administration may be the same or different from each other. The present invention may also provide a kit containing a formulation containing a bispecific anti-PD-1/CD3 antibody of the present invention and a formulation containing another agent. The dose of the other agent may be selected appropriately based on the clinical dose. The other agent may be a combination of two or more types of arbitrary agents in an appropriate ratio. Examples of another tool include not only those that are already known, but also those that will be discovered in the future.

[0098] For example, when the PD-1/CD3 bispecific antibody of the present invention is used to prevent, suppress symptoms of progression or recurrence, and/or treat type I diabetes mellitus, it can be used in combination, for example, with any one or more agent(s) selected from an insulin preparation (for example, human insulin, insulin glargine, insulin lispro, insulin detemir, insulin aspart, and the like), a sulfonylurea preparation (for example, glibenclamide, gliclazide, or glimepiride), a fast-acting insulin secretion promoter (eg, nateglinide), a biguanide drug (eg, metformin), an agent that affects insulin resistance (eg, pioglitazone), an α-glucosidase inhibitor (eg, acarbose, voglibose, and the like), an agent for treating diabetic neuropathy (eg, epalrestat, mexiletine, imidapril, and the like), a GLP-1 analog drug (eg, liraglutide, exenatide, lixisenatide, and the like), and a DPP-4 inhibitor (eg, sitagliptin, vildagliptin, alogliptin, and the like), and the like.

[0099] In addition, for example, when the PD-1/CD3 bispecific antibody of the present invention is used to prevent, suppress symptoms of progression or relapse, and/or treat multiple sclerosis, it can be used in combination with any one or more agent(s) selected from a steroid agent (e.g., 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 sodium succinate, triamcinolone, triamcinolone acetate, triamcinolone acetonide, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone palmitate, paramethasone acetate or betamethasone), interferon β-1a, interferon β-1b, glatiramer acetate, mitoxantrone, azathioprine, cyclophosphamide, cyclosporine, methotrexate, cladribine, adrenocorticotropic hormone (ACTH), corticotropin, mizor ibin, tacrolimus, fingolimod or alemtuzumab; and the like.

[0100] In addition, for example, when the PD-1/CD3 bispecific antibody of the present invention is used to prevent, suppress symptoms of progression or recurrence, and/or treat systemic lupus erythematosus, it can be used in combination with any one or more of the agent(s) selected from a steroid agent (for example, the steroid agents mentioned above), an immunosuppressive agent (for example, cyclosporine, tacrolimus, or fingolimod), and belimumab .

[0101] For example, when the PD-1/CD3 bispecific antibody of the present invention is used to prevent, suppress symptoms of progression or recurrence, and/or treat rheumatoid arthritis, it can be used in combination with any one or more of the agent(s) selected from a steroid agent (e.g., the steroid agents mentioned above), an antirheumatic agent (e.g., methotrexate, sulfazalazine, bucillamine, leflunomide, mizoribine, tacrolimus, and the like), an anti-cytokine agent (eg, infliximab, adalimumab, tocilizumab, etanercept, golimumab, sertolizumab, and the like), and abatacept, and the like.

[0102] In the case of using the PD-1/CD3 bispecific antibody of the present invention to prevent, suppress symptoms of progression or recurrence, and/or treat another autoimmune disease, the PD-1/CD3 bispecific antibody of the present invention may be used in combination with any one or more of the other agent(s) mentioned above.

[0103] Herein, the present invention will be described in more detail using the following examples, but the scope of the present invention is not limited to them. A person skilled in the art can make various changes and modifications based on the description of the present invention, and such changes and modifications are also included in the present invention.

Examples

[0104] Example 1: Immunization of MeMo mice using recombinant human PD-1-Fc fusion protein

As a method for obtaining the first arm specifically binding to PD-1 according to the present invention, the method of immunizing MeMo mice (see WO2009/157771) with recombinant human PD-1 protein is selected. MeMo mice are mice genetically modified to link a gene fragment containing a non-recombinant region of the human V heavy chain gene, a D region of the gene, and a J region of the gene, as well as a recombinant human IgVκ1-39*01/IGJκ1*01 germline κ light chain gene, to the mouse constant region gene. By direct immunization, a variety of MeMo mice can be produced with antibody target protein, antibodies composed of heavy chains and conventional light chains.

[0105] Twelve 12-16 week old MeMo MS5B/MS9 mice were immunized with recombinant human PD-1-Fc fusion protein (R&D systems, serial no. 1086-PD), which was emulsified using Gerbu's MM adjuvant (Gerbu Biotechnik, serial #3001) at 14 day intervals. On days 0, 14 and 28 post-immunization, recombinant human PD-1-Fc fusion protein was administered subcutaneously. Later, recombinant human PD-1-Fc fusion protein dissolved in PBS was administered subcutaneously. At 21, 35, 56, 77 and 98 days post-immunization, serum antibody titer was assessed by flow cytometry using human PD-1 forced HEK293 T cell lines. When HEK293 T cell lines forced to express human PD-1 were stained in 1000-fold diluted serum, mouse lymphatic tissue whose MFI value was at least three times higher than that of HEK293 T cell lines not expressing human PD-1 was used as a control to construct a phage display library. Mice meeting the library construct criteria were further immunized with recombinant PD-1-Fc fusion protein within three days of the date of antibody titer assessment, and their spleens and inguinal lymph nodes were harvested. Mice whose serum antibody titer against human PD-1 and cynomolgus PD-1 was 1/100 or more and did not increase in antibody titer with supplemental immunization were also subjected to the same method, and then their spleens and inguinal lymph nodes were harvested. RNA was extracted from these lymphoid tissues, and then cDNA synthesis was carried out.

[0106] Example 2: Construction of a phage display library to generate an anti-PD-1 antibody having a first arm that specifically binds to PD-1 (protein immunization)

The PCR reaction was performed using the DNA prepared in Example 1 and primers specific for the immunoglobulin heavy chain variable region family. The resulting PCR product was digested with restriction enzymes SfiII and XhoI and introduced into the MV1473 phagemid vector [having a gene (human IgVκ1-39*01/IGJκ1*01 κ light chain germline gene) encoding a conventional light chain] cleaved with the same restriction enzymes to construct a library.

[0107] Example 3: Screening for an Anti-PD-1 Antibody Having a First Arm Specifically Binding to PD-1

Phage selection based on PD-1 binding property was performed using plates coated with human PD-1-Fc fusion protein, human PD-1-His tagged fusion protein, cynomolgus PD-1-His tagged fusion protein, or mouse PD-1-His tagged fusion protein. When using human PD-1-Fc fusion protein, human IgG (SIGMA, serial number I4506) was added during incubation with phage to absorb Fc-reactive clones. Binding phages that can bind to human PD-1, cynomolgus PD-1 and mouse PD-1 were enriched. Phages that can bind to cynomolgus PD-1 were enriched using selection of HEK293 T cell lines expressing cynomolgus PD-1. To obtain a reference plate, TG1 clones of Escherichia coli strain were obtained, transformed with phages obtained by selection.

[0108] In addition, based on the PD-1 binding property on the plate coated with the human PD-1-Fc fusion protein, phage selection was performed using an extract from the periplasmic space of the clones obtained in the above selection. It should be noted here that, as selection criteria, clones with signals three times greater than the signals (OD ₄₅₀ value) obtained from the negative control (PBS) wells were defined as positive clones.

[0109] Example 4: DNA sequence for candidate anti-PD-1 antibody clones having a first arm that specifically binds to PD-1

The DNA sequence for the heavy chain variable region gene of the positive clones obtained by screening in Example 3 was obtained. The analyzed DNA sequences were classified into superclusters (a group having CDR3 of the same length and the amino acid sequence of CDR3 is 70% or more homologous) and clusters (a group in which the amino acid sequences of the heavy chain CDR3 are the same). Nine hundred and twenty-four clones were obtained, which were classified into superclusters of 146 types and clusters of 194 types.

[0110] Example 5: Screening based on the evaluation of the property of binding to cells expressing PD-1

From each of the classified superclusters, anti-PD-1 monoclonal antibody clones were screened for isolation, satisfying the following conditions:

(1) somatic mutation(s) were introduced into the CDR region(s) at a high frequency;

(2) the VH germline gene is included with a high frequency of use, and

(3) received a high signal when screening the binding properties of the human fusion protein PD-1-Fc.

[0111] The evaluation was performed by detecting binding to CHO-S cell lines expressing human PD-1 and CHO-S cell lines expressing cynomolgus macaque PD-1 with anti-mouse IgG polyclonal antibodies using the Fab fragments contained in these extracts from the periplasmic space. Among the 117 clones (105 cluster types) evaluated, 22 anti-PD-1 monoclonal antibody-containing clones, PD1-1, PD1-2, PD1-3 and PD1-4 clones, were found to be able to bind to CHO-S cell lines expressing human PD-1.

[0112] Example 6: Preparation of an amino acid substituted anti-PD-1 monoclonal antibody product having a first arm that specifically binds to PD-1

Clone PD1-1 and PD1-4 contain a deamidation motif (Asn-Gly) in framework 4 of the heavy chain variable region, respectively. To obtain the first arm with a reduced risk of deamidation, a variant was obtained in which the deamidation motif was transformed. Clone PD1-5 was obtained such that the asparagine (Asn) at position 119 according to the EC numbering system of clone PD1-4 was changed to glutamine using the well-known site-specific mutation method, which was isolated. The binding property to human PD-1 expressing CHO-S cells of this clone was the same as clone PD1-4.

[0113] Example 7: Screening for Anti-CD3 Monoclonal Antibodies Having a Second Arm Specifically Binding to CD3

In order to obtain a CD3-binding Fab having greater stability and furthermore reduced charge heterogeneity, an anti-CD3 antibody having a "second arm specifically binding to CD3" of the present invention was obtained by the following method using an anti-CD3 antibody clone having the VH of the anti-CD3 antibody clone 15C3 described in WO2005/118635 and the conventional IGVK1-39/JK1 light chain.

[0114] By converting the underlined 55th glycine in the 15C3 VH amino acid sequence shown in FIG. 10, an anti-CD3 antibody CD3-1 clone having the same human CD3 binding property as the clone was obtained in alanine and having improved charge heterogeneity.

[0115] In addition, in order to obtain a set of Fabs that bind to CD3, to improve the interaction of VH/VL with a conventional light chain (IgVκ1-39*01/IGJκ1*01), based on the VH clone CD3-1, a phage display library was constructed expressing a set of Fabs containing VH variants of the CD3-1 clone in which this amino acid residue was replaced. This phage library was screened using HBP-ALL cells or recombinant human CD3ε-Fc protein. Phages binding to recombinant human CD3ε-Fc protein were chemically eluted and used to re-infect the bacteria. Many surviving bacterial colonies were removed, then phages were removed from them, and then screened using flow cytometry based on binding to CD3 expressed on the cell surface. All CD3-binding phages were subjected to PCR to select colonies for amplification of the cDNA encoding their VH and their DNA sequences were determined.

[0116] As a result, the resulting CD3-2 clone of anti-CD3 antibodies having a VH containing the amino acid sequence of SEQ ID NO:36 exhibited CD3 binding property and charge uniformity equivalent to clone CD3-1.

[0117] Example 8: Obtaining a bispecific monoclonal antibody against PD-1/CD3

An expression vector expressing each heavy chain of the first arm specifically binding to PD-1 was obtained by linking the DNAs encoding the heavy chain variable regions of the anti-PD-1 monoclonal antibody clones PD1-1-PD1-5 and PD1-6, respectively, selected in Example 5, to the DNAs encoding the IgG ₁ heavy chain constant regions, respectively. On the other hand, an expression vector expressing the heavy chain of the second arm specifically binding to CD3 was obtained by linking the DNA encoding the heavy chain variable region of the CD3-2 clone of the anti-CD3 monoclonal antibody selected in Example 7 to the DNA encoding the IgG ₁ heavy chain constant region. Herein, as genes expressing heavy chain constant regions such as the first arm specifically binding to PD-1, a gene expressing an Fc region having the L351D/L368E variant (DE variant) was used. As for the second arm specifically binding to CD3, a gene expressing an Fc region having the L351K/T366K variant (KK variant) was used. These expression vectors were designed to further contain a gene encoding a conventional IGVK1-39/JK1 light chain so as to be expressed together. In addition, the genes expressing these heavy chain constant regions were modified to express those in which the leucine at position 235 was replaced by glycine, and in addition the glycine at position 236 was replaced by arginine at the heavy chain constant region to abolish Fc effector activity, and furthermore, the lysine at position 447 at the C-terminus of the heavy chain constant region was deleted to avoid post-translational processing. Both of these expression vectors transferred genes to Free Style 293F cells to generate antibodies in culture supernatants. Culture supernatants were harvested and then processed by protein A affinity chromatography to purify clone PD1-1(Bi), clone PD1-2(Bi), clone PD1-3(Bi), clone PD1-4(Bi), and clone PD1-5(Bi) of the bispecific anti-PD-1/CD3 monoclonal antibodies of the present invention, respectively. In addition, clone PD1-6(Bi) was also obtained using the same method. It should be noted here that these anti-PD-1/CD3 bispecific monoclonal antibody clones have first arms specifically binding to PD-1 derived from anti-PD-1 monoclonal antibody: clones PD1-1, PD1-2, PD1-3, PD1-4 and PD1-5, and clone PD1-6 used in their preparation, they correspond to these clones of anti-PD-1 monoclonal antibodies, respectively.

[0118] Example 9: Evaluation of the binding property of an anti-PD-1/CD3 bispecific monoclonal antibody

By Biacore assay using human extracellular recombinant PD-1 protein fused to human IgG1-Fc or labeled with 6×His cynomolgus monkey extracellular recombinant fusion protein PD-1, the binding affinity for each first arm recombinant PD-1 protein of the anti-PD-1/CD3 bispecific monoclonal antibodies prepared in Example 8 was evaluated. It should be noted here that a Series S Sensor Chip CM5 (GE Health Care, serial number 29-1049-88) was used to immobilize the recombinant proteins. Similarly, the CD3 binding affinity of the second arm of the antibody was assessed by the Biacore assay using extracellular recombinant CD3δ/CD3ε protein fused to human IgG1-Fc. In FIG. 11 shows the binding affinity (Kd value) for PD-1 of the first arm and the binding affinity for CD3δ/CD3ε of the second arm for each clone.

[0119] Example 10: Confirmation of the binding property of an anti-PD-1/CD3 bispecific monoclonal antibody

Confirmed that bispecific monoclonal antibodies against PD-1/CD3, obtained in example 8, can specifically bind to PD-1 and CD3, simultaneously.

[0120] First, clones PD1-1(Bi) - PD1-6(Bi) were added to human PD-1 deficient Jurka T cell lines (human T cell line) expressing human CD3, respectively, and which were incubated on ice for 15 minutes. After washing these cells, soluble recombinant PD-1 proteins (R&D systems, serial number 1086-PD-050) labeled with 3x biotin were added and incubated on ice for 15 minutes. After washing these cells, 100 μl of Alexa Fluor 488 labeled streptavidin 1.25 μg/ml (BioLegend, serial number 405235) was added to them and incubated on ice for 15 minutes. After washing these cells, the binding amount of soluble recombinant PD-1 protein was assessed by flow cytometry. In FIG. 12 shows the results of the analysis.

[0121] All clones contacted PD-1 and CD3 simultaneously. It should be noted here that no non-specific binding was found in this binding system.

[0122] Example 11: Evaluation of the first arm binding property of an anti-PD-1/CD3 bispecific monoclonal antibody

To evaluate the results of binding to PD-1/PD-L1 of the first arm of the anti-PD-1/CD3 bispecific monoclonal antibodies obtained in Example 8, competitive binding assays were performed for binding to PD-1 of clones of bispecific monoclonal antibodies and soluble recombinant PD-L1 proteins. First, clones PD1-1(Bi) - PD1-6(Bi) were added to CHO-S cell lines expressing human PD-1, respectively, and incubated on ice for 30 minutes. Soluble recombinant PD-L1 proteins (R&D systems, serial number 156-B7-100) labeled with 1/20 biotin were added to them and incubated on ice for 30 minutes. After washing these cells, PE labeled streptavidin (BD Pharmingen, serial number 554061) was added to them and incubated on ice for 30 minutes. After washing these cells, the binding amount of soluble recombinant PD-L1 proteins was assessed by flow cytometry. In FIG. 13 presents the results of the analysis.

[0123] Although clones PD1-1(Bi) - PD1-5(Bi) are present at 20 times the amount of soluble recombinant PD-L1 protein, they allow binding of soluble recombinant PD-L1 protein to PD-1. On the other hand, PD1-6(Bi) completely inhibited the binding of soluble recombinant PD-L1 protein to PD-1 under the same conditions.

[0124] Example 12: In vitro suppressive effect of an anti-PD-1/CD3 bispecific monoclonal antibody on activated CD4 T cells

The suppressive effect of activated T cells on IFN-γ production was evaluated using CD4-positive T cells derived from healthy human peripheral blood (LONZA, serial number 2W-200) (human T cells). Human T cells were seeded on a cell culture plate in which anti-human TCRVβ8 antibodies (Thermo Scientific, serial number TCR1750) were in solid phase, anti-human CD28 antibodies (BioLegend, serial number 302923) were added to them, and subjected to activation treatment for 72 hours. Here, human T cells that had undergone an activation treatment were cultured overnight in fresh medium containing 100 U/ml human IL-2 (R&D system, serial number 202-IL). In addition, the harvested human T cells were seeded on another cell culture plate in which anti-human TCRVβ8 antibodies were in solid phase, and anti-human CD28 antibodies were also added thereto, and then activated again. At this time, clones PD1-1(Bi) to PD1-6(Bi), respectively, were added thereto, and then 96 hours after reactivation treatment, IFN-γ contained in the culture supernatant (pg/ml) was quantified by ELISA. In FIG. 14 shows the results. The suppressive activity of IFN-γ production of clones PD1-1(Bi) - PD1-5(Bi) was confirmed. The suppressive activity of IFN-γ production of clone PD1-6(Bi) tends to decrease more than other clones. As a control antibody, an antibody obtained by immunization with tetanus toxoid in the same manner as for the first arm, which is a non-specific antibody, was used.

[0125] Example 13: In vivo results of an anti-PD-1/CD3 bispecific monoclonal antibody in an experimental mouse model of allergic encephalomyelitis (EAE model)

In vivo results of the anti-PD-1/CD3 bispecific monoclonal antibody of the present invention were evaluated in the EAE model using human CD3ε/human PD-1 knockin C57BL/6 mice in which the CD3ε gene and PD-1 gene were replaced with human CD3ε gene and human PD-1 gene, respectively. Dead Mycobacterium tuberculosis H37Ra (BD Biosciences, serial number 231141) and incomplete Freund's adjuvant (BD Biosciences, serial number 263910) were mixed with each other to obtain a complete Freund's adjuvant (CFA) containing 4 mg/ml of dead Mycobacterium tuberculosis H37Ra. 1 mg/ml of MOG peptide (ANASPEC, serial number AS-60130) and an equal amount of CFA were mixed together to form an emulsion as an EAE model stimulant. 200 μl of the stimulant was injected subcutaneously into the root of the base of the tail of C57BL/6 mice. On the day of immunization and on day 2, 200 μl of 1 μg/ml pertussis toxin (SIGMA-ALDRICH, serial number P7208) were injected into the tail vein, respectively. Here, on days 6 and 7 of immunization, C57BL/6 mice were intraperitoneally injected with 2 mg/kg clones PD1-1 (Bi) to PD1-6 (Bi) once a day, respectively. Neurological symptoms after immunization were assessed according to the method of Onuki, et al. (Onuki M, et al. Microsc Res Tech 2001; 52: 731-9). The scores assessed the degree of neurological symptoms (normal: 0 points, tail relaxation: 1 point, partial hind limb paralysis: 2 points, hind limb paralysis: 3 points, forelimb paralysis: 4 points, and dying or death: 5 points). If multiple neurological symptoms were observed, the higher score was used as the neurological symptom on the day of assessment. The neurological symptom of deceased mice was scored up to 5 until the end of the observation. In FIG. 15-17 show the results of the evaluations. It should be noted here that C57BL/6 mice with a human CD3ε knockin/human PD-1 knockin were obtained by crossing a mouse with a human CD3ε knockin obtained according to the method described in Genesis. 2009 Jun; 47(6): 414-22, with a human PD-1 knockin mouse using a well known method.

[0126] In the EAE model, the onset of neurological symptoms was markedly suppressed against any of the PD1-1-PD1-5(Bi) clones, but no significant suppressive effect was observed against the PD1-6(Bi) clone.

[0127] Example 14: Evaluation of the in vitro effect of an anti-PD-1/CD3 bispecific monoclonal antibody on cytokine release from a human peripheral blood monocyte

In order to analyze the cytokine-releasing activity of the anti-PD-1/CD3 bispecific monoclonal antibody, an experiment was conducted in which the anti-PD-1/CD3 bispecific monoclonal antibody clones of the present invention or the anti-PD-1/CD3 bispecific scDb (J110×UCHT1) disclosed in Patent Document 2 were added to human peripheral blood mononuclear cells (herein referred to as "human PBMC"). a clone of anti-PD-1/CD3 and J110×UCHT1 bispecific monoclonal antibodies was added to human PBMC (LONZA, serial number CC-2702) so that their concentrations were 30 μg/ml and 10 μg/ml, respectively, and then cultured for 24 hours. After culturing for 24 hours, each IL-2 contained in their culture supernatants was quantified by multiplex immunoassay (BIO-RAD, serial number M50000007A). In FIG. 18 shows the results. It should be noted here that the amount of IL-2 production (pg/ml) in the figure is represented as value ± standard error (N=3).

[0128] J110xUCHT1 markedly induced IL-2 production. However, the production of IL-2 against any clone of bispecific anti-PD-1/CD3 monoclonal antibodies was low.

[0129] Example 15: Evaluation of cross-competitive activity for binding to PD-1 of each clone of anti-PD-1/CD3 bispecific monoclonal antibodies with PD1-5(Bi)

Conducted cross-competitive analysis to assess the cross-competitive activity of binding to PD-1 each clone PD1-1(Bi) - PD1-5(Bi) bispecific monoclonal antibodies against PD-1/CD3 obtained in example 8, with PD1-5(Bi).

[0130] First, clone PD1-5(Bi) was added to CHO-S cells expressing human PD-1, then incubated on ice for twenty minutes. In addition, one hundredth of the number of biotin-labeled clones PD1-1(Bi) to PD1-5(Bi) compared with the clone PD1-5(Bi) already added, respectively, was added to these cells, then incubated on ice for 20 minutes. Those cells to which PE labeled streptavidin (BD Pharmingen, model number 554061) was added were washed and then incubated on ice for 20 minutes. After washing these cells, the binding amount of biotin-labeled PD1-1(Bi)-PD1-5(Bi) clones was measured using flow cytometry. In FIG. 19 shows this result.

[0131] It has been shown that clone PD1-5(Bi) can inhibit each binding of clones PD1-1(Bi) - PD1-5(Bi) to PD-1, and thus clone PD1-5(Bi) can cross-compete with their binding to PD-1.

Industrial Applicability

[0132] The bispecific anti-PD-1/CD3 antibody or antibody fragment of the present invention is useful for preventing, suppressing symptoms of progression or relapse, and/or treating autoimmune diseases or graft-versus-host disease (GVHD).

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SEQUENCE LIST

<110> ONO PHARMACEUTICAL CO., LTD.

<120> Bispecific antibody

<130> P18-199WO

<150> JP 2018-021498

<151> 2018-02-09

<150> JP 2018-153149

<151> 2018-08-16

<160> 41

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Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Lys Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Lys Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 25

<211> 107

<212> Protein

<213> Homo sapiens

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 26

<211> 11

<212> Protein

<213> Homo sapiens

<400> 26

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 27

<211> 7

<212> Protein

<213> Homo sapiens

<400> 27

Ala Ala Ser Ser Leu Gln Ser

15

<210> 28

<211> 9

<212> Protein

<213> Homo sapiens

<400> 28

Gln Gln Ser Tyr Ser Thr Pro Pro Thr

15

<210> 29

<211> 107

<212> Protein

<213> Homo sapiens

<400> 29

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 30

<211> 381

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1)..(381)

<400> 30

cag gtg cag ctg gtg caa tct ggg tct gag ttg aag cag cct ggg gcc 48

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Gln Pro Gly Ala

1 5 10 15

tca gtg aag gtt tcc tgt aag gct tct gga tac acc ttc act cac tat 96

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr

20 25 30

ggt ttg cat tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg 144

Gly Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

gga tgg ctc aac acc aac act gag aac cca acg ttt gcc cag ggc ttc 192

Gly Trp Leu Asn Thr Asn Thr Glu Asn Pro Thr Phe Ala Gln Gly Phe

50 55 60

aca gga cgg ttt gtc ttc tcc ttg gac acc tct gtc acc acg gca tat 240

Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Thr Thr Ala Tyr

65 70 75 80

ctg cag atc agc agc ctg aag gct gag gac act gcc gta tat tac tgt 288

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

gcg aga ggg gat atg gta gta ccg act act ata tgg aac tac tac cac 336

Ala Arg Gly Asp Met Val Val Pro Thr Thr Ile Trp Asn Tyr Tyr His

100 105 110

ttc atg gac gtc tgg ggc aac ggc acc ctg gtc acc gtc tcg agt 381

Phe Met Asp Val Trp Gly Asn Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 31

<211> 381

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1)..(381)

<400> 31

cag gtg cag ctg gtg caa tct ggg tct gag ttg aag cag cct ggg gtc 48

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Gln Pro Gly Val

1 5 10 15

tca gtg aag gtt tcc tgc aag gct tct gga tac acc ttc act cac tat 96

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr

20 25 30

ggt tta cat tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg 144

Gly Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

gga tgg atc aac acc aac act ggg aac cca acg tat gcc cag ggc ttc 192

Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe

50 55 60

aca gga cgg ttt gtc ttc tcc ttg gac acc tct gtc agc acg gca tat 240

Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

ctg cag atc agc agc cta aag gct gaa gac act gcc gtg tat tac tgt 288

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

gcg aga ggg gat tta gta gta cca act act ata tgg aac tac tac cac 336

Ala Arg Gly Asp Leu Val Val Pro Thr Thr Ile Trp Asn Tyr Tyr His

100 105 110

tac atg gag gtc tgg ggc aaa ggc acc ctg gtc acc gtc tcg agt 381

Tyr Met Glu Val Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 32

<211> 381

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1)..(381)

<400> 32

cag gtg cag ctg gtg caa tct ggg tct gag ttg aag aag cct ggg gcc 48

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

tca gtg atg gtt tcc tgc aag gct tct gga tac acc ttc act cac tat 96

Ser Val Met Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr

20 25 30

gct ttg cat tgg gtg cgc cag gcc cct gga caa ggg ctt gag tgg atg 144

Ala Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

gga tgg ctc aat acc aac act gag aat cca acg tat gcc cag ggc ttc 192

Gly Trp Leu Asn Thr Asn Thr Glu Asn Pro Thr Tyr Ala Gln Gly Phe

50 55 60

aca gga cgg ttt gtc ttc tcc ttg gac acc tct gtc acc acg gca tat 240

Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Thr Thr Ala Tyr

65 70 75 80

ctg cag atc aac agc cta aag gct gag gac act gcc gtg tat tac tgt 288

Leu Gln Ile Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

gcg aga ggg gat atg gta gta cca act act ata tgg aac tac tac tac 336

Ala Arg Gly Asp Met Val Val Pro Thr Thr Ile Trp Asn Tyr Tyr Tyr

100 105 110

tac atg gac gtc tgg ggc aaa ggg acc acg gtc acc gtc tcg agt 381

Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 33

<211> 381

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1)..(381)

<400> 33

cag gtg cag ctg gtg caa tct ggg tct gag ttg aag cag cct ggg gcc 48

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Gln Pro Gly Ala

1 5 10 15

tca gtg aag gtt tcc tgt aag gct tct gga tac acc ttc act cac tat 96

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr

20 25 30

gct ttg cat tgg ttg cga cag gcc cct gga caa ggg ctt gag tgg atg 144

Ala Leu His Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

gga tgg ctc aac acc aac act gag aac cca acg ttt gcc cag ggc ttc 192

Gly Trp Leu Asn Thr Asn Thr Glu Asn Pro Thr Phe Ala Gln Gly Phe

50 55 60

aca gga cgt ttt gtc ttc tct ttg gac acc tct gtc acc acg gca tat 240

Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Thr Thr Ala Tyr

65 70 75 80

ctg cag atc agc agc ctg aag gct gag gac act gcc gta tat tac tgt 288

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

gcg aga ggg gat atg gta gta ccg act act ata tgg aac tac tac cac 336

Ala Arg Gly Asp Met Val Val Pro Thr Thr Ile Trp Asn Tyr Tyr His

100 105 110

ttc atg gac gtc tgg ggc aac ggg acc acg gtc acc gtc tcg agt 381

Phe Met Asp Val Trp Gly Asn Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 34

<211> 381

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1)..(381)

<400> 34

cag gtg cag ctg gtg caa tct ggg tct gag ttg aag cag cct ggg gcc 48

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Gln Pro Gly Ala

1 5 10 15

tca gtg aag gtt tcc tgt aag gct tct gga tac acc ttc act cac tat 96

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr

20 25 30

gct ttg cat tgg ttg cga cag gcc cct gga caa ggg ctt gag tgg atg 144

Ala Leu His Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

gga tgg ctc aac acc aac act gag aac cca acg ttt gcc cag ggc ttc 192

Gly Trp Leu Asn Thr Asn Thr Glu Asn Pro Thr Phe Ala Gln Gly Phe

50 55 60

aca gga cgt ttt gtc ttc tct ttg gac acc tct gtc acc acg gca tat 240

Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Thr Thr Ala Tyr

65 70 75 80

ctg cag atc agc agc ctg aag gct gag gac act gcc gta tat tac tgt 288

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

gcg aga ggg gat atg gta gta ccg act act ata tgg aac tac tac cac 336

Ala Arg Gly Asp Met Val Val Pro Thr Thr Ile Trp Asn Tyr Tyr His

100 105 110

ttc atg gac gtc tgg ggc cag ggg acc acg gtc acc gtc tcg agt 381

Phe Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 35

<211> 321

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1)..(321)

<400> 35

gac atc cag atg acc cag tct cca tcc tcc ctg tct gca tct gta gga 48

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

gac aga gtc acc atc act tgc cgg gca agt cag agc att agc agc tac 96

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

tta aat tgg tat cag cag aaa cca ggg aaa gcc cct aag ctc ctg atc 144

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

tat gct gca tcc agt ttg caa agt ggg gtc cca tca agg ttc agt ggc 192

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

agt gga tct ggg aca gat ttc act ctc acc atc agc agt ctg caa cct 240

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

gaa gat ttt gca act tac tac tgt caa cag agt tac agt acc cct cca 288

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

acg ttc ggc caa ggg acc aag gtg gag atc aaa 321

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 36

<211> 118

<212> Protein

<213> Homo sapiens

<400> 36

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Trp Tyr Asn Ala Arg Lys Gln Glu Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Thr Gly Tyr Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 37

<211> 5

<212> Protein

<213> Homo sapiens

<400> 37

Ser Tyr Gly Met His

15

<210> 38

<211> 17

<212> Protein

<213> Homo sapiens

<400> 38

Gln Ile Trp Tyr Asn Ala Arg Lys Gln Glu Tyr Ser Asp Ser Val Lys

1 5 10 15

gly

<210> 39

<211> 9

<212> Protein

<213> Homo sapiens

<400> 39

Gly Thr Gly Tyr Asn Trp Phe Asp Pro

15

<210> 40

<211> 354

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1)..(354)

<400> 40

cag gtg cag ctg gtg cag tct ggc ggc gga gtg gtg cag ccc ggc aga 48

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

agc ctg aga ctg agc tgc gtg gcc agc ggc ttc acc ttc agc agc tac 96

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

ggc atg cac tgg gtc cgc cag gcc cct ggc aag gga ctg gaa tgg gtg 144

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

gcc cag atc tgg tac aac gcc cgg aag cag gaa tac tct gac agc gtg 192

Ala Gln Ile Trp Tyr Asn Ala Arg Lys Gln Glu Tyr Ser Asp Ser Val

50 55 60

aag ggc cgg ttc acc atc agc cgg gac aac agc aag aac acc ctg tac 240

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

ctg cag atg aac agc ctc cgg gcc gag gac acc gcc gtg tac tac tgt 288

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

acc cgg ggc acc ggc tac aat tgg ttc gac cct tgg ggc cag ggc acc 336

Thr Arg Gly Thr Gly Tyr Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr

100 105 110

ctg gtc acc gtc tcc agt 354

Leu Val Thr Val Ser Ser

115

<210> 41

<211> 11

<212> Protein

<213> Homo sapiens

<400> 41

Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser

1 5 10

<---

Claims (151)

1. A bispecific anti-PD-1/CD3 antibody or a fragment of this antibody containing a first arm specifically binding to PD-1 and a second arm specifically binding to CD3, where the first arm specifically binding to PD-1 contains any VH selected from: (A) VH having (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:18; (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:19; And (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:20; (B) VH having (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:6; (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:7; And (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:8; (C) VH having (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:9; (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:10; And (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:11; (D) VH having (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:12; (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:13, and (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:14; And (E) VH having (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:15; (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:16; And (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:17; And (F) VL having (a) VL-CDR1 containing the amino acid sequence of SEQ ID NO:26; (b) VL-CDR2 containing the amino acid sequence of SEQ ID NO:27; And (c) VL-CDR3 containing the amino acid sequence of SEQ ID NO:28; And where the second arm specifically binding to CD3 contains: (A) VH having (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:37; (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:38; And (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:39, and (B) VL having (a) VL-CDR1 containing the amino acid sequence of SEQ ID NO:26; (b) VL-CDR2 containing the amino acid sequence of SEQ ID NO:27; And (c) VL-CDR3 containing the amino acid sequence of SEQ ID NO:28. 2. Bispecific antibody against PD-1/CD3 or a fragment of this antibody according to claim 1, where (i) The VH of the first arm specifically binding to PD-1 has: (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:18; (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:19; And (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:20; And (ii) VH of the second arm specifically binding to CD3 has: (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:37; (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:38; And (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:39. 3. Bispecific antibody against PD-1/CD3 or a fragment of this antibody according to claim 1, where (i) The VH of the first arm specifically binding to PD-1 has: (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:6, (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:7, and (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:8; And (ii) VH of the second arm specifically binding to CD3 has: (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:37, (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:38, and (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:39. 4. Bispecific antibody against PD-1/CD3 or a fragment of this antibody according to claim 1, where (i) The VH of the first arm specifically binding to PD-1 has: (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:9, (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:10, and (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:11; And (ii) VH of the second arm specifically binding to CD3 has: (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:37, (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:38, and (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:39. 5. Bispecific antibody against PD-1/CD3 or a fragment of this antibody according to claim 1, where: (i) The VH of the first arm specifically binding to PD-1 has (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:12, (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:13, and (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:14, and (ii) VH of the second arm specifically binding to CD3 has: (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:37, (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:38, and (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:39. 6. Bispecific antibody against PD-1/CD3 or a fragment of this antibody according to claim 1, where (i) The VH of the first arm specifically binding to PD-1 has: (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:15, (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:16, and (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:17; And (ii) VH of the second arm specifically binding to CD3 has: (a) VH-CDR1 containing the amino acid sequence of SEQ ID NO:37, (b) VH-CDR2 containing the amino acid sequence of SEQ ID NO:38, and (c) VH-CDR3 containing the amino acid sequence of SEQ ID NO:39. 7. Bispecific anti-PD-1/CD3 antibody or fragment of this antibody according to any one of claims 1-6, where the FR1, FR2 and FR3 regions of the VH of the first arm specifically binding to PD-1 correspond to the amino acid sequence encoded by the V germline gene IGHV7-4-1 with the somatic mutation(s), respectively, and framework region 4 contains the amino acid sequence encoded by the JH6c germline J gene with the somatic mutation(s) (excluding the amino acid sequence included in the VH-CDR3 region). 8. A bispecific anti-PD-1/CD3 antibody or a fragment of this antibody according to any one of claims 1 to 7, wherein the VH of the first arm specifically binding to PD-1 contains any amino acid sequence of SEQ ID NO:5, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or an amino acid sequence that is at least 80% identical to the amino acid sequence of VH. 9. A bispecific anti-PD-1/CD3 antibody or fragment thereof according to any one of claims 1 to 8, wherein the VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36, or an amino acid sequence that is at least 80% identical to the amino acid sequence of the VH. 10. Bispecific antibody against PD-1/CD3 or a fragment of this antibody according to claim 1, where The VH of the first arm specifically binding to PD-1 contains any amino acid sequence of SEQ ID NO:5, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; And The VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36. 11. Bispecific antibody against PD-1/CD3 or a fragment of this antibody according to claim 1 or 2, where The VH of the first arm specifically binding to PD-1 contains the amino acid sequence of SEQ ID NO:5; And The VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36. 12. Bispecific antibody against PD-1/CD3 or a fragment of this antibody according to claim 1 or 3, where The VH of the first arm specifically binding to PD-1 contains the amino acid sequence of SEQ ID NO:1; And The VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36. 13. Bispecific antibody against PD-1/CD3 or a fragment of this antibody according to claim 1 or 4, where The VH of the first arm specifically binding to PD-1 contains the amino acid sequence of SEQ ID NO:2; And The VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36. 14. Bispecific antibody against PD-1/CD3 or a fragment of this antibody according to claim 1 or 5, where The VH of the first arm specifically binding to PD-1 contains the amino acid sequence of SEQ ID NO:3; And The VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36. 15. Bispecific antibody against PD-1/CD3 or a fragment of this antibody according to claim 1 or 6, where The VH of the first arm specifically binding to PD-1 contains the amino acid sequence of SEQ ID NO:4; And The VH of the second arm specifically binding to CD3 contains the amino acid sequence of SEQ ID NO:36. 16. The PD-1/CD3 bispecific antibody or antibody fragment of any one of claims 1 to 15, wherein the first PD-1 specific binding arm and/or the CD3 specific binding second arm have a VL comprising the amino acid sequence of SEQ ID NO:25. 17. An anti-PD-1/CD3 bispecific antibody, or a fragment thereof, having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3, wherein (A) The first arm specifically binding to PD-1 has VH containing any amino acid sequence from SEQ ID NO:5, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and VL containing the amino acid sequence of SEQ ID NO:25; And (B) The second arm specifically binding to CD3 has VH containing the amino acid sequence of SEQ ID NO:36; And VL containing the amino acid sequence of SEQ ID NO:25. 18. An anti-PD-1/CD3 bispecific antibody or antibody fragment according to any one of claims 1-17, wherein the first arm specifically binding to PD-1 provides interaction between PD-1 and PD-L1. 19. The anti-PD-1/CD3 bispecific antibody or antibody fragment of any one of claims 1 to 18, wherein the production of IL-2, IFN-γ, or TNF-α in blood or tissue is not increased or only increased to the extent that it can be suppressed by administration of steroids. 20. The anti-PD-1/CD3 bispecific antibody or antibody fragment of any one of claims 1 to 19, wherein the anti-PD-1/CD3 bispecific antibody is an IgG antibody. 21. An anti-PD-1/CD3 bispecific antibody or fragment thereof according to claim 20, which is an IgG ₁ antibody or an IgG ₄ antibody. 22. The bispecific anti-PD-1/CD3 antibody or fragment thereof according to claim 20, which is an IgG ₁ antibody. 23. A bispecific anti-PD-1/CD3 antibody or a fragment of this antibody according to claim 22, wherein there is no or reduced binding to the Fc receptor. 24. A bispecific anti-PD-1/CD3 antibody or a fragment of this antibody according to claim 23, where in two constant regions of the heavy chains of the bispecific IgG ₁ antibody against PD-1/CD3 the leucine at position 235 according to the EU numbering system has been replaced by glycine, and/or glycine in position 236 according to the EU numbering system is replaced by arginine. 25. A bispecific anti-PD-1/CD3 antibody or a fragment of this antibody according to any one of claims 22-24, where in the heavy chain constant region having the VH of the first arm specifically binding to PD-1, the leucine at position 351 according to the EU numbering system is replaced by lysine, and the threonine at position 366 according to the EU numbering system is replaced by lysine; And in the constant region of the heavy chain, which has a VH of the second arm that specifically binds to CD3, the leucine at position 351 according to the EU numbering system has been replaced by aspartic acid, and the leucine at position 368 according to the EU numbering system has been replaced by glutamic acid. 26. A bispecific anti-PD-1/CD3 antibody or a fragment of this antibody according to any one of claims 22-24, where in the heavy chain constant region having the VH of the first arm specifically binding to PD-1, the leucine at position 351 according to the EU numbering system has been replaced by aspartic acid, and leucine at position 368 is replaced by glutamic acid, and in the constant region of the heavy chain, which has a VH of the second arm that specifically binds to CD3, the leucine at position 351 according to the EU numbering system has been replaced by a lysine, and threonine at position 366 has been replaced by lysine according to the EU numbering system. 27. The anti-PD-1/CD3 bispecific antibody or antibody fragment of any one of claims 22-26, wherein in the two heavy chain constant regions of the anti-PD-1/CD3 bispecific antibody, the lysine at position 447 according to the EU numbering system is deleted. 28. The PD-1/CD3 bispecific antibody or antibody fragment of claim 21, wherein the PD-1/CD3 bispecific antibody is an IgG ₄ antibody, and wherein in the two heavy chain constant regions, serine at position 228 according to the EU numbering system is replaced by proline, respectively. 29. The anti-PD-1/CD3 bispecific antibody or antibody fragment of any one of claims 1 to 23, wherein the heavy chain having a first arm VH specifically binding to PD-1 has a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:23. 30. The anti-PD-1/CD3 bispecific antibody or antibody fragment of any one of claims 1-23 and 29, wherein the heavy chain having a second arm VH specifically binding to CD3 has a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:24. 31. A bispecific anti-PD-1/CD3 antibody or a fragment of this antibody according to any one of claims 1 to 30, wherein the light chain having a first arm VL specifically binding to PD-1 and/or a light chain having a second arm VL specifically binding to CD3 have a light chain constant region containing the amino acid sequence of SEQ ID NO:29. 32. A bispecific antibody against PD-1/CD3 or a fragment of this antibody, having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3, where: (A) a heavy chain having a first arm VH specifically binding to PD-1 has a VH containing any of the amino acid sequences of SEQ ID NO:5, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 and a heavy chain constant region containing the amino acid sequence of SEQ ID NO:23; (B) a light chain having a first arm VL specifically binding to PD-1 has a VL containing the amino acid sequence of SEQ ID NO:25 and a light chain constant region containing the amino acid sequence of SEQ ID NO:29; (C) a heavy chain having a second arm VH specifically binding to CD3 has a VH containing the amino acid sequence of SEQ ID NO:36 and a heavy chain constant region containing the amino acid sequence of SEQ ID NO:24; And (D) A light chain having a second arm VL specifically binding to CD3 has a VL containing the amino acid sequence of SEQ ID NO:25 and a light chain constant region containing the amino acid sequence of SEQ ID NO:29. 33. A bispecific anti-PD-1/CD3 antibody having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3, where: (A) a heavy chain having a first arm VH specifically binding to PD-1 has a VH containing any amino acid sequence from SEQ ID NO:5 and a heavy chain constant region containing the amino acid sequence of SEQ ID NO:23; (B) a light chain having a first arm VL specifically binding to PD-1 has a VL containing the amino acid sequence of SEQ ID NO:25 and a light chain constant region containing the amino acid sequence of SEQ ID NO:29; (C) a heavy chain having a second arm VH specifically binding to CD3 has a VH containing the amino acid sequence of SEQ ID NO:36 and a heavy chain constant region containing the amino acid sequence of SEQ ID NO:24; And (D) A light chain having a second arm VL specifically binding to CD3 has a VL containing the amino acid sequence of SEQ ID NO:25 and a light chain constant region containing the amino acid sequence of SEQ ID NO:29. 34. A pharmaceutical composition for the prevention, suppression of symptoms of progression or recurrence of autoimmune diseases, where the composition contains a bispecific anti-PD-1/CD3 antibody or a fragment of this antibody according to any one of claims 1-33 and a pharmaceutically acceptable carrier. 35. The pharmaceutical composition according to claim 34, wherein the autoimmune disease is Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa, aortic syndrome, rheumatoid arthritis with malignant diseases, rheumatoid arthritis, juvenile idiopathic arthritis , spondyloarthritis, mixed connective tissue disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, large vessel vasculitis, ANCA-associated vasculitis, Cogan's syndrome, RS3PE, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis , IgG4-related disease, Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic steatohepatitis, primary biliary cirrhosis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, a autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease, Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, Addison's disease, idiopathic Addison's disease, type I diabetes mellitus, type I slowly progressive diabetes mellitus, scleroderma localis, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, gest cationic herpes, linear IgA-dependent bullous dermatosis, acquired epidermosis bullosa, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis of the optic nerve, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harad's disease, a autoimmune optic neuropathy, azoospermia of unknown origin, recurrent abortion, inflammatory bowel disease, celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, transplantation immunity, familial Mediterranean fever, eosinophilic chronic rhinosinusitis, dilated cardiomyopathy, systemic mastocytosis, or inclusion body myositis. 36. The pharmaceutical composition according to claim 34 or 35, which is administered together with one or more agent(s) selected from an insulin drug, a sulfonylurea drug, a fast-acting insulin secretion promoter, a biguanide drug, an insulin resistance agent, an α-glucosidase inhibitor, an agent for the treatment of diabetic neuropathy, a GLP-1 analogue drug and a DPP-4 inhibitor, a steroid agent, an interferer she β-1a, interferon β-1b, glatiramer acetate, mitoxantrone, azathioprine, cyclophosphamide, cyclosporine, methotrexate, cladribine, adrenocorticotropic hormone (ACTH), corticotropin, mizoribine, tacrolimus, fingolimod, alemtuzumab, immunosuppressive agent, belimumab, anti a rheumatic drug, an anti-cytokine drug, and abatacept.

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