HK40036202B - Bispecific antibody - Google Patents

Description

Bispecific antibodies

Technical Field

This invention relates to a bispecific antibody (hereinafter abbreviated as "PD-1/CD3 bispecific antibody") capable of specifically binding to PD-1 and CD3 respectively, a pharmaceutical composition containing the bispecific antibody as an active ingredient, and its use in pharmaceutical therapy.

Background Technology

PD-1 is an immunosuppressive receptor belonging to the immunoglobulin family and is a molecule that inhibits immune activation signals in T cells activated by antigen receptor stimulation. Analysis of PD-1 knockout mice and other studies has shown that PD-1 signaling plays an important role in suppressing autoimmune diseases such as autoimmune dilated cardiomyopathy, lupus-like syndrome, autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease, type 1 diabetes, and rheumatoid arthritis. Therefore, it is noted that agents that enhance PD-1 signaling could be used as preventative or therapeutic agents for autoimmune diseases.

Previously, bispecific antibodies recognizing PD-1 were known to be agents that enhance PD-1 signaling (Patent References 1 to 3). Bispecific antibodies consist of antigen recognition sites of antibodies recognizing CD3, a member of the T-cell receptor complex, and antigen recognition sites of antibodies recognizing PD-1, which are linked together through genetic engineering. Bispecific antibodies enhance the inhibitory signal of PD-1 against the T-cell receptor complex by increasing the frequency at which PD-1 is brought near the T-cell receptor complex. Furthermore, the patent documents indicate that PD-1 bispecific antibodies can be used to prevent or treat autoimmune diseases.

Incidentally, in protein formulations, there is already concern about the expression of adverse reactions occurring immediately after administration, known as infusion reactions or cytokine release syndrome. Formulations that reduce or inhibit such reactions are needed.

In the PD-1/CD3 bispecific antibody of the present invention, the stimulation of post-administration cytokine production, which is considered to be the cause of the aforementioned adverse reactions, is significantly reduced. Therefore, the PD-1/CD3 bispecific antibody shows promise as a drug for inhibiting the expression of related adverse reactions.

No bispecific antibodies with such characteristics have been reported to date.

Citation List

Patent References

Patent Reference 1: International Publication No. WO2003/011911

Patent Reference 2: International Publication No. WO2004/072286

Patent Reference 3: International Publication No. WO2013/022091

Summary of the Invention

Technical issues

The purpose of this invention is to provide a new drug for preventing autoimmune diseases, inhibiting the progression or recurrence of symptoms of autoimmune diseases, and/or treating autoimmune diseases.

Solution to the problem

The inventors of this invention have conducted thorough research and focused on the PD-1/CD3 bispecific antibody of this invention as a reagent capable of solving the aforementioned problems. The inventors have further demonstrated that the PD-1/CD3 bispecific antibody can be a reagent for reducing the expression of reactions known as infusion reactions or cytokine release syndrome, and this invention has been completed.

Furthermore, the inventors of this invention have discovered that PD-1/CD3 bispecific antibodies have the characteristic of allowing interaction between PD-1 and PD-L1 as its ligand, and have found that such characteristics help to enhance or maintain the effect of PD-1/CD3 bispecific antibodies.

In other words, the present invention relates to the following:

[1] A PD-1/CD3 bispecific antibody or an antibody fragment thereof, having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3.

The first arm that specifically binds to PD-1 contains a VH selected from any of the following:

(A) Heavy chain variable region (hereinafter, "heavy chain variable region" can be abbreviated as "VH"), which has

(a) The complementarity-determining region 1 of the heavy chain variable region containing the amino acid sequence shown in SEQ ID No. 6 (hereinafter, “complementarity-determining region 1 of the heavy chain variable region” can be abbreviated as “VH-CDR1”);

(b) The complementarity-determining region 2 of the heavy chain variable region containing the amino acid sequence shown in SEQ ID No. 7 (hereinafter, "complementarity-determining region 2 of the heavy chain variable region" may be abbreviated as "VH-CDR2"); and

(c) The complementarity-determining region 3 of the heavy chain variable region containing the amino acid sequence shown in SEQ ID No. 8 (hereinafter, “complementarity-determining region 3 of the heavy chain variable region” may be abbreviated as “VH-CDR3”);

(B)VH, which has

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 9;

(b) VH-CDR2 comprising the amino acid sequence shown in SEQ ID No. 10; and

(c) VH-CDR3 containing the amino acid sequence shown in SEQ ID No. 11;

(C)VH, which has

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 12;

(b) VH-CDR2 comprising the amino acid sequence shown in SEQ ID No. 13; and

(c) VH-CDR3 containing the amino acid sequence shown in SEQ ID No. 14;

(D)VH, which has

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 15;

(b) VH-CDR2 comprising the amino acid sequence shown in SEQ ID No. 16; and

(c) VH-CDR3 comprising the amino acid sequence shown in SEQ ID No. 17; and

(E)VH, which has

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 18;

(b) VH-CDR2 comprising the amino acid sequence shown in SEQ ID No. 19; and

(c) VH-CDR3 comprising the amino acid sequence shown in SEQ ID No. 20; and

The second arm, which specifically binds to CD3, contains VH, which has...

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 37;

(b) VH-CDR2 comprising the amino acid sequence shown in SEQ ID No. 38; and

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

[2] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1], wherein in any one or more VH-CDRs selected from VH-CDR1, VH-CDR2 and VH-CDR3 in the first arm that specifically binds to PD-1, one to five arbitrary amino acid residues may be replaced with other amino acids (preferably their conserved amino acids); and/or in any one or more VH-CDRs selected from VH-CDR1, VH-CDR2 and VH-CDR3 in the second arm that specifically binds to CD3, one to five arbitrary amino acid residues may be replaced with other amino acids (preferably their conserved amino acids).

[3] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [2], wherein (i) the VH of the first arm that specifically binds to PD-1 has:

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 6,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 7, and

(c) VH-CDR3 comprising the amino acid sequence shown in SEQ ID No. 8; and

(ii) The VH of the second arm that specifically binds to CD3 has:

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 37,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 38, and

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

[4] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [2], wherein (i) the VH of the first arm that specifically binds to PD-1 has:

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 9,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 10, and

(c) VH-CDR3 comprising the amino acid sequence shown in SEQ ID No. 11; and

(ii) The VH of the second arm that specifically binds to CD3 has:

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 37,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 38, and

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

[5] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [2], wherein (i) the VH of the first arm that specifically binds to PD-1 has:

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 12,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 13, and

(c) VH-CDR3 comprising the amino acid sequence shown in SEQ ID No. 14; and

(ii) The VH of the second arm that specifically binds to CD3 has:

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 37,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 38, and

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

[6] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [2], wherein (i) the VH of the first arm that specifically binds to PD-1 has:

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 15,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 16, and

(c) VH-CDR3 comprising the amino acid sequence shown in SEQ ID No. 17; and

(ii) The VH of the second arm that specifically binds to CD3 has:

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 37,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 38, and

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

[7] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [2], wherein (i) the VH of the first arm that specifically binds to PD-1 has:

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 18,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 19, and

(c) VH-CDR3 comprising the amino acid sequence shown in SEQ ID No. 20; and

(ii) The VH of the second arm that specifically binds to CD3 has:

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 37,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 38, and

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

[8] A PD-1/CD3 bispecific antibody or an antibody fragment thereof, having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3.

The first arm, which specifically binds to PD-1, contains VH, which has:

(a) VH-CDR1, which contains an amino acid sequence represented by HYJ ¹ LH [where J ¹ represents G (glycine) or A (alanine), and J ¹ or other letters represent one-letter amino acid abbreviations respectively],

(b) VH-CDR2, comprising the amino acid sequence represented by WJ ² NTNTU ² NPTX ² AQGFTG [where J ² represents L (leucine) or I (isoleucine), U ² represents E (glutamic acid) or G (glycine), X ² represents F (phenylalanine) or Y (tyrosine), and the letters represented by J ² , U ² or X ² or others represent the same as above], and

(c) VH-CDR3, comprising the amino acid sequence represented by GDJ ³ VVPTTIWNYYU ³ X ³ MZ ³ V [where J ³ represents M (methionine) or L (leucine), U ³ represents H (histidine) or Y (tyrosine), X ³ represents F (phenylalanine) or Y (tyrosine), Z ³ represents D (aspartic acid) or E (glutamic acid), and the letters represented by J ³ , U ³ , X ³ or Z ³ or other letters represent the same as above], and;

The second arm, which specifically binds to CD3, contains VH, which has:

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 37,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 38, and

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

[9] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [8], wherein

(a) ^J1 represents G (glycine), ^J2 represents L (leucine), ^U2 represents E (glutamic acid), ^X2 represents F (phenylalanine), ^J3 represents M (methionine), ^U3 represents H (histidine), ^X3 represents F (phenylalanine), and ^Z3 represents D (aspartic acid).

(b) ^J1 represents G (glycine), ^J2 represents I (isoleucine), U2 represents G (glycine), ^X2 represents Y (tyrosine), ^J3 represents L (leucine), ^U3 represents H (histidine), ^X3 represents Y (tyrosine), and ^Z3 represents E ⁽ glutamic acid).

(c) ^J1 represents A (alanine), ^J2 represents L (leucine), ^U2 represents E (glutamic acid), ^X2 represents Y (tyrosine), ^J3 represents M (methionine), ^U3 represents Y (tyrosine), ^X3 represents Y (tyrosine), and ^Z3 represents D (aspartic acid); or

(d) ^J1 represents A (alanine), ^J2 represents L (leucine), ^U2 represents E (glutamic acid), ^X2 represents F (phenylalanine), ^J3 represents M (methionine), ^U3 represents H (histidine), ^X3 represents F (phenylalanine), and ^Z3 represents D (aspartic acid).

[10] According to any of the preceding items [1] to [9], the PD-1/CD3 bispecific antibody or antibody fragment thereof, wherein frame region 1 (hereinafter, may be abbreviated as "FR1"), frame region 2 (hereinafter, may be abbreviated as "FR2") and frame region 3 (hereinafter, may be abbreviated as "FR3") in the frame region of the first arm that specifically binds to PD-1 correspond to the amino acid sequence encoded by germline V gene IGHV7-4-1 or its gene with somatic mutation.

[11] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [10], wherein the frame 4 (hereinafter, “frame 4” may be abbreviated as “FR4”) region in the VH of the first arm that specifically binds to PD-1 includes an amino acid sequence encoded by germline J gene JH6c or a gene of which has a somatic mutation (excluding the amino acid sequence included in the VH-CDR3 region).

[12] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [10] or [11], wherein the FR region in the VH of the first arm that specifically binds to PD-1 is encoded by the germline V gene IGHV7-4-1 with a somatic mutation, and wherein the FR region contains the FR1 region, wherein, in the amino acid sequence shown in SEQ ID No. 21, lysine at position 13 is or may be replaced by glutamine, alanine at position 16 is or may be replaced by valine, or lysine at position 19 is or may be replaced by methionine, or any combination thereof, through somatic mutations.

[13] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [10] to [12], wherein the FR region of VH in the first arm that specifically binds to PD-1 is encoded by the germline V gene IGHV7-4-1 with a somatic mutation, and wherein the FR region contains the FR2 region, wherein the valine at position 37 in the amino acid sequence shown in SEQ ID No. 21 is replaced by or may be replaced by leucine through a somatic mutation.

[14] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [10] to [13], wherein the FR region of VH in the first arm that specifically binds to PD-1 is encoded by the germline V gene IGHV7-4-1 with a somatic mutation, and wherein the FR region contains the FR3 region, wherein, in the amino acid sequence shown in SEQ ID No. 21, serine at position 77 is or may be replaced by threonine, or cysteine at position 84 is or may be replaced by serine or asparagine, or may be replaced by any combination of these, respectively, through somatic mutation.

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

[16] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to [15], wherein the VH of the first arm that specifically binds to PD-1 comprises an amino acid sequence selected from any one 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 having at least 80%, 90%, 95%, 98% or 99% identity with its amino acid sequence.

[17] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] and [3] to [7], wherein the VH of the first arm that specifically binds to PD-1 contains an amino acid sequence selected from any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5.

[18] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to [17], wherein the VH of the second arm that specifically binds to CD3 contains the amino acid sequence shown in SEQ ID No. 36, or an amino acid sequence having at least 80%, 90%, 95%, 98% or 99% identity with its amino acid sequence.

[19] A PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] and [3] to [18], wherein the VH of the second arm that specifically binds to CD3 contains the amino acid sequence shown in SEQ ID No. 36.

[20] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1], wherein the VH of the first arm that specifically binds to PD-1 contains an amino acid sequence selected from any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5; and the VH of the second arm that specifically binds to CD3 contains an amino acid sequence shown in SEQ ID No. 36.

[21] A PD-1/CD3 bispecific antibody or an antibody fragment thereof, having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3, wherein the VH of the first arm that specifically binds to PD-1 contains an amino acid sequence selected from any one 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 having at least 80%, 90%, 95%, 98% or 99% identity with its amino acid sequence; and wherein the VH of the second arm that specifically binds to CD3 contains an amino acid sequence shown in SEQ ID No. 36, or an amino acid sequence having at least 80%, 90%, 95%, 98% or 99% identity with its amino acid sequence.

[22] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [3], wherein the VH of the first arm that specifically binds to PD-1 contains the amino acid sequence shown in SEQ ID No. 1; and the VH of the second arm that specifically binds to CD3 contains the amino acid sequence shown in SEQ ID No. 36.

[23] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [4], wherein the VH of the first arm that specifically binds to PD-1 contains the amino acid sequence shown in SEQ ID No. 2; and the VH of the second arm that specifically binds to CD3 contains the amino acid sequence shown in SEQ ID No. 36.

[24] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [5], wherein the VH of the first arm that specifically binds to PD-1 contains the amino acid sequence shown in SEQ ID No. 3; and the VH of the second arm that specifically binds to CD3 contains the amino acid sequence shown in SEQ ID No. 36.

[25] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [6], wherein the VH of the first arm that specifically binds to PD-1 contains the amino acid sequence shown in SEQ ID No. 4; and the VH of the second arm that specifically binds to CD3 contains the amino acid sequence shown in SEQ ID No. 36.

[26] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [1] or [7], wherein the VH of the first arm that specifically binds to PD-1 contains the amino acid sequence shown in SEQ ID No. 5; and the VH of the second arm that specifically binds to CD3 contains the amino acid sequence shown in SEQ ID No. 36.

[27] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to [26], wherein the first arm specifically binding to PD-1 and/or the second arm specifically binding to CD3 each have a light chain variable region (hereinafter, "light chain variable region" may be abbreviated as "VL"), which has

(a) Complementarity-determining region 1 of the light chain variable region containing the amino acid sequence shown in SEQ ID No. 26 (hereinafter, “complementarity-determining region 1 of the light chain variable region” may be abbreviated as “VL-CDR1”);

(b) The complementarity-determining region 2 of the light chain variable region containing the amino acid sequence shown in SEQ ID No. 27 (hereinafter, "complementarity-determining region 2 of the light chain variable region" may be abbreviated as "VL-CDR2"); and

(c) The complementarity-determining region 3 of the light chain variable region containing the amino acid sequence shown in SEQ ID No. 28 (hereinafter, “complementarity-determining region 3 of the light chain variable region” may be abbreviated as “VL-CDR3”).

[28] A PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to [27], wherein the first arm that specifically binds to PD-1 and/or the second arm that specifically binds to CD3 each has a VL containing the amino acid sequence shown in SEQ ID No. 25.

[29] A PD-1/CD3 bispecific antibody or an antibody fragment thereof, having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3, wherein (A) the first arm that specifically binds to PD-1 has a VH comprising an amino acid sequence selected from any one 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 comprising an amino acid sequence shown in SEQ ID No. 25; and

(B) The second arm that specifically binds to CD3 has a VH containing the amino acid sequence shown in SEQ ID No. 36; and a VL containing the amino acid sequence shown in SEQ ID No. 25.

[30] A PD-1/CD3 bispecific antibody or an antibody fragment thereof, having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3.

The first arm (1) that specifically binds to PD-1 cross-competes with a first arm that specifically binds to PD-1 having VH and VL, wherein VH contains an amino acid sequence selected from any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, and VL contains an amino acid sequence of SEQ ID No. 25, or (2) cross-competes with the variable region of a monoclonal antibody that specifically binds to PD-1 having the same VH and VL for binding to PD-1.

[31] A PD-1/CD3 bispecific antibody or an antibody fragment thereof having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3, wherein the following cross-competitive first arm that specifically binds to PD-1: (1) a first arm that specifically binds to PD-1 having VH and VL, wherein VH contains an amino acid sequence selected from any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, and VL contains an amino acid sequence of SEQ ID No. 25, or (2) a variable region of a monoclonal antibody that specifically binds to PD-1 having the same VH and VL.

[32] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to item [30] or [31], wherein the second arm that specifically binds to CD3 further (1) cross-competes with a second arm that specifically binds to CD3 having VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID No. 36 and the VL comprises the amino acid sequence shown in SEQ ID No. 25, or (2) cross-competes with the variable region of a monoclonal antibody that specifically binds to CD3 having the same VH and VL for binding to CD3.

[33] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to item [30] or [31], wherein the second arm that specifically binds to CD3 contains VH, which has:

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 37,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 38, and

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

[34] A PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [30], [31] and [33], wherein the VH of the second arm that specifically binds to CD3 contains the amino acid sequence shown in SEQ ID No. 36, or an amino acid sequence having at least 80%, 90%, 95%, 98% or 99% identity with its amino acid sequence.

[35] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [30] or [31], wherein the VH of the second arm that specifically binds to CD3 contains the amino acid sequence shown in SEQ ID No. 36.

[36] A PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [30], [31] and [33] to [35], wherein the second arm that specifically binds to CD3 has a VL, which has:

(a) VL-CDR1 containing the amino acid sequence shown in SEQ ID No. 26;

(b) VL-CDR2 comprising the amino acid sequence shown in SEQ ID No. 27; and

(c) VL-CDR3 containing the amino acid sequence shown in SEQ ID No. 28.

[37] A PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [30], [31] and [33] to [35], wherein the second arm that specifically binds to CD3 has a VL comprising the amino acid sequence shown in SEQ ID No. 25.

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

[39] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [38], wherein the IgG antibody in the preceding item [38] is an IgG ₁ or IgG ₄ antibody.

[40] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [38], wherein the IgG antibody in the preceding item [38] is an IgG ₁ antibody.

[41] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [38], wherein the IgG antibody in the preceding item [38] is an IgG ₄ antibody.

[42] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [40], wherein in the two heavy chain constant regions of the IgG ₁ antibody in the preceding item [40], leucine at position 235 according to the EU numbering system is replaced by glycine, and/or glycine at position 236 according to the EU numbering system is replaced by arginine.

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

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

[45] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [40] and [42] to [44], wherein the lysine at position 447 according to the EU numbering system is missing in the two heavy chain constant regions of the IgG ₁ antibody according to any one of the preceding items [40] and [42] to [44].

[46] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [41], wherein in the two heavy chain constant regions of the IgG ₄ antibody in the preceding item [41], the serine at position 228 according to the EU numbering system is replaced by proline.

[47] A PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to [38], [40], [42] and [44], wherein the heavy chain having a VH having a first arm that specifically binds to PD-1 has a heavy chain constant region comprising the amino acid sequence shown in SEQ ID No. 23.

[48] A PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to [38], [40], [42], [44] and [47], wherein the heavy chain having a VH with a second arm that specifically binds to CD3 has a heavy chain constant region comprising the amino acid sequence shown in SEQ ID No. 24.

[49] A PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to [48], wherein the light chain of the VL having a first arm that specifically binds to PD-1 and/or the light chain of the VL having a second arm that specifically binds to CD3 has a light chain constant region comprising the amino acid sequence shown in SEQ ID No. 29.

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

(A) A heavy chain having a VH with a first arm that specifically binds to PD-1 has a VH and a heavy chain constant region, wherein the VH contains an amino acid sequence selected from any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, and the heavy chain constant region contains an amino acid sequence shown in SEQ ID No. 23;

(B) A light chain of a VL having a first arm that specifically binds to PD-1 has a VL containing the amino acid sequence shown in SEQ ID No. 25 and a light chain constant region containing the amino acid sequence shown in SEQ ID No. 29;

(C) The heavy chain having a VH with a second arm that specifically binds to CD3 has a VH comprising the amino acid sequence shown in SEQ ID No. 36, and a heavy chain constant region comprising the amino acid sequence shown in SEQ ID No. 24; and

(D) A light chain having a VL with a second arm that specifically binds to CD3 has a VL containing the amino acid sequence shown in SEQ ID No. 25 and a light chain constant region containing the amino acid sequence shown in SEQ ID No. 29.

[51] A PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to [50], wherein the first arm that specifically binds to PD-1 allows for interaction between PD-1 and PD-L1.

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

[53] A PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to [50], wherein the first arm that specifically binds to PD-1 allows for interaction between PD-1 and PD-L1, and wherein cytokine production is sufficiently reduced.

[54] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to the preceding item [52] or [53], wherein the cytokines include at least IL-2, IFN-γ or TNF-α.

[55] The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to [54], wherein PD-1 is human PD-1 and CD3 is human CD3.

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

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

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

[1-1] A pharmaceutical composition comprising a PD-1/CD3 bispecific antibody or an antibody fragment thereof according to any one of the preceding items [1] to [58] and a pharmaceutically acceptable carrier.

[2-1] An agent for the prevention of autoimmune diseases, the suppression of the progression or recurrence of symptoms of autoimmune diseases and/or the treatment of autoimmune diseases, wherein the agent contains a PD-1/CD3 bispecific antibody or an antibody fragment thereof as an active ingredient according to any one of the preceding items [1] to [58].

[2-2] According to the drugs in item [2-1] above, autoimmune diseases include 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), aortitis syndrome (Takayasu's arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, and Sjogren's syndrome. Omega-1), adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, large vessel vasculitis, ANCA-associated vasculitis (e.g., granulomatous disease with polyangiitis and eosinophilic granulomatous disease with polyangiitis), Cogan's syndrome, RS3PE, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related diseases (e.g., primary sclerosing cholangitis, autoimmune pancreatitis), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic steatohepatitis, primary biliary cirrhosis, pulmonary Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Basedow's disease (Graves' disease, hyperthyroidism), Hashimoto's disease, autoimmune adrenocortical insufficiency, primary hypothyroidism, Addison's disease (chronic adrenocortical insufficiency), idiopathic Addison's disease, type 1 diabetes mellitus, slowly progressive type 1 diabetes mellitus (adult latent autoimmune diabetes mellitus), focal scleroderma. scleroderma), psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatitis, acquired epidermolysis bullosa, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent miscarriage, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, transplant immunosuppression, familial Mediterranean fever, eosinophilic chronic rhinosinusitis, dilated cardiomyopathy, systemic mastocytosis, or inclusion body myositis.

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

[2-4] Medications for the prevention of type 1 diabetes, the suppression of the progression or recurrence of symptoms of type 1 diabetes and/or the treatment of type 1 diabetes, comprising, as an active ingredient, a PD-1/CD3 bispecific antibody or an antibody fragment thereof according to any one of the preceding items [1] to [58], and administered together with any one or more of the following: insulin formulations (e.g., human insulin, insulin glargine, insulin lispro, insulin detemir, insulin aspart, etc.), sulfonylureas (e.g., glibenclamide, gliclazide or glimepiride), rapid-acting insulin secretagogues (e.g., nateglinide), biguanide formulations (e.g., metformin), insulin resistance modifiers Drugs (e.g., pioglitazone), alpha-glucosidase inhibitors (e.g., acarbose, voglibose, etc.), drugs for the treatment of diabetic neuropathy (e.g., epalrestat, mexiletine, imidapril, etc.), GLP-1 analogs (e.g., liraglutide, exenatide, lixisenatide, etc.) and DPP-4 inhibitors (sitagliptin, vildagliptin, alogliptin, etc.).

[2-5] An agent for the prevention of multiple sclerosis, the inhibition of the progression or relapse of symptoms of multiple sclerosis and/or the treatment of multiple sclerosis, comprising as an active ingredient a PD-1/CD3 bispecific antibody or an antibody fragment thereof according to any one of the preceding items [1] to [58], and administered together with any one or more of the following: steroid agents (e.g., cortisone acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, flucortisone acetate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisolone butyl acetate, prednisolone sodium phosphate, prednisolone acetate haloacetate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, Triamsinolone, triamsinolone acetate, triamsinolone acetonide, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone palmitate, peramisone acetate or betamethasone, etc.), interferon β-1a, interferon β-1b, glatirameracetate, mitoxantrone, azathioprine, cyclophosphamide, cyclosporine, methotrexate, cladribine, adrenocorticotropic hormone (ACTH), corticotropin, mizoribine, tacrolimus, fingolimod, and alenzusumab.

[2-6] An agent for the prevention of systemic lupus erythematosus, the suppression of the progression or relapse of systemic lupus erythematosus symptoms and/or the treatment of systemic lupus erythematosus, comprising as an active ingredient a PD-1/CD3 bispecific antibody or an antibody fragment thereof according to any one of the preceding items [1] to [58], and administered together with any one or more of the following: steroids (e.g., steroids mentioned in the preceding items [2-5]), immunosuppressants (e.g., cyclosporine, tacrolimus or fingolimod) and belimumab.

[2-7] An agent for the prevention of rheumatoid arthritis, the suppression of the progression or recurrence of rheumatoid arthritis symptoms and/or the treatment of rheumatoid arthritis, comprising as an active ingredient a PD-1/CD3 bispecific antibody or an antibody fragment thereof according to any one of the preceding items [1] to [58], and administered together with any one or more of the following: steroids (e.g., steroids mentioned in the preceding items [2-5]), antirheumatic agents (e.g., methotrexate, sulfasalazine, buccilamine, leflunomide, mizoribine, tacrolimus, etc.), anticytokine agents (e.g., infliximab, adalimumab, tocilizumab, etanercept, golimumab, sertolizumab) and abatacept.

[2-8] An agent for the prevention of autoimmune diseases, the suppression of the progression or recurrence of symptoms of autoimmune diseases and/or the treatment of autoimmune diseases, comprising as an active ingredient a PD-1/CD3 bispecific antibody or an antibody fragment thereof according to any one of the preceding items [1] to [58], and administered together with any one or more of the reagents described in the preceding items [2-4] to [2-7].

[2-9] An agent for preventing the diseases mentioned in [2-4] to [2-8] above, inhibiting the progression or recurrence of symptoms of the diseases mentioned in [2-4] to [2-8] above, and/or treating the diseases mentioned in [2-4] to [2-8] above, is administered to a patient who is given any one or more of the drugs mentioned in [2-4] to [2-7] above.

[2-10] An agent for preventing the diseases mentioned in [2-4] to [2-8] above, inhibiting the progression or recurrence of symptoms of the diseases mentioned in [2-4] to [2-8] above, and/or treating the diseases mentioned in [2-4] to [2-8] above, shall be administered after the administration of any one or more of the drugs mentioned in [2-4] to [2-7] above.

[2-11] An agent for preventing the diseases mentioned in [2-4] to [2-8] above, inhibiting the progression or recurrence of symptoms of the diseases mentioned in [2-4] to [2-8] above, and/or treating the diseases mentioned in [2-4] to [2-8] above, shall be applied before the application of any one or more of the drugs mentioned in [2-4] to [2-7] above.

[3-1] An intravenous injection preparation comprising any of the preceding items [1] to [58] a PD-1/CD3 bispecific antibody or an antibody fragment thereof and a pharmaceutically acceptable carrier.

[3-2] The intravenous injection preparation according to [3-1] above is used to prevent autoimmune diseases, suppress the progression of symptoms of autoimmune diseases, suppress the recurrence of autoimmune diseases and/or treat autoimmune diseases.

[3-3] The intravenous injection preparation according to the preceding item [3-1] or [3-2] is used for infusion.

[4-1] An isolated polynucleotide or fragment thereof encoding a heavy chain of VH having a first arm that specifically binds to PD-1, wherein the first arm that specifically binds to PD-1 constitutes any one of the PD-1/CD3 bispecific antibodies selected from the preceding items [1] to [58].

[4-2] A polynucleotide or fragment thereof isolated therefrom, which encodes a VH of a first arm that specifically binds to PD-1, wherein the first arm that specifically binds to PD-1 constitutes any one of the PD-1/CD3 bispecific antibodies selected from the preceding items [1] to [58].

[4-3] The isolated polynucleotide or fragment thereof according to the preceding item [4-1] or [4-2], wherein the VH of the first arm that specifically binds to PD-1 is encoded by a polynucleotide comprising a sequence selected from any one of SEQ ID No. 30 to 34.

[4-4] Isolated polynucleotides or fragments thereof, having a polynucleotide comprising a sequence selected from any one of SEQ ID No. 30 to 34.

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

[6-1] Animal cells, said animal cells being transfected with the expression vector of the preceding item [5-1], or said animal cells being transformed by the transfected vector.

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

[8-1] PD-1/CD3 bispecific antibodies or antibody fragments thereof selected from the preceding items [1] to [58] are used to prevent autoimmune diseases, inhibit the progression or recurrence of symptoms of autoimmune diseases and/or treat autoimmune diseases.

[9-1] Use of PD-1/CD3 bispecific antibodies or antibody fragments thereof selected from the preceding items [1] to [58] in the preparation of medicaments for the prevention of autoimmune diseases, the suppression of the progression or recurrence of symptoms of autoimmune diseases and/or the treatment of autoimmune diseases.

[10-1] An isolated anti-PD-1 monoclonal antibody or antibody fragment thereof, which cross-competes with an antibody having VH and VL that specifically binds to PD-1, wherein the VH comprises an amino acid sequence selected from any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, and the VL comprises the amino acid sequence of SEQ ID No. 25.

[10-2] An isolated anti-PD-1 monoclonal antibody or an antibody fragment thereof, wherein an antibody having VH and VL that specifically binds to PD-1 cross-competes with the isolated anti-PD-1 monoclonal antibody or an antibody fragment thereof, wherein the VH comprises an amino acid sequence selected from any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, and the VL comprises the amino acid sequence of SEQ ID No. 25.

[10-3] An isolated anti-PD-1 monoclonal antibody or antibody fragment thereof, having a VH selected from any of the following:

(A)VH, which has

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 6,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 7, and

(c) VH-CDR3 containing the amino acid sequence shown in SEQ ID No. 8,

(B)VH, which has

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 9,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 10, and

(c) VH-CDR3 containing the amino acid sequence shown in SEQ ID No. 11,

(C)VH, which has

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 12,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 13, and

(c) VH-CDR3 containing the amino acid sequence shown in SEQ ID No. 14,

(D)VH, which has

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 15,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 16, and

(c) VH-CDR3 comprising the amino acid sequence shown in SEQ ID No. 17, and (E) VH, which has

(a) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 18,

(b) VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 19, and

(c) VH-CDR3 and VL, comprising the amino acid sequence shown in SEQ ID No. 20, having

(a) VL-CDR1 containing the amino acid sequence shown in SEQ ID No. 26,

(b) VL-CDR2 containing the amino acid sequence shown in SEQ ID No. 27, and

(c) VL-CDR3 containing the amino acid sequence shown in SEQ ID No. 28.

[10-4] The isolated anti-PD-1 monoclonal antibody or antibody fragment thereof according to the preceding item [10-3], wherein one to five arbitrary amino acid residues in any one or more CDRs selected from VH-CDR1, VH-CDR2 and VH-CDR3 may be replaced by other amino acids (preferably their conserved amino acids).

[10-5] An isolated anti-PD-1 monoclonal antibody or antibody fragment thereof according to the preceding item [10-3] or [10-4], having a VH comprising an amino acid sequence selected from any one 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 comprising an amino acid sequence having at least 80%, 90%, 95%, 98% or 99% identity with its amino acid sequence, and a VL comprising the amino acid sequence shown in SEQ ID No. 25.

[10-6] An isolated anti-PD-1 monoclonal antibody or an antibody fragment thereof according to any one of the preceding items [10-1] to [10-5], wherein PD-1 is human PD-1.

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

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

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

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

Invention Effects

Because the PD-1/CD3 bispecific antibody of the present invention reduces the inducibility of cytokine production, the occurrence of infusion reactions or cytokine release syndrome after administration is suppressed. Furthermore, the PD-1/CD3 bispecific antibody allows for interaction between PD-1 and PD-L1 and is expected to enhance or maintain its effects on the prevention of autoimmune diseases, the suppression of symptom progression or relapse of autoimmune diseases, and/or the treatment of autoimmune diseases.

Attached Figure Description

Figure 1 shows the sequence of each amino acid in the VL and constant region of the common light chain.

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

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

Figure 4 shows the sequence alignment of the VH of each clone of the PD-1-specific antibody (hereinafter referred to as "anti-PD-1 antibody") with the germline genes IGHV7-4-1 and JH6c. In the figure, in the amino acid sequence of each clone, "-" indicates an amino acid that is the same as the corresponding germline gene IGHV7-4-1 or JH6c, while the portion with simplified characters for each amino acid indicates an amino acid that is different from the germline gene.

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

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

Figure 7 shows the VH amino acid sequence of the CD3-2 clone as an antibody that specifically binds to CD3 (hereinafter, it can be abbreviated as "anti-CD3 antibody").

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

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

Figure 10 shows the VH amino acid sequence of the anti-CD3 antibody clone 15C3 as described in WO2005/118635. Note that the underlined amino acid represents the 55th glycine, which was converted to alanine during the production of clone CD3-1.

Figure 11 shows the Biacore measurement results, which demonstrate the binding activity of each PD-1/CD3 bispecific monoclonal antibody clone with PD-1 and CD3.

Figure 12 shows flow cytometry, which demonstrated the simultaneous binding of each PD-1/CD3 bispecific monoclonal antibody clone to PD-1 and CD3.

Figure 13 shows flow cytometry, which demonstrates the effect of each PD-1/CD3 bispecific monoclonal antibody clone on PD-1/PD-L1 interaction.

Figure 14 shows the effect of each PD-1/CD3 bispecific monoclonal antibody clone on IFN-γ production by activated human T cells. Note that in the figure, "Ctrl" represents the control group.

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

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

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

Figure 18 shows the results of the effect of each PD-1/CD3 bispecific monoclonal antibody clone on the production of cytokines by human peripheral blood mononuclear cells.

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

Detailed Implementation

Human PD-1 (programmed cell death-1) is a membrane-bound protein consisting of the amino acid sequence represented by GenBank accession number NP_005009. In this specification, unless explicitly defined otherwise, the term "PD-1" may be used to include all its isotypes and variants, wherein the epitope of "the first arm that specifically binds to PD-1" in this invention is conserved. In this invention, PD-1 is preferably human PD-1.

CD3 is a membrane-bound protein that forms a T-cell receptor complex by associating with the T-cell receptor. In this specification, unless explicitly defined otherwise, the term "CD3" may be used to mean including subtypes (ε, δ, γ, and ζ subtypes) and their variants, wherein the epitope of the "second arm that specifically binds to CD3" of this invention is conserved. In this invention, CD3 is preferably CD3ε, and human CD3, and more preferably human CD3ε.

In this specification, the term "isolation" means a single, essentially pure component obtained by identification, separation, and/or purification from impurities containing multiple or numerous components extracted from host cells.

In this specification, the term "monoclonal antibody" means an antibody obtained from a substantially homogeneous population of antibodies that bind to the same specific antigen.

In this specification, the term "bispecific antibody" means an antibody that has binding specificity to two different antigen molecules or epitopes in molecules. In addition, the term "bispecific monoclonal antibody" means a bispecific antibody obtained from a substantially homogeneous population of antibodies.

This invention relates to bispecific antibodies capable of specifically binding to PD-1 and CD3 (hereinafter referred to as "PD-1/CD3 bispecific antibodies"). In this invention, the PD-1/CD3 bispecific antibody is preferably a PD-1/CD3 bispecific monoclonal antibody, more preferably an isolated PD-1/CD3 bispecific monoclonal antibody, and even more preferably an isolated human PD-1/human CD3 bispecific monoclonal antibody. In this document, "isolated human PD-1/human CD3 bispecific monoclonal antibody" means an isolated bispecific monoclonal antibody of human PD-1 and human CD3.

Exemplary implementations of PD-1 bispecific antibodies include, for example, diabodies, bispecific sc(Fv) ₂ , bispecific minibodies, bispecific F(ab') ₂ , bispecific hybrid antibodies, covalently coupled diabodies (bispecific DART), bispecific (FvCys) ₂ , bispecific F(ab'-zipper) ₂ , bispecific (Fv-zipper) ₂ , bispecific triple-chain antibodies, and bispecific mAb ² , etc.

Biantibodies are dimers of single-chain peptides in which VH and VL, which recognize different antigens, are linked together by peptide linkers (Proc. Natl. Acad. Sci. USA, 1993, Vol. 90, No. 14, pp. 6444-6448).

Bispecific sc(Fv) ₂ is a low-molecular-weight antibody modified in such a way that two pairs of VH/VL antibodies that recognize different antigens are linked together by peptide linkers in a continuous single-chain form (see J. Biological Chemistry, 1994, 269: pp. 199-206).

Bispecific F(ab') ₂ is a low molecular weight antibody in which the Fab' fragment of an antibody that recognizes two different antigens is covalently bonded, for example, by a disulfide bond.

Bispecific microantibodies are low-molecular-weight antibodies in which low-molecular-weight antibody fragments modified in such a way that the constant region CH3 domain of the antibody is linked to scFv that recognizes different antigens are covalently bonded, for example, by disulfide bonds on CH3 (see Biochemistry, 1992, Vol. 31, No. 6, pp. 1579-1584).

Bispecific hybrid antibodies are complete antibodies in which heavy chain/light chain complexes that recognize two different antigens are covalently bound to each other by, for example, disulfide bonds.

In this invention, examples of preferred forms of bispecific antibodies include bispecific hybrid antibodies.

Bispecific hybrid antibodies can be generated from hybridomas, for example, using a hybridoma hybridization approach (see US4474893). Alternatively, bispecific hybrid antibodies can be generated by secretion from mammalian cells that co-express four heavy and light chain cDNAs encoding antibodies that recognize different antigens.

The monoclonal antibodies used in this invention can be produced by hybridoma methods (see, for example, Kohler and Milstein et al., Natur (1975), Vol. 256, pp. 495-97; Hongo et al., Hybridoma (1995), Vol. 14, No. 3, pp. 253-260; Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press (1988), No. 2) and Hammerling et al., Monoclonal Antibodies and T-Cell Hy Bridomas, pp. 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, for example, US4816567), phage display methods (see, for example, Ladner et al., US5223409, US5403484 and US5571698, Dower et al., US5427908 and US5580717, McCafferty et al., US5969108 and US6172197, and Griffiths et al., US5885793, US6521404, US6544731, US6555313, US6582915 and US6593081) are used to generate phages.

When administered to humans, antibodies or monoclonal antibodies can be produced in the form of chimeric antibodies, humanized antibodies, or fully human antibodies to reduce or eliminate their antigenicity.

The term "chimeric antibody" refers to an antibody whose constant region sequence and variable region sequence are derived from different mammals. Examples of chimeric antibodies include antibodies whose variable region sequence is derived from mouse antibodies and whose constant region sequence is derived from human antibodies. Chimeric antibodies can be generated by linking a gene encoding the variable region of an antibody isolated from a hybridoma that produces the antibody with a gene encoding the constant region of a human-derived antibody using well-known methods (see, for example, Cabilly et al., US4816567), wherein the hybridoma is isolated using well-known techniques such as hybridoma methods, recombinant DNA methods, or phage display methods.

The term "humanized antibody" refers to an antibody prepared by, for example, grafting the complementarity-determining region (CDR) sequence of an antibody derived from a lineage of a mammal other than humans (such as a mouse) into the human framework sequence of a human antibody. Alternatively, humanized antibodies can be generated by linking a gene encoding the CDR of an antibody isolated from a hybridoma (isolated by the aforementioned method) with a gene encoding the framework region of a human-derived antibody using well-known methods (see, for example, Winter, US5225539 and US5530101; Queen et al., US5585089 and US6180370).

The term "human antibody" or "complete human antibody" refers to an antibody in which both the variable region (comprised of the frame region and the CDR region) and the constant region are derived from human germline immunoglobulin sequences. The human antibodies to be used in this invention can be generated using mice converted to produce human antibodies, such as Humab mice (see, for example, Lonberg and Kay et al. US5545806, US5569825, US5625126, US5633425, US5789650, US5877397, US5661016, US5814318, US5874299 and US5770). 429), 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 antibodies can also be prepared using SCID mice in which human immune cells have been reconstituted to produce a human antibody response after immunization (see, e.g., Wilson et al., US5476996 and US5698767). Furthermore, the human antibodies used in this invention can also be generated by the phage display method described above.

In this specification, the term "antibody fragment" for PD-1/CD3 bispecific antibodies refers to a portion of a full-length antibody and is an antibody having an antigen-binding portion for PD-1 and an antigen-binding portion for CD3. Examples include F(ab′) ₂ , etc. Herein, the antigen-binding portion refers to the smallest unit of the antibody that can bind to its antigen, and for example, it consists of three CDRs in each of VH and VL and a frame region for arranging the CDRs such that the target antigen can be recognized by a combination of those CDRs.

In this specification, the term "common light chain" means a light chain that can associate with two or more different heavy chains and can exhibit binding ability with 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 common light chains include light chains encoded by the germline gene of the human κ light chain IgVκ1-39*01/IGJκ1*01 (the nomenclature of the IMGT database) (hereinafter abbreviated as "IGVK1-39/JK1 common light chain"). More preferred examples include light chains containing a VL having a CDR1 containing the amino acid sequence shown in SEQ ID No. 26, a CDR2 containing the amino acid sequence shown in SEQ ID No. 27, and a CDR3 containing the amino acid sequence shown in SEQ ID No. 28. Further preferred examples include light chains containing a VL containing the amino acid sequence shown in SEQ ID No. 25. Additionally, preferred examples of constant regions of the common light chain include light chain constant regions containing the amino acid sequence shown in SEQ ID No. 29. The VL of the common light chain and each amino acid sequence of the constant region used in this invention are shown in Figure 1, and each CDR amino acid sequence of the variable region is shown in Figure 2.

In this specification, the term "isotype" refers to an antibody class (e.g., IgM or IgG) encoded by a gene in the heavy chain constant region. Preferred examples of isotypes of the PD-1/CD3 bispecific antibodies of the present invention include IgG, and more preferably IgG ₁ or IgG _4. IgG ₁ as used herein is preferably characterized by the elimination or reduction of its binding to the Fc receptor. Specifically, IgG ₁ antibodies with eliminated or reduced binding to the Fc receptor can be obtained by substituting, deleting, or inserting any amino acid in its heavy chain constant region. Examples include antibodies in which leucine at position 235 according to the EU numbering system is replaced by glycine, and/or glycine at position 236 according to the EU numbering system is replaced by arginine, in each of the two heavy chain constant regions or their hinge regions. Furthermore, to reduce antibody heterogeneity, antibodies in which an amino acid at the C-terminus has been deleted, such as lysine at position 447 according to the EU numbering system, are preferred. Additionally, when the bispecific antibody is IgG ₄ , variants in which any amino acid in its heavy chain constant region is substituted, deleted, or inserted are preferred to inhibit exchange in the antibody molecule. For example, antibodies in which the serine residue at position 228 according to the EU numbering system is replaced by proline are preferred. Note here that in this specification, the amino acid positions assigned to the CDR and framework in the variable region of the antibody are specified according to the Kabat numbering system (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)). Furthermore, the amino acids in the constant region are represented according to the EU numbering system based on Kabat amino acid positions (see Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242).

In the Fc region of the PD-1/CD3 bispecific antibody of the present invention, any amino acid therein can be substituted, making it easy for the two different heavy chains to associate with each other. An example of a preferred embodiment includes a PD-1/CD3 bispecific antibody in which, in the constant region of the heavy chain having a VH arm that specifically binds to PD-1, leucine at position 351 according to the EU numbering system is replaced by lysine and threonine at position 366 according to the EU numbering system is replaced by lysine; and in the constant region of the heavy chain having a VH arm that specifically binds to CD3, leucine at position 351 according to the EU numbering system is replaced by aspartic acid and leucine at position 368 according to the EU numbering system is replaced by glutamic acid. Examples also include PD-1/CD3 bispecific antibodies, wherein in the constant region of the heavy chain of the VH having a first arm that specifically binds to PD-1, leucine at position 351 according to the EU numbering system is replaced by aspartic acid and leucine at position 368 is replaced by glutamic acid; and in the constant region of the heavy chain of the VH having a second arm that specifically binds to CD3, leucine at position 351 according to the EU numbering system is replaced by lysine and threonine at position 366 according to the EU numbering system is replaced by lysine.

The first arm that specifically binds to PD-1

In this specification, "the first arm that specifically binds to PD-1" (hereinafter, it may be abbreviated as "first arm") means a portion of an antibody (hereinafter, it may be abbreviated as "anti-PD-1 antibody") containing at least a VH of an antibody that specifically binds to PD-1, regardless of whether it is contained in an antibody or a fragment thereof, or exists as a monomolecule rather than as a portion. For example, such a first arm consists of a VH of an anti-PD-1 antibody and a VL of a common light chain constituting the same anti-PD-1 antibody. Furthermore, examples of first arms also include the Fab portion of an antibody containing both VH and VL. In this document, the term "specifically binds to PD-1" is used to describe ^an antibody that can bind directly to PD-1 with a binding activity of at least 1 × ^10⁻⁵ M, preferably 1 × ^10⁻⁷ M, and more preferably exceeding 1 × ^10⁻⁹ M affinity (dissociation constant (Kd value)), and substantially does not bind to other receptor members belonging to the so-called CD28 family of receptors (e.g., at least CD28, CTLA-4, and ICOS). Furthermore, the term "antibody" in "antibody that specifically binds to PD-1" or "anti-PD-1 antibody" refers to a full-length antibody consisting of two heavy chains and two light chains linked by disulfide bonds, preferably a monoclonal antibody.

Examples of "the first arm that specifically binds to PD-1" in this article include:

(a) VH-CDR1, which contains an amino acid sequence represented by HYJ ¹ LH [where J ¹ represents G (glycine) or A (alanine), and in this document, the letters represented by J ¹ or others represent one-letter amino acid abbreviations respectively];

(b) VH-CDR2, comprising the amino acid sequence represented by WJ ² NTNTU ² NPTX ² AQGFTG [where J ² represents L (leucine) or I (isoleucine), U ² represents E (glutamic acid) or G (glycine), X ² represents F (phenylalanine) or Y (tyrosine), and in this document, letters represented by J ² , U ² , or X ² or others are represented as above]; and

(c) VH-CDR3, which contains the amino acid sequence represented by GDJ ³ VVPTTIWNYYU ³ X ³ MZ ³ V [where J ³ represents M (methionine) or L (leucine), U ³ represents H (histidine) or Y (tyrosine), X ³ represents F (phenylalanine) or Y (tyrosine), Z ³ represents D (aspartic acid) or E (glutamic acid), and in this document, the letters represented by J ³ , U ³ , X ³ or Z ³ or other letters are represented as above].

In this document, preferred embodiments of the "first arm that specifically binds to PD-1" include those comprising:

(1a) VH having each VH-CDR, wherein ^J1 in the HYJ ¹ LH sequence as VH-CDR1 represents G (glycine); in the WJ ² NTNTU ² NPTX ² AQGFTG sequence as VH-CDR2, ^J2 represents L (leucine), ^U2 represents E (glutamic acid) and ^X2 represents F (phenylalanine), respectively; in the GDJ ³ VVPTTIWNYYU ³ X ³ MZ ³ V sequence as VH-CDR3, ^J3 represents M (methionine), ^U3 represents H (histidine), ^X3 represents F (phenylalanine) and ^Z3 represents D (aspartic acid), respectively.

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

(3a) VH having each VH-CDR, wherein ^J1 in the HYJ ¹ LH sequence as VH-CDR1 represents A (alanine); in the WJ ² NTNTU ² NPTX ² AQGFTG sequence as VH-CDR2, respectively, ^J2 represents L (leucine), ^U2 represents E (glutamic acid) and ^X2 represents Y (tyrosine); in the GDJ ³ VVPTTIWNYYU ³ X ³ MZ ³ V sequence as VH-CDR3, respectively, ^J3 represents M (methionine), ^U3 represents Y (tyrosine), ^X3 represents Y (tyrosine) and ^Z3 represents D (aspartic acid); and

(4a) VH having each VH-CDR, wherein ^J1 in the HYJ ¹ LH sequence as VH-CDR1 represents A (alanine); in the WJ ² NTNTU ² NPTX ² AQGFTG sequence as VH-CDR2, ^J2 represents L (leucine), ^U2 represents E (glutamic acid) and ^X2 represents F (phenylalanine), respectively; in the GDJ ³ VVPTTIWNYYU ³ X ³ MZ ³ V sequence as VH-CDR3, ^J3 represents M (methionine), ^U3 represents H (histidine), ^X3 represents F (phenylalanine) and ^Z3 represents D (aspartic acid), respectively.

Furthermore, as a preferred example of another implementation, the "first arm that specifically binds to PD-1" includes any of the following VHs:

(1b) VH, which has VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 6, VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 7 and VH-CDR3 containing the amino acid sequence shown in SEQ ID No. 8;

(2b) VH, which has VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 9, VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 10 and VH-CDR3 containing the amino acid sequence shown in SEQ ID No. 11;

(3b) VH, which has VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 12, VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 13 and VH-CDR3 containing the amino acid sequence shown in SEQ ID No. 14;

(4b) VH, having VH-CDR1 comprising the amino acid sequence shown in SEQ ID No. 15, VH-CDR2 comprising the amino acid sequence shown in SEQ ID No. 16, and VH-CDR3 comprising the amino acid sequence shown in SEQ ID No. 17; and

(5b) VH, which has VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 18, VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 19 and VH-CDR3 containing the amino acid sequence shown in SEQ ID No. 20.

Furthermore, the "PD-1-specific binding first arm" of the present invention includes those in which, in each VH-CDR of any VH selected from (1a) to (4a) or (1b) to (5b) above, one to five arbitrary amino acid residues are substituted with other amino acids (preferably their conserved amino acids), and which have substantially the same PD-1 binding activity as the original first arm without any amino acid substitutions. For example, for VH-CDR1, one amino acid residue is substituted with its conserved amino acid. For VH-CDR2 or VH-CDR3, one or two amino acid residues are substituted with their conserved amino acids, respectively. Herein, "conserved amino acid substitution" means interchangeability with residues having similar side chains. For example, a group of amino acids with aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic hydroxyl side chains includes serine and threonine; a group of amino acids with amide-containing side chains includes asparagine and glutamine; a group of amino acids with aromatic side chains includes phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains includes lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains includes cysteine and methionine. Preferred examples of substitution with conserved amino acids include substitutions between valine, leucine, and isoleucine; substitutions between phenylalanine and tyrosine; substitutions between lysine and arginine; substitutions between alanine and valine; and substitutions between asparagine and glutamine. Furthermore, in this document, the phrase “having substantially the same binding activity with PD-1 as the original first arm without any amino acid substitutions” means that the binding activity with PD-1 of the first arm with the same amino acid substitutions is 95% or higher, preferably 98% or higher, and more preferably 99% or higher, compared to the binding activity with PD-1 of the original first arm without any same amino acid substitutions.

Furthermore, the “first arm that specifically binds to PD-1” in this invention includes those containing VHs having each VH-CDR, having the specific amino acid sequence described above, and wherein the framework of the VH is encoded by a specific germline gene or a gene of which has a somatic mutation. For example, the VH shown in any of (1a) to (4a) or (1b) to (5b) above can be encoded by a VDJ recombinant gene wherein the germline V gene is IGHV7-4-1 and the germline J gene is JH6c or a gene of which has a somatic mutation. In this document, the amino acid sequence encoded by the germline V gene IGHV7-4-1 corresponds to the amino acid sequence shown in SEQ ID No. 21 (Figure 3).

The framework of the VH in the first arm of the present invention, which specifically binds to PD-1, can be encoded by a germline VDJ recombinant gene with somatic mutations. For example, since FR1, FR2, and FR3 of the VH represented by any of (1a) to (4a) or (1b) to (5b) above, wherein the germline V gene is IGHV7-4-1, differs from the amino acid sequence encoded by the IGHV7-4-1 gene at the positions shown in FIG4, they have undergone somatic mutations at each position. For example, regarding the FR1 region, in the amino acid sequence shown in SEQ ID No. 21, respectively, 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, or they can be replaced by any combination thereof. Regarding the FR2 region, valine at position 37 in the amino acid sequence shown in SEQ ID No. 21 is replaced by leucine. Regarding the FR3 region, in the amino acid sequence shown in SEQ ID No. 21, respectively, serine at position 77 can be replaced by threonine, or cysteine at position 84 can be replaced by serine or asparagine, or they can be replaced by any combination thereof. Furthermore, regarding the FR4 region of VH selected from any of (1a) to (4a) or (1b) to (5b) above, in the amino acid sequence of the FR4 region derived from the J gene JH6c (Trp-Gly-Lys-Gly-Thr-Thr*-Val-Thr-Val-Ser-Ser) (SEQ ID No. 41), lysine (Lys) can be replaced by glutamine or asparagine, and/or the asterisked threonine (Thr) can be replaced by leucine. Each of FR1, FR2, FR3, and FR4, including any combination of the above amino acid substitutions, has no substantial effect on the function of the first arm that specifically binds to PD-1 and can be used as a framework.

Furthermore, the "first arm that specifically binds to PD-1" of the present invention includes those having a CDR containing the above-described amino acid sequence, wherein the FR amino acid sequence in the VH is encoded by a specific germline gene or a gene having a somatic mutation therein. Examples of such first arms include those having a VH containing an amino acid sequence selected from any one of SEQ ID No. 1 to 5.

Furthermore, examples of such "first arms that specifically bind to PD-1" include those having a VH containing an amino acid sequence having at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, further preferably at least 98% identity, and even more preferably at least 99% identity, with respect to an amino acid sequence selected from any of SEQ ID Nos. 1 to 5, and wherein the difference in amino acid sequence from the VH in the original first arm has no substantial effect on the binding activity with PD-1 (hereinafter, may be abbreviated as "homologous first arm"). In this document, the term "% identity" used to compare the identity of amino acid sequences is defined as the percentage of amino acid sequences identical to a reference amino acid sequence (in this document, a nicked reference amino acid sequence has been introduced where necessary to maximize sequence identity) when two sequences are aligned. Furthermore, in this document, the sentence “the difference in the amino acid sequence of VH in the original first arm has no substantial effect on the binding activity with PD-1” means that the binding activity of the homologous first arm with PD-1 is 95% or higher, preferably 98% or higher, and more preferably 99% or higher, compared with the binding activity of the original first arm.

In yet another embodiment, the “first arm that specifically binds to PD-1” of the present invention further includes those having a variable region (in this document, the variable region comprises the VH and VL constituting thereof) having an anti-PD-1 antibody, which (1) cross-competes with a first arm having a VH represented by any one of (1a) to (4a) or (1b) to (5b) above, or a VH comprising an amino acid sequence represented by any one of SEQ ID No. 1 to 5 and a VL of a common light chain, or (2) has the same VH and VL as the first arm that specifically binds to PD-1. The variable regions of combined monoclonal antibodies cross-compete for binding to PD-1, and further include those having variable regions of anti-PD-1 antibodies that cross-compete for binding to PD-1 with: (3) a first arm having a VH represented by any one of (1a) to (4a) or (1b) to (5b) above, or a VH containing an amino acid sequence shown in any one of SEQ ID No. 1 to 5 and a common light chain, or (4) a variable region of a monoclonal antibody that specifically binds to PD-1 having the same VH and VL. In this document, the phrase “cross-competing for binding to PD-1” means that it inhibits the binding of the first arm to PD-1, to any extent, by binding an epitope that is the same as or partially overlaps with the epitope of the first arm exemplified in this specification, or means that it inhibits the binding of an antibody to PD-1 bound to an epitope by the first arm exemplified in this specification, to any extent, where the epitope is the same as or partially overlaps with the epitope of the first arm exemplified in this specification. Cross-competition can be assessed by a competitive binding assay. For example, it can be measured using Biacore analysis, ELISA assay, flow cytometry, enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer assay (FRET), and fluorescence microvolume assay (FMAT (registered trademark)).

Examples of a first arm that cross-competes with the first arm of a VL having the VH and common light chain shown in (5b) above include a VL having the VH and common light chain shown in any of (1b) to (4b) above (preferably, a VL having the amino acid sequence shown in SEQ ID No. 26, a VL having the amino acid sequence shown in SEQ ID No. 27, and a VL having the amino acid sequence shown in SEQ ID No. 28), and further having a VL having the amino acid sequence shown in any of SEQ ID No. 1 to 4 and the common light chain (preferably, a VL having the amino acid sequence shown in SEQ ID No. 25).

Furthermore, examples of a first arm that cross-competes with a first arm having a VH selected from any of (1b) to (4b) above or a VL containing an amino acid sequence selected from any of SEQ ID No. 1 to 4 and a common light chain include a VL having a VH and a common light chain as shown in (5b) above (preferably, having a VL-CDR1 containing an amino acid sequence shown in SEQ ID No. 26, a VL-CDR2 containing an amino acid sequence shown in SEQ ID No. 27, and a VL-CDR3 containing an amino acid sequence shown in SEQ ID No. 28), and further having a first arm containing a VH and a common light chain containing an amino acid sequence selected from SEQ ID No. 5 (preferably, having a VL containing an amino acid sequence shown in SEQ ID No. 25).

In this document, preferred examples of the “first arm that specifically binds to PD-1” of the present invention include a first arm having a VH selected from any of (1b) to (5b) above. Furthermore, as described above, preferred first arms also include those in which 1 to 5 arbitrary amino acid residues in each CDR of the VH are substituted with other amino acids (preferably conserved amino acids), and the substitution of amino acids substantially does not affect the binding activity with PD-1. Furthermore, as described above, preferred first arms include those having a VH whose frame amino acid sequence is encoded by germline V gene IGHV7-4-1 or J gene JH6c or a gene of the same type having a somatic mutation. Then, more preferred examples of the first arm include those having a VH comprising an amino acid sequence selected from any of SEQ ID No. 1 to 5.

Furthermore, the first arm of the present invention that specifically binds to PD-1 preferably comprises a common light chain, and preferred examples of such a common light chain include a light chain encoded by the IGVK1-39/JK1 common light chain. More preferred examples include a light chain containing a VL having a CDR1 comprising the amino acid sequence shown in SEQ ID No. 26, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 27, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 28, and further preferred examples include a light chain containing a VL comprising the amino acid sequence shown in SEQ ID No. 25. Additionally, preferred examples of the constant region of the common light chain include a light chain constant region comprising the amino acid sequence shown in SEQ ID No. 29.

Furthermore, preferred examples of "the first arm that specifically binds to PD-1" include allowing interactions between PD-1 and PD-L1, interactions between PD-1 and PD-L2, or both. In this document, the phrase "allowing interactions between PD-1 and PD-L1, interactions between PD-1 and PD-L2, or both" means that even when a 20-fold excess of the present invention's PD-1/CD3 bispecific antibody in soluble form of PD-L1 or PD-L2 is present, compared to the absence of the present invention's PD-1/CD3 bispecific antibody, the interactions between PD-L1 and PD-L1, interactions between PD-L2 and PD-1, or both, are maintained at 50% or more, preferably 70% or more, more preferably 80% or more. Furthermore, the definition of "allowing interactions between PD-1 and PD-L1, interactions between PD-1 and PD-L2, or both" can be synonymous with "substantially not inhibiting interactions between PD-1 and PD-L1, interactions between PD-1 and PD-L2, or both."

Figure 5 shows the correspondence between each clone of the anti-PD-1 monoclonal antibody obtained for constructing the PD-1/CD3 bispecific antibody of the present invention and its VH amino acid sequence and its SEQ ID number. Figure 6 shows the correspondence between each CDR amino acid sequence in the VH of each anti-PD-1 monoclonal antibody clone and its SEQ ID number.

The second arm that specifically binds to CD3

In this specification, "a second arm that specifically binds to CD3" (hereinafter, which may be abbreviated as "second arm") means an antibody portion having at least the VH of an antibody that specifically binds to CD3 (hereinafter, which may be abbreviated as "anti-CD3 antibody"), regardless of whether it is contained in or as part of an antibody or antibody fragment, or exists as a monomolecule rather than as part of an antibody. For example, such a second arm consists of the VH of an anti-CD3 antibody and the VL of a common light chain constituting the same anti-CD3 antibody, and further includes the Fab portion of the antibody containing both the VH and the VL. In this document, the phrase "specifically binds to CD3" is used to describe an antibody that can bind directly to CD3 with a binding activity of at least 1 × ^10⁻⁵ M, preferably 1 × ^10⁻⁷ M, and more preferably exceeding 1 × ^10⁻⁹ M affinity (dissociation constant (Kd value)), and substantially does not bind to other proteins. Furthermore, the term "antibody" in "antibody that specifically binds to CD3" or "anti-CD3 antibody" refers to a full-length antibody consisting of two heavy chains and two light chains linked by disulfide bonds, with monoclonal antibodies being preferred.

In this document, examples of “second arm that specifically binds to CD3” include those containing VH, which have: (1c) VH-CDR1 containing the amino acid sequence shown in SEQ ID No. 37, VH-CDR2 containing the amino acid sequence shown in SEQ ID No. 38, and VH-CDR3 containing the amino acid sequence shown in SEQ ID No. 39.

Furthermore, the "second arm that specifically binds to CD3" of the present invention includes those in which, in each VH-CDR of the VH in (1c) above, 1 to 5 arbitrary amino acid residues are substituted with other amino acids (preferably their conserved amino acids), and that it has substantially the same CD3-binding activity as the original second arm without any identical amino acid substitutions. For example, for CDR1, one amino acid residue is substituted with its conserved amino acid. For CDR2 or CDR3, one or two amino acid residues are substituted with their conserved amino acids, respectively. In this document, the phrase "has substantially the same CD3-binding activity as the original second arm without any identical amino acid substitutions" means that the CD3-binding activity of the amino acid-substituted second arm is 95% or higher, preferably 98% or higher, and more preferably 99% or higher, compared to the CD3-binding activity of the original second arm without any identical amino acid substitutions. Note here that examples of "substituted with conserved amino acids" in each VH-CDR of the second arm include those amino acid substitutions in the first arm described above.

Furthermore, the "second arm that specifically binds to CD3" in this invention includes those containing a VH having each CDR comprising the specific amino acid sequence described above, wherein the FR amino acid sequence of the VH is encoded by a specific germline gene or a gene thereof having a somatic mutation. For example, the VH in (1c) above is encoded by a VDJ recombinant gene wherein the germline V gene is IGHV3-33 or a gene thereof having a somatic mutation. In this document, the amino acid sequence (SEQ ID No. 22) encoded by the germline V gene IGHV3-33 is shown in Figure 3.

Furthermore, the "second arm that specifically binds to CD3" of the present invention also includes those comprising each CDR having the specific amino acid sequence described above, wherein the amino acid sequence of the FR in the VH is encoded by a specific germline gene or a gene having a somatic mutation therein. Examples of such second arms include those having a VH comprising the amino acid sequence shown in SEQ ID No. 36.

Furthermore, examples of such "CD3-specific binding second arms" include those having 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 still even more preferably at least 99% identity with the amino acid sequence shown in SEQ ID No. 36, and wherein the difference in the VH amino acid sequence from the original second arm has no substantial effect on the binding activity with CD3 (hereinafter, may be abbreviated as "homologous second arm"). In this document, the phrase "the difference in the VH amino acid sequence from the original second arm has no substantial effect on the binding activity with CD3" means that the binding activity of the homologous second arm with CD3 is 95% or higher, preferably 98% or higher, and more preferably 99% or higher, compared to the binding activity of the original second arm.

In yet another embodiment, the “second arm that specifically binds to CD3” of the present invention comprises those containing a variable region (hereinafter, the variable region includes the VH and VL constituting thereof) of an anti-CD3 antibody (1) that cross-competes with a second arm having a VH represented by (1c) above or a VH containing the amino acid sequence shown in SEQ ID No. 36 and a VL of a common light chain, or (2) that cross-competes with a variable region of a monoclonal antibody that specifically binds to CD3 having the same VH and VL. Hereinafter, “cross-competing for binding to CD3” means that it inhibits the binding of the second arm to CD3 by binding the same or partially overlapping epitopes as the second arm exemplified in this specification, however to what extent. Hereinafter, cross-competition can be evaluated in a similar manner to that described with respect to the “first arm that specifically binds to PD-1”.

In this document, preferred examples of the "CD3-specific binding second arm" of the present invention include a second arm having the VH shown in (1c) above. Furthermore, as described above, preferred examples of the second arm also include those in which 1 to 5 arbitrary amino acid residues in each CDR of the VH are substituted with other amino acids (preferably conserved amino acids), and the substitution of amino acids substantially does not affect the binding activity with CD3. Furthermore, as described above, preferred examples of the second arm include those having a VH in which the amino acid sequence of the frame is encoded by the germline V gene IGHV3-33 or a gene having a somatic mutation thereof. Then, more preferred examples of the second arm include those having a VH containing the amino acid sequence shown in SEQ ID No. 36.

The correspondence between the VH amino acid sequence of the anti-CD3 antibody clone used to construct the PD-1/CD3 bispecific antibody of the present invention and its SEQ ID number is shown in Figure 7. The correspondence between the CDR amino acid sequence in the VH of the anti-CD3 antibody clone and its SEQ ID number is shown in Figure 8.

The "second arm that specifically binds to CD3" of the present invention preferably contains a common light chain, and preferred examples of such a common light chain include the IGVK1-39/JK1 common light chain. More preferred examples include a light chain containing a VL having a CDR1 containing the amino acid sequence shown in SEQ ID No. 26, a CDR2 containing the amino acid sequence shown in SEQ ID No. 27, and a CDR3 containing the amino acid sequence shown in SEQ ID No. 28. Further preferred examples include a light chain containing a VL containing the amino acid sequence shown in SEQ ID No. 25. Furthermore, preferred examples of the constant region of the common light chain include a light chain constant region containing the amino acid sequence shown in SEQ ID No. 29.

A preferred example of the "second arm that specifically binds to CD3" of the present invention includes a second arm that specifically binds to CD3ε.

Examples of preferred isotypes of the PD-1/CD3 bispecific antibody of the present invention include IgG antibodies, more preferably IgG ₁ antibodies or IgG ₄ antibodies, and even more preferably IgG ₁ antibodies.

In another aspect, a preferred embodiment of the PD-1/CD3 bispecific antibody of the present invention is, for example, a PD-1/CD3 bispecific antibody.

The first arm, which specifically binds to PD-1, contains:

(A) VH, wherein in any one or more CDRs of VH-CDR1, VH-CDR2, and VH-CDR3 selected from any of the VHs represented by (1a) to (4a) or (1b) to (5b) above, 1 to 5 arbitrary amino acid residues may be substituted with other amino acids (preferably their conserved amino acids); and

(B) A VL with a common light chain, having a CDR1 comprising the amino acid sequence shown in SEQ ID No. 26, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 27, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 28; and

The second arm, which specifically binds to CD3, contains:

(C)VH, wherein in any one or more CDRs selected from VH-CDR1, VH-CDR2, and VH-CDR3 of the VH represented in (1c) above, 1 to 5 arbitrary amino acid residues may be substituted by other amino acids (preferably their conserved amino acids); and

(D) A VL with a common light chain having a CDR1 containing the amino acid sequence shown in SEQ ID No. 26, a CDR2 containing the amino acid sequence shown in SEQ ID No. 27, and a CDR3 containing the amino acid sequence shown in SEQ ID No. 28.

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

The first arm, which specifically binds to PD-1, contains:

(A)VH, which is represented by any one of (1a) to (4a) or (1b) to (5b) above; and

(B) A VL with a common light chain, having a CDR1 comprising the amino acid sequence shown in SEQ ID No. 26, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 27, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 28; and

The second arm, which specifically binds to CD3, contains:

(C)VH, which is represented by (1c) above; and

(D) A VL with a common light chain having a CDR1 containing the amino acid sequence shown in SEQ ID No. 26, a CDR2 containing the amino acid sequence shown in SEQ ID No. 27, and a CDR3 containing the amino acid sequence shown in SEQ ID No. 28.

Furthermore, another preferred embodiment of the PD-1/CD3 bispecific antibody of the present invention is, for example, a PD-1/CD3 bispecific antibody.

The first arm, which specifically binds to PD-1, contains:

(A) VH, comprising an amino acid sequence selected from any one of SEQ ID No. 1 to 5, or VH comprising an amino acid sequence having at least 80% identity with an amino acid sequence of the same VH; and

(B) A VL of a common light chain comprising the amino acid sequence shown in SEQ ID No. 25; and wherein the second arm that specifically binds to CD3 contains:

(C) VH, which contains the amino acid sequence shown in SEQ ID No. 36, or VH, which contains an amino acid sequence having at least 80% identity with the amino acid sequence of the same VH; and

(D) A VL of a common light chain containing the amino acid sequence shown in SEQ ID No. 25.

Other preferred embodiments include, for example, PD-1/CD3 bispecific antibodies.

The first arm, which specifically binds to PD-1, contains:

(A)VH, which contains an amino acid sequence selected from any one of SEQ ID No. 1 to 5;

and

(B) A VL of a common light chain comprising the amino acid sequence shown in SEQ ID No. 25; and wherein the second arm that specifically binds to CD3 contains:

(C)VH, which contains the amino acid sequence shown in SEQ ID No. 36; and

(D) A VL of a common light chain containing the amino acid sequence shown in SEQ ID No. 25.

In the PD-1/CD3 bispecific antibody of the present invention, when the antibody is an _IgG1 antibody, the following _IgG1 antibody is preferred: wherein leucine at position 235 according to the EU numbering system is replaced by glycine, and/or glycine at position 236 according to the EU numbering system is replaced by arginine in both heavy chain constant regions or hinge regions. Furthermore, bispecific antibodies in which the C-terminal amino acid of the heavy chain is missing, such as lysine at position 447 according to the EU numbering system, are preferred. Furthermore, when the PD-1/CD3 bispecific antibody is an _IgG4 antibody, antibodies in which serine located in the hinge region and at position 228 according to the EU numbering system is replaced by proline are preferred.

Furthermore, when the PD-1/CD3 bispecific antibody is an IgG ₁ antibody, preferred embodiments include those where, in the constant region of the heavy chain of the VH having a first arm specifically binding to PD-1, leucine at position 351 is replaced by lysine and threonine at position 366 is replaced by lysine according to the EU numbering system; and in the constant region of the heavy chain of the VH having a second arm specifically binding to CD3, leucine at position 351 is replaced by aspartic acid and leucine at position 368 is replaced by glutamate. Additionally, the following IgG ₁ antibody is also preferred: where, in the constant region of the heavy chain of the VH having a first arm specifically binding to PD-1, leucine at position 351 is replaced by aspartic acid and leucine at position 368 is replaced by glutamate according to the EU numbering system; and in the constant region of the heavy chain of the VH having a second arm specifically binding to CD3, leucine at position 351 is replaced by lysine and threonine at position 366 is replaced by lysine.

Examples of preferred embodiments of a PD-1/CD3 bispecific _IgG1 antibody in which all of the aforementioned amino acid substitutions in the heavy chain constant region are employed include those in which the heavy chain having a VH of a first arm specifically binding to PD-1 contains a heavy chain constant region comprising the amino acid sequence shown in SEQ ID No. 23, and the heavy chain having a VH of a second arm specifically binding to CD3 contains a heavy chain constant region comprising the amino acid sequence shown in SEQ ID No. 24. Those amino acid sequences are shown in Figure 9.

The most preferred embodiment of the PD-1/CD3 bispecific antibody of the present invention is the PD1-1(Bi) clone, PD1-2(Bi) clone, PD1-3(Bi) clone, PD1-4(Bi) clone and PD1-5(Bi) clone generated by the method described in Example 8 of this specification.

Preferred features of the PD-1/CD3 bispecific antibody of the present invention include: (1) allowing interaction between PD-1 and PD-L1, interaction between PD-1 and PD-L2, or both; and/or (2) sufficiently reducing cytokine production. In this document, the phrase “allowing interaction between PD-1 and PD-L1, interaction between PD-1 and PD-L2, or both” has the same meaning as described regarding “the first arm that specifically binds to PD-1.” On the other hand, the phrase “sufficiently reducing cytokine production” means, for example, that during intravenous administration or within 24 hours after such administration, the concentration of cytokines containing IL-2, IFN-γ, and/or TNF-α in blood or tissue does not increase, or only increases to the extent that it can be inhibited by steroid administration.

Methods for generating and purifying PD-1/CD3 bispecific antibodies

The PD-1/CD3 bispecific antibody and its antibody fragment of the present invention can also be manufactured by the methods disclosed in WO2014/051433, WO2013/157953 or WO2013/157954.

Specifically, the PD-1/CD3 bispecific antibody and its antibody fragment of the present invention can be prepared by transferring expression vector genes that have been inserted into mammalian animal cells to transform them, and then by allowing them to express and secrete both the heavy chain and the common light chain: (1) a polynucleotide encoding a heavy chain containing a VH that specifically binds to PD, (2) a polynucleotide encoding a heavy chain containing a VH that specifically binds to CD3, and (3) a polynucleotide encoding a common light chain.

In this document, any host cells expressing the PD-1/CD3 bispecific antibody of the present invention can be used, provided they can be expressed using an expression vector that allows gene transfer. Host cells are preferably insect cells, such as SF-9 and SF-21, more preferably mammalian cells, such as mouse 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, hybridomas, tumor cells, immortalized primary cells, W138, HepG2, HeLa, HEK293, HT1080, or embryonic retinal cells such as PER.C6. It is noted here that, in selecting the expression system, the expression vector used for the mammalian host cell and the host used as the mammalian host cell can be used to ensure that the antibody is properly glycosylated. Advantageously, human cell lines (preferably PER.C6) are used to obtain antibodies whose glycosylation pattern corresponds to the human glycosylation pattern.

For example, references can be made to Current Protocols in Protein Science (1995), Coligan JE, Dunn BM, Ploegh HL, Speicher DW, Wingfield PT, ISBN 0-471-11184-8, Bendig, 1988, regarding protein production in host cells transformed via gene transfer using expression vectors. Additionally, references can be made to Mammalian Cell Biotechnology: A Practical Approach (edited by M. Butler, IRL Press, 1991) for general guidelines, procedures, and practical methods to maximize the productivity of host cell culture. Antibody expression in host cells is described in publications such as EP0120694, EP0314161, EP0481790, EP0523949, US4816567, WO2000/63403, etc.

In this paper, the culture conditions of host cells can be optimized using well-known methods, thereby optimizing protein production. Examples of cultures include batch culture, feed culture, continuous culture, and hollow fiber culture in culture dishes, roller flasks, or reaction chambers. For large-scale and continuous production of recombinant proteins through cell culture, cell proliferation in suspension is preferred. Furthermore, cell culture under conditions free of animal or human serum or containing certain components of animal or human serum is more preferred.

Antibodies expressed in host cells and then recovered from cells or cell cultures using well-known methods can be purified. Examples of purification methods include immunoprecipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and/or anion exchange chromatography, hydrophobic interaction chromatography, etc. Furthermore, protein A or protein G affinity chromatography is preferably used (see, for example, US4801687 and US5151504).

Anti-PD-1 monoclonal antibody

This invention includes a "monoclonal antibody that specifically binds to PD-1" (hereinafter, it may be abbreviated as "anti-PD-1 monoclonal antibody") to construct the PD-1/CD3 bispecific antibody and its antibody fragment of the present invention.

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 associating the VH and VL of the common light chain of the present invention. In this document, the phrase "specifically binds to PD-1" is used to describe an antibody that can directly bind to PD-1 with a binding activity (dissociation constant (Kd value)) of at least 1 × ^10⁻⁵ M, preferably 1 × ^10⁻⁷ M, and more preferably exceeding 1 × ^10⁻⁹ M, and substantially does not bind to receptor members belonging to the so-called CD28 family of receptors (e.g., at least CD28, CTLA-4, and ICOS). In this document, "antibody" in "monoclonal antibody specifically binding 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. Furthermore, "fragment of a monoclonal antibody specifically binding to PD-1" is a part of a full-length antibody and is an antibody that at least includes an antigen-binding portion, examples of which include Fab, Fab′, Fv, scFv, F(ab′) _₂ , etc.

Examples of anti-PD-1 monoclonal antibodies of the present invention include those having a VH constituting a "first arm that specifically binds to PD-1" selected from any of (1a) to (4a) or (1b) to (5b) above, or a VH containing an amino acid sequence selected from any of SEQ ID No. 1 to 5 and a common light chain as described in this specification.

Furthermore, the anti-PD-1 monoclonal antibodies of the present invention include those in which, in each CDR of any VH selected from (1a) to (4a) or (1b) to (5b) above, 1 to 5 arbitrary amino acid residues are substituted with other amino acids (preferably their conserved amino acids), and they have substantially the same PD-1 binding activity as the original anti-PD-1 monoclonal antibody having no identical amino acid substitutions. For example, for CDR1, one amino acid residue is substituted with its conserved amino acid. For CDR2 or CDR3, one or two amino acid residues are substituted with their conserved amino acids, respectively. In this document, the phrase "has substantially the same PD-1 binding activity as the original anti-PD-1 monoclonal antibody having no identical amino acid substitutions" means that the binding activity of the amino acid-substituted anti-PD-1 monoclonal antibody with PD-1 is 95% or higher, preferably 98% or higher, and more preferably 99% or higher, compared to the binding activity of the original anti-PD-1 monoclonal antibody having no identical amino acid substitutions.

Furthermore, examples of the anti-PD-1 monoclonal antibody of the present invention include those that contain a VH having each CDR comprising the specific amino acid sequence described above, wherein the framework amino acid sequence of the VH is encoded by a specific germline gene or a gene thereof having a somatic mutation, and examples of such antibodies include a specific VH encoded by a specific germline gene or a gene thereof having a somatic mutation, as described above regarding the "first arm that specifically binds to PD-1".

Furthermore, examples of the anti-PD-1 monoclonal antibodies of the present invention that have a VH containing each CDR selected from any of the VHs (1a) to (4a) or (1b) to (5b) described above, and wherein the FR amino acid sequence is encoded by a specific germline gene or a gene having a somatic mutation thereof, include those having a VH containing an amino acid sequence selected from any of SEQ ID No. 1 to 5. Furthermore, examples of such anti-PD-1 monoclonal antibodies include those having a VH amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, further preferably at least 98%, and further preferably at least 99% identity with the amino acid sequence selected from any of SEQ ID No. 1 to 5, and having substantially the same PD-1 binding activity as the anti-PD-1 monoclonal antibody having the original VH. In this document, the sentence “having substantially the same PD-1 binding activity as an anti-PD-1 monoclonal antibody with the original VH” means that it has 95% or more, preferably 98% or more, and more preferably 99% or more of PD-1 binding activity compared to an anti-PD-1 monoclonal antibody with the original VH.

Furthermore, other embodiments of the anti-PD-1 monoclonal antibody of the present invention include an anti-PD-1 monoclonal antibody that cross-competes with an anti-PD-1 monoclonal antibody having an amino acid sequence selected from any one of (1a) to (4a) or (1b) to (5b) above or a VH amino acid sequence selected from any one of SEQ ID No. 1 to 5, and an anti-PD-1 monoclonal antibody that cross-competes with an anti-PD-1 monoclonal antibody having an amino acid sequence selected from any one of (1a) to (4a) or (1b) to (5b) above or a VH containing an amino acid sequence selected from any one of SEQ ID No. 1 to 5.

Preferred features of the anti-PD-1 monoclonal antibody of the present invention include allowing interaction between PD-1 and PD-L1, interaction between PD-1 and PD-L2, or both, as well as the PD-1/CD3 bispecific antibody of the present invention.

Polynucleotide encoding PD-1/CD3 bispecific antibodies

The polynucleotide encoding the PD-1/CD3 bispecific antibody comprises: (1) a polynucleotide encoding a heavy chain of VH having a first arm that specifically binds to PD-1, (2) a polynucleotide encoding a heavy chain of VH having a second arm that specifically binds to CD3, and (3) a polynucleotide encoding a common light chain. In this document, the polynucleotide encoding the heavy chain of VH having a first arm that specifically binds to PD-1 comprises a polynucleotide encoding the VH of the first arm that specifically binds to PD-1 and a polynucleotide encoding a constant region of the heavy chain having VH. Similarly, the polynucleotide encoding the heavy chain of VH having a second arm that specifically binds to CD3 comprises a polynucleotide encoding the VH of the second arm that specifically binds to CD3 and a polynucleotide encoding a constant region of the heavy chain having VH.

The polynucleotide encoding the PD-1/CD3 bispecific antibody can be any polynucleotide as long as it encodes each part that constitutes the PD-1/CD3 bispecific antibody, and can be any of genomic DNA, cDNA, synthetic DNA, RNA, and DNA-RNA hybrids. One to six types of codons are known as codons encoding a single amino acid. For example, respectively, Phe corresponds to TTT or TTC, Leu corresponds to TTA, TTG, CTT, CTC, CTA, or CTG, Ile corresponds to ATT, ATC, or ATA, Met corresponds to ATG, Val corresponds to GTT, GTC, GTA, or GTG, Ser corresponds to TCT, TCC, TCA, or TCG, Pro corresponds to CCT, CCC, CCA, or CCG, Thr corresponds to ACT, ACC, ACA, or ACG, Ala corresponds to GCT, GCC, GCA, or GCG, Tyr... Corresponding to TAT or TAC, His corresponds to CAT or CAC, Gln corresponds to CAA or CAG, Asn corresponds to AAT or AAC, Lys corresponds to AAA or AAG, Asp corresponds to GAT or GAC, Glu corresponds to GAA or GAG, Cys corresponds to TGT or TGC, Trp corresponds to TGG, Arg corresponds to CGT, CGC, CGA, or CGG, Ser corresponds to AGT or AGC, Arg corresponds to AGA or AGG, and Gly corresponds to GGT, GGC, GGA, or GGG. Therefore, the polynucleotide encoding a PD-1/CD3 bispecific antibody comprises a polynucleotide in which any combination of codons corresponding to each amino acid is used. Preferred examples of polynucleotides encoding a VH of the first arm that specifically binds to PD-1 include polynucleotides having the base sequence shown in any one of SEQ ID Nos. 30 to 34, respectively encoding VHs of PD1-1 to PD1-5 clones. Preferred examples of polynucleotides encoding the VH of the second arm that specifically binds to CD3 include polynucleotides having the base sequence shown in SEQ ID No. 40. Furthermore, preferred examples of polynucleotides encoding the variable region of the common light chain include polypeptides having the base sequence shown in SEQ ID No. 35.

Drug Use

The PD-1/CD3 bispecific antibody of the present invention can be used to prevent autoimmune diseases or graft-versus-host disease (GVHD), suppress the progression or recurrence of symptoms of autoimmune diseases or graft-versus-host disease (GVHD), and/or treat autoimmune diseases or graft-versus-host disease (GVHD).

Examples of diseases that can be prevented, inhibited, or treated by the PD-1/CD3 bispecific antibody of the present invention include Behçet'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), aortitis syndrome (high-pressure arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Sjögren's syndrome, and adult-onset Still's disease. Vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, large vessel vasculitis, ANCA-associated vasculitis (e.g., granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis), Kurgan's syndrome, RS3PE, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related diseases (e.g., primary sclerosing cholangitis, autoimmune pancreatitis), Gurren Lahm-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic steatohepatitis, primary biliary cirrhosis, pulmonary hemorrhage nephritis Syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Bartholomew's disease (Graves' disease (hyperthyroidism)), Hashimoto's disease, autoimmune adrenocortical insufficiency, primary hypothyroidism, Addison's disease (chronic adrenocortical insufficiency), idiopathic Addison's disease, type 1 diabetes mellitus, slowly progressive type 1 diabetes mellitus (adult latent autoimmune diabetes mellitus), focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid Herpes gestationis, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent miscarriage, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, transplant immunosuppression, familial Mediterranean fever, eosinophilic chronic rhinosinusitis, dilated cardiomyopathy, systemic mastocytosis, or inclusion body myositis.

In this invention, the term "treatment" means curing or improving a disease or its symptoms. The term "prevention" means preventing or delaying the onset of a disease or its symptoms within a certain period of time. The term "suppressing symptom progression" means suppressing the progression or worsening of symptoms to stop the progression of the disease condition. "Prevention" includes suppressing recurrence. The term "suppressing recurrence" means preventing the recurrence of certain diseases or syndromes or reducing the likelihood of recurrence.

The PD-1/CD3 bispecific antibody of the present invention is typically administered systemically or locally via parenteral administration. Specific examples of such administration methods include injection, intranasal administration, pulmonary administration, and percutaneous administration. Examples of injection administration include intravenous injection, intramuscular injection, and intraperitoneal injection. For intravenous injection, intravenous infusion is preferred. The dosage varies depending on age, weight, symptoms, treatment effect, method of administration, and duration of treatment. For an adult patient, a single dose is typically administered once or several times daily via parenteral administration, in the range of 0.1 μg/kg to 300 mg/kg, and particularly preferably in the range of 0.1 mg/kg to 10 mg/kg, or via intravenous maintenance administration in the range of one hour to 24 hours daily. Needless to say, as mentioned above, since the dosage varies depending on various conditions, a sufficient dose may be lower than the above dose, or it may be necessary to exceed the above administration.

Preparations

When the PD-1/CD3 bispecific antibody of the present invention is formulated into an injectable or infusion solution for use as a drip infusion, the injectable or infusion solution may be in any form of aqueous solution, suspension, or emulsion, or may be formulated as a solid reagent with a pharmaceutically acceptable carrier such that the reagent will dissolve, suspend, or emulsify in a solvent upon use. Examples of solvents for injectable or infusion solutions for use as a drip infusion include distilled water for injection, physiological saline, glucose solution, and isotonic solutions (e.g., wherein sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, borax, propylene glycol, etc., are soluble).

Examples of pharmaceutically acceptable carriers in this article include stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, disinfectants, pH adjusters, and antioxidants. As stabilizers, various amino acids, albumins, globulins, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, polyethylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, butylated hydroxytoluene, etc., can be used. As solubilizers, alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (e.g., Polysorbate 20 (registered trademark), Polysorbate 80 (registered trademark), HCO-50, etc.), etc., can be used. As suspending agents, glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate, etc., can be used. As emulsifiers, gum arabic, sodium alginate, tragacanth gum, etc., can be used. As a soothing agent, benzyl alcohol, chlorobutanol, and sorbitol can be used. As a buffer, phosphate buffers, acetate buffers, borate buffers, carbonate buffers, citrate buffers, Tris buffers, glutamate buffers, and epsilon aminocaproic acid buffers can be used. As a preservative, methylparaben, ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax can be used. As a disinfectant, benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol can be used. As a pH adjuster, hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid can be used. As antioxidants, (1) water-based antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite and sodium sulfite, (2) oil-soluble antioxidants such as ascorbate palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate and α-tocopherol, and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid, sorbitol, tartaric acid and phosphoric acid.

Injectable or infusion solutions for intravenous infusion can be produced by sterilization or aseptic processing in the final process, such as sterilization by filtration with a filter, followed by filling with a sterile container. Injectable or infusion solutions for intravenous infusion can be used by dissolving vacuum-dried or lyophilized sterile powder (which may include pharmaceutically acceptable carrier powder) in a suitable solvent at the time of use.

Used in combination or in combination formulations

Furthermore, the PD-1/CD3 bispecific antibody of the present invention can be used in combination with other agents for the prevention of autoimmune diseases, the suppression of symptom progression or relapse of autoimmune diseases, and/or the treatment of autoimmune diseases. Examples of administration methods (combined use) in this invention, when used in combination with other agents, can include a combined formulation containing both components in one formulation, or administration in a separate formulation. Such combined use can supplement the preventive, symptom progression suppression, relapse suppression, and/or therapeutic effects of another agent, or can maintain or reduce the dosage or frequency of administration of another agent. In this invention, when administering the PD-1/CD3 bispecific antibody and another agent separately, both agents can be administered simultaneously for a certain period of time, followed by administration of either the PD-1/CD3 bispecific antibody or the other agent alone. Alternatively, the PD-1/CD3 bispecific antibody of the present invention can be administered first, and the other agent can be administered after the administration is completed. The other agent can be administered first, followed by administration of the PD-1/CD3 bispecific antibody of the present invention. Administration methods can be the same or different from each other. The present invention may also provide a kit comprising a formulation containing the PD-1/CD3 bispecific antibody of the present invention and a formulation containing another reagent. The dosage of the other reagent may be appropriately selected based on the dosage for clinical use. The other reagent may be a combination of two or more reagents in an appropriate ratio. Examples of the other reagent include not only known reagents but also reagents to be discovered in the future.

For example, when the PD-1/CD3 bispecific antibody of the present invention is applied to prevent type 1 diabetes, inhibit the progression or recurrence of type 1 diabetes symptoms, and/or treat type 1 diabetes, it can be used, for example, in combination with any one or more agents selected from: insulin preparations (e.g., human insulin, insulin glargine, insulin lispro, insulin detemir, insulin aspart, etc.), sulfonylureas (e.g., glibenclamide, gliclazide, or glimepiride), rapid-acting insulin secretion stimulants (e.g., nateglinide), biguanide preparations (e.g., metformin), and insulin resistance modifiers (e.g., pioglitazone). Itazone), α-glucosidase inhibitors (e.g., acarbose, voglibose, etc.), diabetic neuropathy treatments (e.g., epalrestat, mexiletine, imidapril, etc.), GLP-1 analogs (e.g., liraglutide, exenatide, lixisenatide, etc.), and DPP-4 inhibitors (sitagliptin, vildagliptin, alogliptin, etc.).

Furthermore, for example, when the PD-1/CD3 bispecific antibody of the present invention is applied to prevent multiple sclerosis, inhibit the progression or relapse of multiple sclerosis symptoms, and/or treat multiple sclerosis, it can be used in combination with any one or more of the following agents: steroid agents (e.g., cortisone acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, flucortisone acetate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisolone acetate butyl acetate, prednisolone sodium phosphate, prednisolone acetate haloacetate). Methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, triamcinolone, triamcinolone acetate, triamcinolone acetonide, dexamethasone, dexamethasone acetate sodium phosphate, dexamethasone palmitate, peramisone acetate or betamethasone), interferon β-1a, interferon β-1b, glatiramer acetate, mitoxantrone, azathioprine, cyclophosphamide, cyclosporine, methotrexate, cladribine, adrenocorticotropic hormone (ACTH), corticotropin, imidazolidinedin, tacrolimus, fingolimod or alenzusumab, etc.

Furthermore, for example, when the PD-1/CD3 bispecific antibody of the present invention is used to prevent systemic lupus erythematosus, inhibit the progression or relapse of systemic lupus erythematosus symptoms and/or treat systemic lupus erythematosus, it can be used in combination with any one or more of the following agents: steroids (e.g., the above-mentioned steroids), immunosuppressants (e.g., cyclosporine, tacrolimus or fingolimod) and belimumab.

For example, when the PD-1/CD3 bispecific antibody of the present invention is used to prevent rheumatoid arthritis, inhibit the progression or recurrence of rheumatoid arthritis symptoms, and/or treat rheumatoid arthritis, it can be used in combination with any one or more of the following agents: steroids (e.g., the above-mentioned steroids), antirheumatic agents (e.g., methotrexate, sulfasalazine, buccilamine, leflunomide, imidazolidinedione, tacrolimus, etc.), anticytokine agents (e.g., infliximab, adalimumab, tocilizumab, etanercept, golimumab, sertolizumab, etc.), and abatacept, etc.

When applying the PD-1/CD3 bispecific antibody of the present invention to prevent other autoimmune diseases, suppress the progression or recurrence of symptoms of other autoimmune diseases, and/or treat other autoimmune diseases, the PD-1/CD3 bispecific antibody of the present invention can be used in any one or more combinations with the other reagents described above.

The present invention will now be described in more detail through the following embodiments, but the scope of the invention is not limited thereto. Based on the description of the invention, those skilled in the art can make various changes and modifications, and such changes and modifications are also included in the present invention.

Example

Example 1: Immunization of MeMo mice with recombinant human PD-1-Fc fusion protein

As a first arm of the method for obtaining the PD-1-specific binding of the present invention, the method of immunizing MeMo mice with recombinant human PD-1 protein was chosen (see WO2009/157771). MeMo mice are genetically modified mice in which gene fragments including the non-recombinant human heavy chain V, D, and J gene regions, as well as the germline gene of the recombinant human κ light chain IgVκ1-39*01/IGJκ1*01, are linked to the mouse constant region gene. By directly immunizing MeMo mice with the target protein of the antibody, antibodies of diverse composition consisting of heavy chains and common light chains can be produced.

Twelve MeMo MS5B/MS9 mice aged 12 to 16 weeks were immunized at 14-day intervals with recombinant human PD-1-Fc fusion protein (R&D Systems, SEQ ID NO. 1086-PD), which had been emulsified using Gerbu adjuvant MM (Gerbu Biotechnik, SEQ ID NO. #3001). The recombinant human PD-1-Fc fusion protein was administered subcutaneously on days 0, 14, and 28 post-immunization. Subsequently, the recombinant human PD-1-Fc fusion protein dissolved in PBS was administered subcutaneously. Serum antibody titers were assessed by flow cytometry using a human PD-1-forced HEK293 T cell line on days 21, 35, 56, 77, and 98 post-immunization. When HEK293 T cell lines expressing human PD-1 were stained in serum diluted 1000 times, the MFI value of mice at least three times higher than that of the control HEK293 T cell line not expressing human PD-1 was used to construct phage display libraries. Mice meeting the criteria for library construction were immunized three days after antibody titer assessment with recombinant PD-1-Fc fusion protein, and their spleens and inguinal lymph nodes were collected. The same procedure was performed on mice with serum antibody titers of 1/100 or higher against human PD-1 and cynomolgus monkey PD-1, and whose antibody titers did not increase with further immunization; their spleens and inguinal lymph nodes were then collected. RNA was extracted from these lymph tissues, and then cDNA synthesis was performed.

Example 2: Constructing a phage display library to obtain anti-PD-1 antibodies with a first arm that specifically binds to PD-1. Antibody (protein immunity)

PCR was performed using the DNA prepared in Example 1 and primers specific to the immunoglobulin heavy chain variable region family. The obtained PCR products were digested with restriction enzymes SfiII and XhoI and inserted into the MV1473 phage vector [containing the gene encoding the common light chain (human κ light chain IgVκ1-39*01/IGJκ1*01 germline gene)] digested with the same restriction enzymes to construct a library.

Example 3: Screening for anti-PD-1 antibodies with a first arm that specifically binds to PD-1

Plates coated with human PD-1-Fc fusion protein, human PD-1-His-tagged fusion protein, cynomolgus monkey PD-1-His-tagged fusion protein, or mouse PD-1-His-tagged fusion protein were used for phage selection based on PD-1 binding properties. When using human PD-1-Fc fusion protein, human IgG (SIGMA, SEQ ID NO. I4506) was added during phage incubation to absorb Fc-reactive clones. Phages that could bind to human PD-1, cynomolgus monkey PD-1, and mouse PD-1 were enriched. Phages that could bind to cynomolgus monkey PD-1 were enriched by selection on the HEK293 T cell line expressing cynomolgus monkey PD-1. A mother plate was produced using *E. coli* strain TG1 clones transformed with the selected phages.

Furthermore, based on the binding characteristics of PD-1 to PD-1 on plates coated with the human PD-1-Fc fusion protein, phage selection was performed using periplasmic extracts of clones obtained through the above selection process. Note that, as a selection criterion, clones exhibiting a signal (OD ₄₅₀ value) three times higher than that obtained from the negative control wells (PBS) were defined as positive clones.

Example 4: DNA sequence of an anti-PD-1 antibody candidate clone with a first arm that specifically binds to PD-1

DNA sequences of the heavy chain variable region genes from the positive clones obtained through screening in Example 3 were analyzed. The analyzed DNA sequences were divided into superclusters (groups with CDR3 of the same length and 70% or higher amino acid sequence homology of CDR3) and clusters (groups where the amino acid sequences of heavy chain CDR3 are identical). 924 clones were obtained, which were classified into 146 types of superclusters and 194 types of clusters.

Example 5: Screening based on the binding characteristics of cells expressing PD-1

From each supercluster of classification, anti-PD-1 monoclonal antibody clones that meet the following criteria are screened to isolate them:

(1) Introduce somatic mutations into the CDR region at a high frequency;

(2) Includes germline genes for VH that are used frequently, and

(3) High signal was obtained in the screening of the binding characteristics of human PD-1-Fc fusion protein.

Using the Fab fragments contained in these periplasmic space extracts, binding to CHO-S cell lines expressing human PD-1 and CHO-S cell lines expressing cynomolgus monkey PD-1 was evaluated using anti-mouse IgG polyclonal antibodies. Of the 117 clones (105 cluster types) evaluated, 22 clones, including anti-PD-1 monoclonal antibody clones PD1-1, PD1-2, PD1-3, and PD1-4, were identified as binding to CHO-S cell lines expressing human PD-1.

Example 6: Amino acid extraction for generating an anti-PD-1 monoclonal antibody with a first arm that specifically binds to PD-1. Products of the times

Both PD1-1 and PD1-4 clones include a deamidation motif (Asn-Gly) in frame 4 of the heavy chain variable region. To obtain a first arm with reduced deamidation risk, a variant in which the deamidation motif has been transformed was generated. The PD1-5 clone was generated by altering the asparagine (Asn) at position 119 of the PD1-4 clone according to the EU numbering system to glutamine using a well-known site-specific mutagenesis method, and then isolating it. This clone exhibits the same binding characteristics to CHO-S cells expressing human PD-1 as the PD1-4 clone.

Example 7: Screening for anti-CD3 monoclonal antibodies with a second arm that specifically binds to CD3

To obtain a CD3-binding Fab with higher stability and further reduced charge heterogeneity, an anti-CD3 antibody having the "CD3-specific binding second arm" of the present invention was obtained by using an anti-CD3 antibody clone having the VH and IGVK1-39/JK1 common light chain of the anti-CD3 antibody 15C3 clone as described in WO2005/118635.

By converting the underlined glycine at position 55 of the VH amino acid sequence of 15C3 shown in Figure 10 to alanine, an anti-CD3 antibody clone CD3-1 with the same human CD3 binding properties as the clone and improved charge heterogeneity was obtained.

Furthermore, to obtain multiple CD3-binding Fabs to improve VH/VL interactions between CD3-1 clone-based VH and the common light chain (IgVκ1-39*01/IGJκ1*01), a phage display library expressing multiple Fabs was constructed, each Fab comprising a VH variant of the CD3-1 clone in which amino acid residues were substituted. 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 for bacterial reinfection. Multiple surviving bacterial colonies were extracted, and phages were extracted from them, then screened by flow cytometry based on binding to CD3 expressed on the cell surface. All CD3-binding phages were subjected to colony PCR to amplify the cDNA encoding their VH, and their DNA sequences were determined.

As a result, the obtained anti-CD3 antibody CD3-2 clone with VH containing the amino acid sequence shown in SEQ ID No. 36 exhibited the same CD3 binding properties and charge homogeneity as the CD3-1 clone. Example 8: Preparation of PD-1/CD3 bispecific monoclonal antibody

By linking DNA encoding the variable regions of the heavy chains of the anti-PD-1 monoclonal antibodies PD1-1 to PD1-5 and PD1-6 selected in Example 5 to DNA encoding the constant regions of the _IgG1 heavy chain, expression vectors for expressing each heavy chain of the first arm that specifically binds to PD-1 were generated. Conversely, by linking DNA encoding the variable regions of the heavy chains of the anti-CD3 monoclonal antibody CD3-2 selected in Example 7 to DNA encoding the constant regions of the _IgG1 heavy chain, expression vectors for expressing the heavy chains of the second arm that specifically binds to CD3 were generated. In this document, as genes expressing such constant regions of heavy chains, for the first arm that specifically binds to PD-1, a gene expressing the Fc region with the L351D/L368E variant (DE variant) was used. For the second arm that specifically binds to CD3, a gene expressing the Fc region with the L351K/T366K variant (KK variant) was used. These expression vectors were constructed to further include genes encoding the common light chain of IGVK1-39/JK1 for co-expression. Furthermore, the genes expressing these heavy chain constant regions have been modified to express the following: leucine at position 235 in the heavy chain constant region is replaced with glycine, and further, glycine at position 236 is replaced with arginine, and lysine at position 447 at the C-terminus of the heavy chain constant region is deleted to avoid post-translational processing. Both expression vectors were transferred into Free Style 293F cells to generate antibodies in the culture supernatant. The culture supernatant was collected and then processed by protein A affinity chromatography to purify the PD1-1(Bi) clone, PD1-2(Bi) clone, PD1-3(Bi) clone, PD1-4(Bi) clone, and PD1-5(Bi) clone of the PD-1/CD3 bispecific monoclonal antibody of the present invention, respectively. Furthermore, the PD1-6(Bi) clone was also generated using the same method. Note that because these PD-1/CD3 bispecific monoclonal antibody clones have a first arm of PD-1-specific binding derived from anti-PD-1 monoclonal antibodies used for their production: PD1-1, PD1-2, PD1-3, PD1-4 and PD1-5 clones, as well as PD1-6 clone, they correspond to those anti-PD-1 monoclonal antibody clones respectively.

Example 9: Evaluation of the binding properties of PD-1/CD3 bispecific monoclonal antibodies

The binding affinity of the first arm of the PD-1/CD3 bispecific monoclonal antibody obtained in Example 8 to each PD-1 recombinant protein was evaluated using the Biacore assay with human IgG1-Fc fused human PD-1 extracellular recombinant protein or 6×His-tagged fused cynomolgus monkey PD-1 extracellular recombinant protein. Note that the S-series sensor chip CM5 (GE Health Care, serial number 29-1049-88) was used for immobilizing the recombinant proteins. Similarly, the binding affinity of the second arm of the antibody to CD3 was evaluated using the Biacore assay with human IgG1-Fc fused CD3δ/CD3ε extracellular recombinant protein. Figure 11 shows the binding affinity (Kd value) of the first arm to PD-1 and the binding affinity of the second arm to CD3δ/CD3ε for each clone.

Example 10: Verification of the binding properties of PD-1/CD3 bispecific monoclonal antibody

This verifies that the PD-1/CD3 bispecific monoclonal antibody obtained in Example 8 can bind to PD-1 and CD3 simultaneously and specifically.

First, clones PD1-1(Bi) through PD1-6(Bi) were added to human PD-1-deficient Jurkat cell lines (human T cell lines) expressing human CD3 and incubated on ice for 15 minutes. After washing the cells, soluble recombinant PD-1 protein labeled with biotin (R&D systems, SEQ ID NO. 1086-PD-050) was added at 3 times the normal concentration and incubated on ice for 15 minutes. After washing the cells, 100 μL of 1.25 μg/mL Alexa Fluor 488-labeled streptavidin (BioLegend, SEQ ID NO. 405235) was added and incubated on ice for 15 minutes. After washing the cells, the binding amount of soluble recombinant PD-1 protein was assessed by flow cytometry. Figure 12 shows the results of the assay.

All clones bound to both PD-1 and CD3 simultaneously. Note that no nonspecific binding was detected in this binding system.

Example 11: Evaluation of the binding properties of the first arm of a PD-1/CD3 bispecific monoclonal antibody

To assess the effect on PD-1/PD-L1 binding of the first arm of the PD-1/CD3 bispecific monoclonal antibody obtained in Example 8, a competitive binding assay was performed to assess the binding of the bispecific monoclonal antibody clone and soluble PD-L1 recombinant protein to PD-1. First, PD1-1(Bi) through PD1-6(Bi) clones were added to CHO-S cell lines expressing human PD-1 and incubated on ice for 30 minutes. Biotin-labeled soluble PD-L1 recombinant protein (R&D systems, SEQ ID NO. 156-B7-100) at 1/20 of its concentration was added, and the cells were incubated on ice for 30 minutes. After washing the cells, PE-labeled streptavidin (BD Pharmingen, SEQ ID NO. 554061) was added, and the cells were incubated on ice for 30 minutes. After washing the cells, the binding amount of soluble PD-L1 recombinant protein was assessed by flow cytometry. Figure 13 shows the results of the assay.

Although clones PD1-1(Bi) to PD1-5(Bi) exist in amounts 20 times greater than that of soluble PD-L1 recombinant protein, they allow soluble PD-L1 recombinant protein to bind to PD-1. On the other hand, under the same conditions, PD1-6(Bi) completely inhibits the binding of soluble PD-L1 recombinant protein to PD-1.

Example 12: In vitro inhibitory effect of PD-1/CD3 bispecific monoclonal antibody on activated CD4 T cells

The inhibitory effect of activated T cells on IFN-γ production was assessed using CD4-positive T cells (LONZA, SEQ ID NO: 2W-200) derived from peripheral blood of healthy individuals (human T cells). Human T cells were seeded onto cell culture plates immobilized with anti-human TCRVβ8 antibody (Thermo Scientific, SEQ ID NO: TCR1750), and anti-human CD28 antibody (BioLegend, SEQ ID NO: 302923) was added, followed by activation treatment for 72 hours. The activated human T cells were then cultured overnight in fresh medium containing 100 units/mL of human IL-2 (R&D systems, SEQ ID NO: 202-IL). Furthermore, the collected human T cells were seeded onto another cell culture plate immobilized with anti-human TCRVβ8 antibody, and anti-human CD28 antibody was also added, followed by reactivation treatment. At this point, PD1-1(Bi) through PD1-6(Bi) clones were added, and the IFN-γ content in the culture supernatant 96 hours after activation was quantified (pg/mL) by ELISA. Figure 14 shows the results. The inhibitory activity of PD1-1(Bi) through PD1-5(Bi) clones against IFN-γ production was confirmed. The inhibitory activity of PD1-6(Bi) clone against IFN-γ production tended to be more significantly reduced than that of the other clones. An antibody obtained by tetanus toxoid immunization using the same technique as that used to obtain the first arm (which is a non-specific antibody) was used as a control antibody.

Example 13: PD-1/CD3 bispecific monoclonal antibody in an experimental allergic encephalomyelitis mouse model (EAE) In vivo effects of the model

In an EAE model, the in vivo effects of the PD-1/CD3 bispecific monoclonal antibody of the present invention were evaluated using human CD3ε/human PD-1 knock-in C57BL/6 mice, in which the CD3ε and PD-1 genes had been replaced by human CD3ε and human PD-1 genes, respectively. Killed Mycobacterium tuberculosis H37Ra (BD Biosciences, SEQ ID NO. 231141) and incomplete Freund's adjuvant (BD Biosciences, SEQ ID NO. 263910) were mixed to prepare a complete Freund's adjuvant (CFA) containing 4 mg/mL of killed Mycobacterium tuberculosis H37Ra. 1 mg/mL of MOG peptide (ANASPEC, SEQ ID NO. AS-60130) and an equal volume of CFA were mixed to prepare an emulsion used as an initiator in the EAE model. 200 μL of the initiator was subcutaneously administered to the tail and head of C57BL/6 mice. On day 1 and day 2 of immunization, 200 μL of 1 μg/mL pertussis toxin (SIGMA-ALDRICH, serial number P7208) was administered intravenously via the tail vein. Subsequently, on days 6 and 7 of immunization, C57BL/6 mice were administered 2 mg/kg of PD1-1(Bi) to PD1-6(Bi) clones intraperitoneally once daily. Neurological symptoms following immunization were assessed according to the method of Onuki et al. (Onuki M, et al., Microsc Res Tech 2001; 52:731-9). The severity of neurological symptoms was scored (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 dead: 5 points). If multiple neurological symptoms were observed, the higher score was used as the neurological symptom on the assessment day. The neurological symptoms of dead mice were scored as 5 until the observation was completed. Figures 15 to 17 show the assessment results. Note here that the human CD3ε/human PD-1 knock-in C57BL/6 mice were generated by a well-known method, which involved mating human CD3ε knock-in mice (according to the method described in Genesis. 2009 Jun; 47(6):414-22) with human PD-1 knock-in mice.

In the EAE model, the onset of neurological symptoms was significantly suppressed for any of the PD1-1 to PD1-5(Bi) clones, but no significant inhibitory effect was observed for the PD1-6(Bi) clone.

Example 14: Evaluation of the effect of PD-1/CD3 bispecific monoclonal antibody on the release of cytokines from human peripheral blood mononuclear cells In vitro effects of the molecule

To analyze the cytokine release activity of the PD-1/CD3 bispecific monoclonal antibody, the following experiment was performed: The PD-1/CD3 bispecific monoclonal antibody clone of this invention or the PD-1/CD3 bispecific scDb (J110×UCHT1) disclosed in Patent Document 2 was added to human peripheral blood mononuclear cells (hereinafter referred to as "human PBMCs"). Each clone of the PD-1/CD3 bispecific monoclonal antibody and J110×UCHT1 were added to human PBMCs (LONZA, serial number CC-2702) at concentrations of 30 μg/mL and 10 μg/mL, respectively, and then cultured for 24 hours. After 24 hours of culture, the amount of each IL-2 contained in the culture supernatant was quantified by a multiplex immunoassay (BIO-RAD, serial number M50000007A). Figure 18 shows the results. Note that the amount of IL-2 produced (pg/mL) in the figure is expressed as mean ± standard error (N=3).

J110×UCHT1 significantly induces IL-2 production. However, for any clone of the PD-1/CD3 bispecific monoclonal antibody, IL-2 production is low.

Example 15: Evaluation of each clone of the PD-1/CD3 bispecific monoclonal antibody against PD1-5(Bi) and PD-1 Combined cross-competitive activity

A cross-competition assay was performed to evaluate the cross-competitive activity of each PD1-1(Bi) to PD1-5(Bi) clone of the PD-1/CD3 bispecific monoclonal antibody obtained in Example 8 with the binding of PD1-5(Bi) to PD-1.

First, the PD1-5(Bi) clone was added to CHO-S cells expressing human PD-1 and then incubated on ice for 20 minutes. Next, one percent of the amount of biotinylated clones PD1-1(Bi) to PD1-5(Bi) were added to those cells, compared to the amount of the previously added PD1-5(Bi) clone, and then incubated on ice for 20 minutes. The cells were washed, and PE-labeled streptavidin (BD Pharmingen, model 554061) was added, followed by incubation on ice for 20 minutes. After washing the cells, the binding amount of biotinylated clones PD1-1(Bi) to PD1-5(Bi) was measured using flow cytometry. Figure 19 shows the results.

This indicates that the PD1-5(Bi) clone can inhibit the binding of each of the PD1-1(Bi) to PD1-5(Bi) clones to PD-1, thus the PD1-5(Bi) clones can cross-compete for their binding to PD-1.

Industrial applicability

The PD-1/CD3 bispecific antibody or antibody fragment thereof of the present invention may be used to prevent autoimmune diseases or graft-versus-host disease (GVHD), suppress the progression or recurrence of symptoms of autoimmune diseases or graft-versus-host disease (GVHD), and/or treat autoimmune diseases or graft-versus-host disease (GVHD).

sequence list

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<213> people

<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> PRT

<213> people

<400> 26

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 27

<211> 7

<212> PRT

<213> people

<400> 27

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 28

<211> 9

<212> PRT

<213> people

<400> 28

Gln Gln Ser Tyr Ser Thr Pro Pro Thr

1 5

<210> 29

<211> 107

<212> PRT

<213> people

<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> people

<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> people

<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> people

<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> people

<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> people

<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> people

<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> PRT

<213> people

<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> PRT

<213> people

<400> 37

Ser Tyr Gly Met His

1 5

<210> 38

<211> 17

<212> PRT

<213> people

<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> PRT

<213> people

<400> 39

Gly Thr Gly Tyr Asn Trp Phe Asp Pro

1 5

<210> 40

<211> 354

<212> DNA

<213> people

<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> PRT

<213> people

<400> 41

Trp Gly Lys Gly Thr Thr Thr Val Thr Val Ser Ser

1 5 10

Claims (38)

1. A PD-1/CD3 bispecific antibody or an antibody fragment thereof, comprising a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3, wherein... The first arm that specifically binds to PD-1 comprises any of the following VHs: (A) VH, which has (a) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID No. 6; (b) VH-CDR2 consisting of the amino acid sequence shown in SEQ ID No. 7; and (c) VH-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 8; (B) VH, which has (a) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID No. 9; (b) VH-CDR2 consisting of the amino acid sequence shown in SEQ ID No. 10; and (c) VH-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 11; (C) VH, which has (a) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID No. 12; (b) VH-CDR2 consisting of the amino acid sequence shown in SEQ ID No. 13; and (c) VH-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 14; and (D) VH, which has (a) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID No. 18; (b) VH-CDR2 consisting of the amino acid sequence shown in SEQ ID No. 19; and (c) VH-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 20; and (E) VL, which has (a) VL-CDR1 consisting of the amino acid sequence shown in SEQ ID No. 26; (b) VL-CDR2 consisting of the amino acid sequence shown in SEQ ID No. 27; and (c) VL-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 28; and The second arm that specifically binds to CD3 comprises: (A) VH, which has (a) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID No. 37; (b) VH-CDR2 consisting of the amino acid sequence shown in SEQ ID No. 38; and (c) VH-CDR3 composed of the amino acid sequence shown in SEQ ID No. 39, and (B) VL, which has (a) VL-CDR1 consisting of the amino acid sequence shown in SEQ ID No. 26; (b) VL-CDR2 consisting of the amino acid sequence shown in SEQ ID No. 27; and (c) VL-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 28; and The antibody fragment mentioned therein is F(ab') ₂ . 2. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 1, wherein... (i) The VH of the first arm that specifically binds to PD-1 has: (a) The VH-CDR1 composed of the amino acid sequence shown in SEQ ID No. 6, (b) The VH-CDR2 composed of the amino acid sequence shown in SEQ ID No. 7, and (c) The VH-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 8; and (ii) The VH of the second arm that specifically binds to CD3 has: (a) The VH-CDR1 composed of the amino acid sequence shown in SEQ ID No. 37, (b) The VH-CDR2 composed of the amino acid sequence shown in SEQ ID No. 38, and (c) The VH-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 39. 3. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 1, wherein... (i) The VH of the first arm that specifically binds to PD-1 has: (a) The VH-CDR1 composed of the amino acid sequence shown in SEQ ID No. 9, (b) The VH-CDR2 composed of the amino acid sequence shown in SEQ ID No. 10, and (c) The VH-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 11; and (ii) The VH of the second arm that specifically binds to CD3 has: (a) The VH-CDR1 composed of the amino acid sequence shown in SEQ ID No. 37, (b) The VH-CDR2 composed of the amino acid sequence shown in SEQ ID No. 38, and (c) The VH-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 39. 4. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 1, wherein... (i) The VH of the first arm that specifically binds to PD-1 has: (a) The VH-CDR1 composed of the amino acid sequence shown in SEQ ID No. 12, (b) The VH-CDR2 composed of the amino acid sequence shown in SEQ ID No. 13, and (c) The VH-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 14; and (ii) The VH of the second arm that specifically binds to CD3 has: (a) The VH-CDR1 composed of the amino acid sequence shown in SEQ ID No. 37, (b) The VH-CDR2 composed of the amino acid sequence shown in SEQ ID No. 38, and (c) The VH-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 39. 5. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 1, wherein... (i) The VH of the first arm that specifically binds to PD-1 has: (a) The VH-CDR1 composed of the amino acid sequence shown in SEQ ID No. 18, (b) The VH-CDR2 composed of the amino acid sequence shown in SEQ ID No. 19, and (c) The VH-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 20; and (ii) The VH of the second arm that specifically binds to CD3 has: (a) The VH-CDR1 composed of the amino acid sequence shown in SEQ ID No. 37, (b) The VH-CDR2 composed of the amino acid sequence shown in SEQ ID No. 38, and (c) The VH-CDR3 consisting of the amino acid sequence shown in SEQ ID No. 39. 6. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of claims 1 to 5, wherein the VH of the first arm that specifically binds to PD-1 comprises an amino acid sequence selected from any one 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 having at least 80% identity with the amino acid sequence of its VH. 7. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of claims 1 to 5, wherein the VH of the second arm that specifically binds to CD3 comprises the amino acid sequence shown in SEQ ID No. 36, or an amino acid sequence having at least 80% identity with its VH amino acid sequence. 8. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 1, wherein the VH of the first arm specifically binding to PD-1 comprises an amino acid sequence selected from any one of 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 comprises the amino acid sequence shown in SEQ ID No. 36. 9. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 1 or 2, wherein the VH of the first arm specifically binding to PD-1 comprises the amino acid sequence shown in SEQ ID No. 1; and the VH of the second arm specifically binding to CD3 comprises the amino acid sequence shown in SEQ ID No. 36. 10. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 1 or 3, wherein the VH of the first arm that specifically binds to PD-1 comprises the amino acid sequence shown in SEQ ID No. 2; and the VH of the second arm that specifically binds to CD3 comprises the amino acid sequence shown in SEQ ID No. 36. 11. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 1 or 4, wherein the VH of the first arm that specifically binds to PD-1 comprises the amino acid sequence shown in SEQ ID No. 3; and the VH of the second arm that specifically binds to CD3 comprises the amino acid sequence shown in SEQ ID No. 36. 12. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 1 or 5, wherein the VH of the first arm that specifically binds to PD-1 comprises the amino acid sequence shown in SEQ ID No. 4; and the VH of the second arm that specifically binds to CD3 comprises the amino acid sequence shown in SEQ ID No. 36. 13. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 1 or 5, wherein the VH of the first arm that specifically binds to PD-1 comprises the amino acid sequence shown in SEQ ID No. 5; and the VH of the second arm that specifically binds to CD3 comprises the amino acid sequence shown in SEQ ID No. 36. 14. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of claims 1 to 5, wherein the first arm specifically binding to PD-1 and/or the second arm specifically binding to CD3 has a VL comprising the amino acid sequence shown in SEQ ID No. 25. 15. A PD-1/CD3 bispecific antibody or an antibody fragment thereof, having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3, wherein... (A) The first arm that specifically binds to PD-1 has a VH and a VL, wherein the VH consists of an amino acid sequence selected from any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5; and the VL consists of an amino acid sequence shown in SEQ ID No. 25; and (B) The second arm that specifically binds to CD3 has VH and VL, wherein VH consists of the amino acid sequence shown in SEQ ID No. 36; and VL consists of the amino acid sequence shown in SEQ ID No. 25; The antibody fragment mentioned therein is F(ab') ₂ . 16. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of claims 1 to 5, wherein the first arm that specifically binds to PD-1 allows for interaction between PD-1 and PD-L1. 17. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of claims 1 to 5, wherein cytokine production is sufficiently reduced. 18. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 17, wherein the cytokine is at least IL-2, IFN-γ or TNF-α. 19. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of claims 1 and 15, wherein the PD-1/CD3 bispecific antibody is an IgG antibody. 20. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 19, wherein it is an _IgG1 antibody or an _IgG4 antibody. 21. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 19, wherein it is an _IgG1 antibody. 22. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 21, wherein binding to the Fc receptor is eliminated or reduced. 23. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 22, wherein in the two heavy chain constant regions of the PD-1/CD3 bispecific IgG ₁ antibody, leucine at position 235 according to the EU numbering system is replaced by glycine, and/or glycine at position 236 according to the EU numbering system is replaced by arginine. 24. A PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of claims 21 to 23, wherein in the constant region of the heavy chain of the VH having the first arm specifically binding to PD-1, leucine at position 351 according to the EU numbering system is replaced by lysine and threonine at position 366 according to the EU numbering system is replaced by lysine; and in the constant region of the heavy chain of the VH having the second arm specifically binding to CD3, leucine at position 351 according to the EU numbering system is replaced by aspartic acid and leucine at position 368 according to the EU numbering system is replaced by glutamic acid. 25. A PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of claims 21 to 23, wherein in the constant region of the heavy chain of the VH having the first arm specifically binding to PD-1, leucine at position 351 according to the EU numbering system is replaced by aspartic acid and leucine at position 368 according to the EU numbering system is replaced by glutamic acid; and in the constant region of the heavy chain of the VH having the second arm specifically binding to CD3, leucine at position 351 according to the EU numbering system is replaced by lysine and threonine at position 366 according to the EU numbering system is replaced by lysine. 26. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to any one of claims 21 to 23, wherein the lysine at position 447 according to the EU numbering system is missing in both heavy chain constant regions of the PD-1/CD3 bispecific antibody. 27. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 20, wherein the PD-1/CD3 bispecific antibody is an _IgG4 antibody, and in both heavy chain constant regions, the serine at position 228 according to the EU numbering system is replaced by proline. 28. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 1, wherein the heavy chain having the VH of the first arm that specifically binds to PD-1 has a heavy chain constant region comprising the amino acid sequence shown in SEQ ID No. 23. 29. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 1, wherein the heavy chain having the VH of the second arm that specifically binds to CD3 has a heavy chain constant region comprising the amino acid sequence shown in SEQ ID No. 24. 30. The PD-1/CD3 bispecific antibody or antibody fragment thereof according to claim 1, wherein the light chain having the first arm that specifically binds to PD-1 and/or the light chain having the second arm that specifically binds to CD3 has a light chain constant region comprising the amino acid sequence shown in SEQ ID No. 29. 31. A PD-1/CD3 bispecific antibody, which has a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD3, wherein: (A) The heavy chain having the VH of the first arm that specifically binds to PD-1 has a VH and a heavy chain constant region, wherein the VH consists of an amino acid sequence selected from any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, and the heavy chain constant region consists of an amino acid sequence shown in SEQ ID No. 23; (B) A light chain having a VL having a first arm that specifically binds to PD-1 has a VL and a light chain constant region, wherein the VL consists of the amino acid sequence shown in SEQ ID No. 25 and the light chain constant region consists of the amino acid sequence shown in SEQ ID No. 29; (C) The heavy chain having the VH of the second arm specifically binding to CD3 has a VH and a heavy chain constant region, wherein the VH consists of the amino acid sequence shown in SEQ ID No. 36, and the heavy chain constant region consists of the amino acid sequence shown in SEQ ID No. 24; and (D) The light chain having the VL of the second arm that specifically binds to CD3 has a VL and a light chain constant region, wherein the VL consists of the amino acid sequence shown in SEQ ID No. 25 and the light chain constant region consists of the amino acid sequence shown in SEQ ID No. 29. 32. A pharmaceutical composition comprising a PD-1/CD3 bispecific antibody selected from any one of claims 1 to 31 or an antibody fragment thereof and a pharmaceutically acceptable carrier. 33. An agent for the prevention of graft-versus-host disease (GVHD), the suppression of the progression or recurrence of symptoms of graft-versus-host disease (GVHD), and/or the treatment of graft-versus-host disease (GVHD), comprising a PD-1/CD3 bispecific antibody or an antibody fragment thereof as an active ingredient according to any one of claims 1 to 31. 34. A pharmaceutical composition comprising a PD-1/CD3 bispecific antibody or an antibody fragment thereof according to any one of claims 1 to 31 and one or more agents selected from the group consisting of: insulin formulations, sulfonylureas, rapid-acting insulin secretagogues, biguanides, insulin resistance modifiers, α-glucosidase inhibitors, diabetic neuropathy treatments, GLP-1 analogs and DPP-4 inhibitors, steroids, interferon β-1a, interferon β-1b, glatiramer acetate, mitoxantrone, azathioprine, cyclophosphamide, cyclosporine, methotrexate, cladribine, adrenocorticotropic hormone (ACTH), corticotropin, mizoribine, tacrolimus, fingolimod, alenzumab, immunosuppressants, belimumab, antirheumatic drugs, anticytokine drugs, and abatacept. 35. A polynucleotide encoding a PD-1/CD3 bispecific antibody or an antibody fragment thereof according to any one of claims 1 to 31. 36. An expression vector having the polynucleotide according to claim 35. 37. A host cell that has been transfected or transformed by the expression vector according to claim 36. 38. A method for producing a PD-1/CD3 bispecific antibody or an antibody fragment thereof according to any one of claims 1 to 31, comprising transferring an expression vector gene therein inserted into a mammalian animal cell to transform it for: (1) a polynucleotide encoding a heavy chain containing a VH that specifically binds to PD, (2) a polynucleotide encoding a heavy chain containing a VH that specifically binds to CD3, and (3) a polynucleotide encoding a common light chain, and then allowing them to express and secrete both the heavy chain and the common light chain.