RU2816592C2 - Immune tolerance inducing antibodies, induced lymphocytes and therapeutic agent/method of cell therapy using induced lymphocytes - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a combination of antibodies for inducing immune tolerance to a cell obtained from an individual, containing an anti-CD86 antibody and an anti-CD80 antibody. Also disclosed is a nucleic acid molecule coding an anti-CD80 antibody or a variant thereof, a nucleic acid molecule coding an anti-CD86 antibody or a variant thereof, as well as a vector and a cell containing them. Invention also relates to a method of producing a cell involving the use of said combination.

EFFECT: invention is effective for induction of immune tolerance to a cell obtained in an individual.

9 cl, 9 dwg, 1 tbl, 6 ex

Description

TECHNICAL FIELD TO WHICH THE INVENTION RELATES

[0001]

The present invention relates to a new technology related to immune tolerance. More specifically, the present invention relates to an antibody that induces immune tolerance, induced lymphocytes or a therapeutic agent/method of cell therapy using induced lymphocytes.

BACKGROUND OF THE ART

[0002]

Liver transplantation is widely used as definitive treatment for patients with end-stage liver failure. Every year, 20,000 or more liver transplants are performed overseas, and more than 500 in Japan.

[0003]

Transplantation is one of the main methods of treating organ failure of the kidneys, heart, liver, pancreas, etc. in the terminal stage of the disease. Although significant progress has been made in the treatment of graft rejection in recent years, most grafts ultimately fail in the absence of immunosuppressive treatment. The current medical regimen of immunosuppression, which depends on continuous drug therapy, suppresses not only the responses that are clearly directed at the transplanted organs, but also all immune responses, leaving organ transplant patients more vulnerable due to increased susceptibility to infections and cancer.

[0004]

Although regenerative medicine techniques are gaining increasing attention, immune rejection is ultimately possible without the involvement of autologous cells, even if induced pluripotent stem cells (iPS cells) or the like are used. Thus, methods that promote immune tolerance have received attention.

[0005]

Such methods of inducing immune tolerance include inducing an antigen-specific non-immune response (anergy) of T cells. Specific methods have been reported, including directly administering to a transplant patient an antibody that inhibits interactions between CD80/CD86 on antigen-presenting cells and CD28 on naïve T cells, causing donor antigen-specific anergy in the body (Patent Literature 1) , and a method of co-culturing recipient cells and radiation-irradiated donor cells in the presence of the same antibody to induce donor antigen-specific anergic cells ex vivo and return said cells to the recipient (Patent Literature 2, Patent Literature 3 and Non-Patent Literature 1-3),

Literature

Patent literature

[0006]

[PTL 1] Japanese PCT National Phase Publication Released No. 2002-504120

[PTL 2] Japanese National Phase PCT Publication Released No. 2007-131598

[PTL 3] Japanese National Phase PCT Publication Released No. 2016-520081

Non-patent literature

[0007]

[NPL 1] Satoru Todo et al. Hepatorogy, 64 (vol. 2), 632-643 (2016)

[NPL 2] Teraoka S, Koyama I, Bashuda H, Uchida K, Tonsho M, et al. (2017) J Transplant Res 2(1) p1-8

[NPL 3] Bashuda H et al., J. Clin. Invest. 115: 1896-1902 (2005).

SUMMARY OF THE INVENTION

Solution

[0008]

We attempted to induce a non-immune response (anergy) using an antibody with a specific human Fc region that inhibits the interaction between CD80/CD86 and CD28, which was found to bind to cells involved in the immune system such as macrophages, neutrophils or natural killer (NK) cells, activates this cell and induces the release of a humoral factor, such as interleukin or interferon (IFN), which is responsible for immune responses. The inventors have also discovered that the release of these humoral factors causes adverse activation of the immune system, which is the opposite of immune tolerance, therefore, the effect of immune tolerance can be improved or the weakening of the effect of immune tolerance can be suppressed by regulating the induction of non-immune responses (anergy) using an antibody with human Fc region, which does not bind to these cells. Thus, the present invention provides an antibody with a structure capable of improving or not impairing the effect of immune tolerance.

[0009]

Therefore, the following is provided:

(1) An antibody or variant thereof that inhibits the interaction between CD80 and/or CD86 expressed on the surface of a cell and CD28 expressed on the surface of another cell, wherein the antibody or variant thereof does not substantially induce the production of a cytokine by immunostimulation.

(2) An antibody or variant thereof according to the preceding paragraph, wherein the cytokine includes interferon γ.

(3) An antibody or variant thereof according to any of the preceding paragraphs, which is a chimeric antibody.

(4) An antibody or variant thereof according to any of the preceding claims, wherein the antibody is of the IgG1 subclass.

(5) An antibody or a variant thereof as defined in any one of the preceding claims, having a heavy chain having the amino acid sequence set forth in SEQ ID NO: 38 or 42 and a light chain having the amino acid sequence set forth in SEQ ID NO: 40 or 44.

(6) An antibody or variant thereof according to any of the preceding claims, containing:

(a) VH containing CDRH1 with the amino acid sequence given in SEQ ID NO: 53, CDRH2 with the amino acid sequence given in SEQ ID NO: 54, and CDRH3 with the amino acid sequence given in SEQ ID NO: 55, and VL containing CDRL1 having the amino acid sequence given in SEQ ID NO: 56, CDRL2 having the amino acid sequence shown in SEQ ID NO: 57, and CDRL3 given in SEQ ID NO: 58; or

(b) VH containing CDRH1 with the amino acid sequence given in SEQ ID NO: 59, CDRH2 with the amino acid sequence given in SEQ ID NO: 60, and CDRH3 with the amino acid sequence given in SEQ ID NO: 61, and VL containing CDRL1 having the amino acid sequence set forth in SEQ ID NO: 62, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 63, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 64.

(7) An antibody or variant thereof according to any of the preceding claims, containing:

(a) VH having the amino acid sequence shown in SEQ ID NO: 46, or a variant sequence thereof, and VL having the amino acid sequence shown in SEQ ID NO: 48, or a variant sequence thereof; or

(b) VH having the amino acid sequence set forth in SEQ ID NO: 50, or a variant sequence thereof, and VL having the amino acid sequence set forth in SEQ ID NO: 52, or a variant sequence thereof.

(8) An antibody or a variant thereof as claimed in any one of the preceding claims, wherein the Fc portion of the antibody is a fragment that does not substantially induce cytokine production by immunostimulation.

(9) An antibody or variant thereof according to any of the preceding claims, wherein the antibody is of the IgG2 or IgG4 subclass.

(10) An antibody or variant thereof as claimed in any one of the preceding claims, wherein the antibody is of the IgG4 subclass.

(11) The antibody or variant thereof as claimed in any one of the preceding claims, wherein the antibody variant is a variant lacking the Fc portion of the antibody.

(12) An antibody or variant thereof as claimed in any one of the preceding claims, wherein the antibody variant is a Fab antibody, F(ab') ₂ antibody, Fab' antibody, Fv antibody, or scFv antibody.

(13) An antibody or variant thereof according to any of the preceding paragraphs having the ability to induce immune tolerance.

(14) An antibody or a variant thereof as claimed in any one of the preceding claims, wherein the cell expressing CD80 and/or CD86 is an antigen presenting cell and the other cell expressing CD28 is a T cell.

(15) An antibody according to any one of the preceding claims, wherein the antibody or a variant thereof is an anti-CD80 antagonist antibody, an anti-CD86 antagonist antibody, an anti-CD28 antagonist antibody, or a bispecific anti-CD80 and CD86 antagonist antibody, or a variant thereof.

(16) An antibody or a variant thereof according to any of the preceding claims, which is a humanized antibody or a human antibody or a variant thereof.

(17) An antibody or a variant thereof as claimed in any one of the preceding claims, comprising an Fc fragment of an IgG2 or IgG4 human antibody.

(18) An antibody or variant thereof as claimed in any one of the preceding claims, comprising an Fc portion of an IgG4 human antibody.

(19) An antibody or variant thereof according to any of the preceding claims, comprising:

(a) VH containing CDRH1 with the amino acid sequence given in SEQ ID NO: 25, CDRH2 with the amino acid sequence given in SEQ ID NO: 26, and CDRH3 with the amino acid sequence given in SEQ ID NO: 27, and VL containing CDRL1 having the amino acid sequence shown in SEQ ID NO: 28, CDRL2 having the amino acid sequence shown in SEQ ID NO: 29, and CDRL3 given in SEQ ID NO: 30; or

(b) VH containing CDRH1 with the amino acid sequence given in SEQ ID NO: 31, CDRH2 with the amino acid sequence given in SEQ ID NO: 32, and CDRH3 with the amino acid sequence given in SEQ ID NO: 33, and VL containing CDRL1 having the amino acid sequence set forth in SEQ ID NO: 34, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 35, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 36.

(20) An antibody or variant thereof according to any of the preceding claims, comprising:

(a) VH having the amino acid sequence shown in SEQ ID NO: 18, or a variant sequence thereof, and VL having the amino acid sequence shown in SEQ ID NO: 20, or a variant sequence thereof; or

(b) VH having the amino acid sequence shown in SEQ ID NO: 22, or a variant sequence thereof, and VL having the amino acid sequence shown in SEQ ID NO: 24, or a variant sequence thereof.

(21) A nucleic acid molecule encoding the amino acid sequence or part thereof of an antibody or variant thereof according to any of the preceding claims.

(22) A nucleic acid molecule according to any of the preceding claims, containing:

(a) a polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 45, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 47; or

(b) a polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 49, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 51.

(23) A vector containing a nucleic acid molecule according to any of the preceding paragraphs.

(24) A cell containing a nucleic acid molecule according to any of the preceding paragraphs or a vector according to paragraph 23.

(25) A method for producing an antibody or a variant thereof according to any of the preceding claims, comprising culturing a cell according to any of the preceding claims.

(26) A cell with immune tolerance induced by an antibody or a variant thereof according to any one of the preceding claims.

(27) A cell as claimed in any one of the preceding claims, wherein immune tolerance is induced by mixing an antibody or variant thereof, a cell derived from an individual, and an antigen that is not derived from the individual, or material containing the antigen.

(28) A composition for producing a cell with induced immune tolerance, containing at least one of the antibodies or variants thereof according to any of the preceding claims.

(29) A composition as claimed in any one of the preceding claims, comprising an anti-CD80 antagonist antibody, an anti-CD86 antagonist antibody, an anti-CD28 antagonist antibody, or a bispecific anti-CD80 and CD86 antagonist antibody or a variant thereof, or any combination thereof.

(30) A method of producing a cell for treating or preventing a disease, disorder, or condition caused by an antigen that is not derived from an individual, comprising mixing an antibody or variant thereof of any one of the preceding claims, a cell derived from the individual, and an antigen that is not derived from individual or material containing this antigen.

(31) The method as claimed in any one of the preceding claims, wherein the disease, disorder or condition is selected from the group consisting of graft rejection, allergy, autoimmune disease, graft-versus-host disease, and immune rejection caused by iPS cell or ES cell transplantation or ES cells and cells, tissue or organ derived from said cells.

(32) The method as claimed in any one of the preceding claims, wherein the antigen-containing material is a cell.

(33) A cell obtained according to any of the preceding paragraphs.

(34) A cell according to any one of the preceding claims, comprising a T cell.

(35) A cell therapy agent comprising a cell with immune tolerance induced by an antibody or a variant thereof according to any of the preceding claims, a cell produced by a method according to any of the preceding claims, or a cell according to any of the preceding claims.

(36) A composition for the treatment or prevention of a disease, disorder or condition caused by an antigen foreign to the individual, comprising a cell with immune tolerance induced by an antibody or a variant thereof according to any one of the preceding claims, a cell produced by a method according to any one of the preceding claims, or a cell according to any of the previous points.

(37) The composition according to any one of the preceding claims, wherein the disease, disorder or condition is selected from the group consisting of graft rejection, allergy, autoimmune disease, graft-versus-host disease and immune rejection caused by iPS cell or ES cell transplantation and cells, tissue or organ derived from said cells.

(38) The composition according to any of the preceding claims, wherein the rejection is characterized in that the transplanted organ is a kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood, blood cell, including an immune system cell, bone, cartilage, blood vessel, cornea, eyeball, or bone marrow.

(39) A method of treating or preventing a disease, disorder, or condition caused by an antigen that is not originating from an individual in said individual, comprising administering to that individual an effective amount of a cell with immune tolerance induced by an antibody or a variant thereof of any one of the preceding claims, the cell, produced by the method according to any of the preceding paragraphs, or a cell according to any of the previous paragraphs.

(40) The method as claimed in any one of the preceding claims, wherein the disease, disorder or condition is selected from the group consisting of graft rejection, allergy, autoimmune disease, graft-versus-host disease, and immune rejection caused by transplantation of iPS cells or ES cells and cells , tissue or organ derived from said cells.

(41) The method as claimed in any one of the preceding claims, wherein the rejection is characterized in that the transplanted organ is a kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood, blood cell, including an immune system cell, bone, cartilage, blood vessel, cornea, eyeball, or bone marrow.

(39A) Use of a cell with immune tolerance induced by an antibody or variant thereof according to any of the preceding claims, a cell produced by a method according to any of the preceding claims, or a cell according to any of the preceding claims, for the manufacture of a medicament for the treatment or prevention of a disease, disorder or a condition caused by an antigen that does not originate in an individual, in a specified individual.

(40A) Use as claimed in any one of the preceding claims, wherein the disease, disorder or condition is selected from the group consisting of graft rejection, allergy, autoimmune disease, graft-versus-host disease and immune rejection caused by iPS cell or ES cell transplantation and cells, tissue or organ derived from said cells.

(41A) Use as claimed in any of the preceding claims, wherein the rejection is characterized in that the transplanted organ is a kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood, blood cell, including an immune system cell, bone, cartilage, blood vessel, cornea, eyeball, or bone marrow.

(39B) A cell with immune tolerance induced by an antibody or variant thereof according to any of the preceding claims, a cell produced by a method according to any of the preceding claims, or a cell according to any of the preceding claims for the production of a medicament for the treatment or prevention of a disease, disorder or condition, caused by an antigen that does not originate in the individual in that individual.

(40B) The cell of any one of the preceding claims, wherein the disease, disorder or condition is selected from the group consisting of graft rejection, allergy, autoimmune disease, graft-versus-host disease, and immune rejection caused by iPS cell or ES cell transplantation and cells, tissue or organ derived from said cells.

(41B) A cell as claimed in any one of the preceding claims, wherein the rejection is characterized in that the transplanted organ is a kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood, blood cell, including an immune system cell, bone, cartilage, blood vessel, cornea, eyeball, or bone marrow.

The following is also suggested.

(A1) An antibody that inhibits the interaction between CD80 and/or CD86 expressed on the surface of a cell and CD28 expressed on the surface of another cell, wherein the antibody does not substantially induce cytokine production by immunostimulation.

(A2) The antibody of (A1), wherein the cytokine includes interferon γ.

(A3) The antibody of claim (A1) or claim (A2), wherein the Fc portion of the antibody is a fragment that does not substantially induce cytokine production by immunostimulation.

(A4) Antibody according to any one of paragraphs. (A1)-(A3), where the antibody is of the IgG2 or IgG4 subclass.

(A5) The antibody of (A4), wherein the antibody is of the IgG4 subclass.

(A6) Antibody according to item (A1) or item (A2), in which the Fc fragment is missing.

(A7) The antibody of (A6), which is a Fab antibody, F(ab') ₂ antibody, Fab' antibody, Fv antibody, or scFv antibody.

(A8) Antibody according to any one of paragraphs. (A1)-(A7), capable of inducing immune tolerance.

(A9) An antibody according to any one of paragraphs. (A1) to (A8), wherein the cell expressing CD80 and/or CD86 is an antigen presenting cell and the other cell expressing CD28 is a T cell.

(A10) Antibody according to any one of paragraphs. (A1)-(A9), which is an anti-CD80 antagonist antibody, an anti-CD86 antagonist antibody, an anti-CD28 antagonist antibody, or a bispecific anti-CD80 and CD86 antagonist antibody.

(A11) Antibody according to any one of paragraphs. (A1)-(A10), which is a chimeric antibody, a humanized antibody or a human antibody.

(A12) Antibody according to any one of paragraphs. (A1)-(A11) containing the Fc fragment of an IgG2 or IgG4 human antibody.

(A13) Antibody according to any one of paragraphs. (A1)-(A12) containing the Fc fragment of a human IgG4 antibody.

(A14) Antibody according to any one of paragraphs. (A1)-(A13), containing:

(a) VH containing CDRH1 with the amino acid sequence given in SEQ ID NO: 25, CDRH2 with the amino acid sequence given in SEQ ID NO: 26, and CDRH3 with the amino acid sequence given in SEQ ID NO: 27, and VL containing CDRL1 having the amino acid sequence shown in SEQ ID NO: 28, CDRL2 having the amino acid sequence shown in SEQ ID NO: 29, and CDRL3 given in SEQ ID NO: 30; or

(b) VH containing CDRH1 with the amino acid sequence given in SEQ ID NO: 31, CDRH2 with the amino acid sequence given in SEQ ID NO: 32, and CDRH3 with the amino acid sequence given in SEQ ID NO: 33, and VL containing CDRL1 having the amino acid sequence set forth in SEQ ID NO: 34, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 35, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 36.

(A15) Antibody according to claim (A14), containing:

(a) VH having the amino acid sequence shown in SEQ ID NO: 18, or a variant sequence thereof, and VL having the amino acid sequence shown in SEQ ID NO: 20, or a variant sequence thereof; or

(b) VH having the amino acid sequence shown in SEQ ID NO: 22, or a variant sequence thereof, and VL having the amino acid sequence shown in SEQ ID NO: 24, or a variant sequence thereof.

(A16) A nucleic acid molecule encoding the amino acid sequence or part thereof of an antibody according to any one of claims. (A1)-(A15).

(A17) A nucleic acid molecule according to claim (A16), containing:

(a) a polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 17 and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 19; or

(b) a polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 21, and a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 23.

(A18) A vector containing a nucleic acid molecule according to item (A16) or item (A17).

(A19) A cell containing a nucleic acid molecule according to item (A16) or item (A17) or a vector according to item (A18).

(A20) A method for producing an antibody according to any one of claims. (A1)-(A15), comprising culturing the cell according to item (A19).

(A21) A cell with immune tolerance induced by an antibody according to any one of claims. (A1)-(A15).

(A22) A cell according to (A21), the immune tolerance of which is induced by mixing an antibody, a cell derived from an individual, and an antigen that is not derived from the individual, or material containing that antigen.

(A23) A composition for producing a cell with induced immune tolerance, containing at least one of the antibodies according to any one of paragraphs. (A1)-(A15).

(A24) The composition of claim (A23) comprising an anti-CD80 antagonist antibody, an anti-CD86 antagonist antibody, an anti-CD28 antagonist antibody, or a bispecific anti-CD80 and CD86 antagonist antibody, or any combination thereof.

(A25) A method of producing a cell for treating or preventing a disease, disorder or condition caused by an antigen foreign to an individual, comprising mixing an antibody according to any one of claims. (A1)-(A15), a cell derived from an individual, and an antigen that does not originate from the individual, or material containing that antigen.

(A26) The method of claim (A25), wherein the disease, disorder or condition is selected from the group consisting of graft rejection, allergy, autoimmune disease, graft-versus-host disease, and immune rejection caused by iPS cell or ES cell transplantation and cells , tissue or organ derived from said cells.

(A27) The method of item (A25) or item (A26), wherein the antigen-containing material is a cell.

(A28) A cell obtained by the method according to any one of claims. (A25)-(A27).

(A29) A cell according to (A28) containing a T cell.

(A30) A cell therapy agent comprising a cell with immune tolerance induced by an antibody according to any one of claims. (A1)-(A15), a cell produced by the method according to paragraph (A25) or paragraph (A26), or a cell according to paragraphs. (A21), (A22), (A28) or (A29).

(A31) A composition for the treatment or prevention of a disease, disorder or condition caused by an antigen foreign to an individual, comprising a cell with immune tolerance induced by an antibody according to any one of claims. (A1)-(A15), a cell produced by the method according to paragraph (A25) or paragraph (A26), or a cell according to paragraphs. (A21), (A22), (A28) or (A29).

(A32) The composition of claim (A31), wherein the disease, disorder or condition is selected from the group consisting of graft rejection, allergy, autoimmune disease, graft-versus-host disease, and immune rejection caused by iPS cell or ES cell transplantation and cells , tissue or organ derived from said cells.

(A33) The composition according to claim (A32), wherein the rejection is characterized in that the transplanted organ is a kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood, blood cell , including an immune system cell, bone, cartilage, blood vessel, cornea, eyeball, or bone marrow.

(A34) A method of treating or preventing a disease, disorder or condition caused by an antigen that is not originating from an individual in said individual, comprising administering to the individual an effective amount of an antibody-induced immune tolerance cell according to any one of claims. (A1)-(A15), cells produced by the method according to paragraph (A23) or paragraph (A24), or cells according to paragraphs. (A21), (A22), (A28) or (A29).

(A35) The method of claim (A34), wherein the disease, disorder or condition is selected from the group consisting of graft rejection, allergy, autoimmune disease, graft-versus-host disease, and immune rejection caused by iPS cell or ES cell transplantation and cells , tissue or organ derived from said cells.

(A36) The method according to claim (A35), wherein the rejection is characterized in that the transplanted organ is a kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerve, blood, cell blood, including an immune system cell, bone, cartilage, blood vessel, cornea, eyeball, or bone marrow.

[0010]

Additionally, the following is provided.

(B1) An antibody having a human Fc region, where the antibody can inhibit the interaction between CD80 or CD86 expressed on a cell surface and CD28 expressed on another cell surface, and does not promote the production of cytokines by human PBMC.

(B2) The antibody of (B1) which can inhibit the interaction between CD80 or CD86 expressed on the surface of antigen presenting cells and CD28 expressed on the surface of T cells.

(B3) An antibody according to item (B1) or item (B2), which can induce immune tolerance to an antigen by contacting the antibody with a PBMC together with the antigen or a cell bearing the antigen on its surface.

(B4) Antibody according to any one of paragraphs. (B1)-(B3), where the cytokine is IFNγ.

(B5) Antibody to any one of paragraphs. (B1)-(B4), which is an anti-CD80 antibody or an anti-CD86 antibody.

(B6) Antibody according to any one of paragraphs. (B1)-(B5), which is a chimeric antibody, a humanized antibody or a human antibody.

(B7) Antibody according to any one of paragraphs. (B1)-(B6), which is a monoclonal antibody.

(B8) Antibody according to any one of paragraphs. (B1)-(B7), which belongs to the IgG2 or IgG4 subclass.

(B8A) Antibody according to any one of paragraphs. (B1)-(B7), which belongs to the IgG1 subclass.

(B8B) The antibody of claim (B8A) having a heavy chain with the amino acid sequence shown in SEQ ID NO. selected from SEQ ID NO: 38 and 42.

(B8C) An antibody according to claim (B8B) having:

a heavy chain having the amino acid sequence set forth in SEQ ID NO: 38, and a light chain having the amino acid sequence set forth in SEQ ID NO: 40, or

a heavy chain having the amino acid sequence set forth in SEQ ID NO: 42; and a light chain having the amino acid sequence set forth in SEQ ID NO: 44.

(B8D) Antibody according to any one of paragraphs. (B1)-(B7) and (B8A)-(B8C) having a CDRH3 with the amino acid sequence shown in SEQ ID NO: 55 or 61.

(B8E) Antibody (B8D), which is:

(Bi) an antibody having a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 53, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 54, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 55, or

(Bii) an antibody having a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 59, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 60, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 61.

(B8F) Antibody (Bi) according to claim (B8E) having CDRL1 having the amino acid sequence given in SEQ ID NO: 56, CDRL2 having the amino acid sequence given in SEQ ID NO: 57, and CDRL3 having the amino acid sequence given in SEQ ID NO: 58, or

antibody (Bii) according to claim (B8E), having CDRL1 with the amino acid sequence given in SEQ ID NO: 62, CDRL2 with the amino acid sequence given in SEQ ID NO: 63, and CDRL3 with the amino acid sequence given in SEQ ID NO: 64.

(B8G) The antibody of claim (B8D) having a heavy chain variable region (VH) with the amino acid sequence set forth in SEQ ID NO: 46 or 50.

(B8H) An antibody according to claim (B8G) having a VH with the amino acid sequence set forth in SEQ ID NO: 46 and a light chain variable region (VL) with the amino acid sequence set forth in SEQ ID NO: 48, or having a VH with the amino acid the sequence given in SEQ ID NO: 50, and VL with the amino acid sequence given in SEQ ID NO: 52.

(B8I) An antibody according to claim (B8G) having a heavy chain with the amino acid sequence shown in SEQ ID NO. selected from SEQ ID NO: 38 and 42.

(B8J) An antibody according to claim (B8I) having:

a heavy chain having the amino acid sequence set forth in SEQ ID NO: 38, and a light chain having the amino acid sequence set forth in SEQ ID NO: 40, or

a heavy chain having the amino acid sequence set forth in SEQ ID NO: 42; and a light chain having the amino acid sequence set forth in SEQ ID NO: 44.

(B9) An antibody according to any one of paragraphs. (B1)-(B8) having a CDRH3 with the amino acid sequence set forth in SEQ ID NO 27 or 33.

(B10) Antibody (B9), which is:

(Bi) an antibody having a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 25, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 26, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 27, or

(Bii) an antibody having a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 31, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 32, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 33.

(B11) Antibody (Bi) according to claim (B10), having CDRL1 having the amino acid sequence given in SEQ ID NO: 28, CDRL2 having the amino acid sequence given in SEQ ID NO: 29, and CDRL3 having the amino acid sequence given in SEQ ID NO: 30, or

antibody (Bii) according to claim (B10), having CDRL1 with the amino acid sequence given in SEQ ID NO: 34, CDRL2 with the amino acid sequence given in SEQ ID NO: 35, and CDRL3 with the amino acid sequence given in SEQ ID NO: 36.

(B12) The antibody of (B9) having a heavy chain variable region (VH) with the amino acid sequence set forth in SEQ ID NO: 18 or 22.

(B13) The antibody of claim (B12) having a VH with the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region (VL) with the amino acid sequence set forth in SEQ ID NO: 20, or having a VH with the amino acid the sequence shown in SEQ ID NO: 22, and VL with the amino acid sequence shown in SEQ ID NO: 24.

(B14) An antibody according to claim (B12) having a heavy chain with the amino acid sequence shown in SEQ ID NO. selected from SEQ ID NOs: 2, 6, 10 and 14.

(B15) An antibody according to claim (B14) having:

a heavy chain having the amino acid sequence shown in SEQ ID NO: 2, and a light chain having the amino acid sequence shown in SEQ ID NO: 4;

a heavy chain having the amino acid sequence shown in SEQ ID NO: 6, and a light chain having the amino acid sequence shown in SEQ ID NO: 8;

a heavy chain having the amino acid sequence shown in SEQ ID NO: 10, and a light chain having the amino acid sequence shown in SEQ ID NO: 12; or

a heavy chain having the amino acid sequence given in SEQ ID NO: 14, and a light chain having the amino acid sequence given in SEQ ID NO: 16.

(B16) A medical composition containing an antibody according to any one of paragraphs. (B1)-(B15).

(B17) The medical composition according to claim (B16), containing an anti-CD80 antibody and an anti-CD86 antibody.

(B18) Medical composition according to item (B16) or item (B17), used for the induction of immune tolerance.

(B19) The medical composition according to claim (B18), used for inducing immune tolerance in a cell obtained from a patient receiving an organ transplant.

(B20) The medical composition according to claim (B19) or claim (B20), wherein the organ to be transplanted is a heart, a kidney or a liver.

(B21) A medicinal composition according to claim (B18), used for inducing immune tolerance in a patient with an autoimmune disease or an allergic disease.

(B22) A nucleic acid molecule encoding the amino acid sequence of an antibody according to any one of claims. (B12)-(B15).

(B23) The nucleic acid molecule of claim (B22), comprising a polynucleotide encoding VH with a nucleotide sequence selected from: SEQ ID NOs: 1, 5, 9 and 13, and a polynucleotide encoding VL with a nucleotide sequence selected from : SEQ ID NO: 3, 7, 11 and 15.

(B24) A vector containing a nucleic acid molecule according to item (B22) or item (B23).

(B25) A host cell having the vector of (B24).

(B26) A method for producing an antibody according to any one of claims. (B1) to (B15), comprising culturing a host cell according to claim (B25).

(B27) A method for inducing immune tolerance to a PBMC antigen, comprising contacting an antibody according to any one of claims. (B1)-(B15), with the antigen to which immune tolerance is to be induced, or a cell bearing the antigen on its surface, and a PBMC.

(B28) The induction method according to (B27), which is carried out ex vivo .

(B29) The method of claim (B27) or claim (B28), wherein the PBMC is a cell obtained from a patient receiving an organ transplant.

(B30) The method of claim (B27) or claim (B28), wherein the PBMC is a cell obtained from a patient with an autoimmune disease or an allergic disease.

(B31) PBMC with immune tolerance induced by the method according to any one of claims. (B27)-(B30).

(B32) A cell therapy agent containing PBMC according to (B30) as an active ingredient.

(B33) Cell therapy agent according to claim (B32) for suppressing rejection in organ transplantation.

(B34) The cell therapy agent of claim (B33), wherein the transplanted organ is a kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, colon, nerve, blood, blood cell, including a cell immune system, bone, cartilage, blood vessel, cornea, eyeball or bone marrow.

(B35) A cell therapy agent according to claim (B32) for treating an autoimmune disease or an allergic disease.

(B36) A method for suppressing rejection in organ transplantation, comprising administering an antibody according to any one of claims. (B1)-(B15) to a patient receiving an organ transplant.

(B37) The method of claim (B36), wherein the transplanted organ is a kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, colon, nerve, blood, blood cell, including an immune system cell , bone, cartilage, blood vessel, cornea, eyeball or bone marrow.

(B37) A method of treating an autoimmune disease or an allergic disease, comprising administering an antibody according to any one of claims. (B1)-(B15) to a patient with an autoimmune disease or allergic disease.

(B38) A method for suppressing rejection in organ transplantation, comprising administering a cell therapy agent according to any one of claims. (B32)-(B34) to a patient receiving an organ transplant.

(B39) A method of treating an autoimmune disease or allergic disease, comprising administering a cell therapy agent according to claim (B32) or claim (B35) to a patient with an autoimmune disease or allergic disease.

(B40) The method according to any one of paragraphs. (B27)-(B30), further comprising administering the induced immune tolerance PBMC to the patient.

(B41) The method of claim (B40), comprising administering to a patient receiving an organ transplant a PBMC obtained from a patient with induced immune tolerance to the organ to be transplanted.

(B42) The method of claim (B40), comprising administering to a patient with an autoimmune disease or allergic disease a PBMC obtained from a patient with immune tolerance to an antigen that is the cause of the autoimmune disease or induced allergic disease.

[0011]

The present description should be considered as disclosing one or more of the features described above not only in the form of combinations explicitly disclosed, but also in the form of other combinations of these features. Additional embodiments and advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description, as appropriate.

[Advantage of the present invention]

[0012]

The antibody of the present invention inhibits the production of IFNγ while retaining the ability to induce immune tolerance. In particular, the use of antibodies of the IgG2 or IgG4 subclass of the present invention allows one to obtain a cell with induced immune tolerance that does not produce IFNγ.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]

[Fig. 1] In FIG. Figure 1 shows the results of the reaction of a mixed culture of lymphocytes (incorporation of ³ H-thymidine) in the presence of antibodies to human CD80 and anti-CD86 antibodies (IgG1). ; no donor added, no antibody added, Allo; donor added, no antibody added. The concentrations of each added antibody are 10, 1 and 0.1 (µg/ml). The vertical axis represents the amount of ³ H-thymidine incorporation based on the absence of added antibodies (Allo).

[Fig. 2] In FIG. Figure 2 shows the results of the reaction of a mixed culture of lymphocytes (production of IFNγ) in the presence of antibodies to human CD80 and anti-CD86 antibodies (IgG1). The vertical axis shows the concentration in the medium (pg/ml).

[Fig. 3] In FIG. Figure 3 shows the results of the reaction of a mixed culture of lymphocytes (incorporation of ³ H-thymidine) in the presence of antibodies to human CD80 and anti-CD86 antibodies (IgG1). The vertical axis represents the amount of ³ H-thymidine incorporation based on the absence of added antibodies (Allo).

[Fig. 4] In FIG. Figure 4 shows the results of the reaction of a mixed culture of lymphocytes (production of IFNγ) in the presence of antibodies to human CD80 and anti-CD86 antibodies (IgG1). Top row: with donor stimulation, bottom row: without donor stimulation. * represents 50,000 pg/ml or more. The vertical axis shows the concentration in the medium (pg/ml).

[Fig. 5] In FIG. Figure 5 shows the results of a mixed reaction of lymphocytes (inclusion of ³ H-thymidine) in the presence of anti-CD80 and anti-CD86 antibodies (IgG2, IgG4). The vertical axis represents the amount of ³ H-thymidine incorporation based on the absence of added antibodies (Allo). The concentrations of each added antibody are 10, 3 and 1 (µg/ml).

[Fig. 6] In FIG. Figure 6 shows the results of the reaction of a mixed culture of lymphocytes (production of IFNγ) in the presence of anti-CD80 and anti-CD86 antibodies (IgG1, IgG2, IgG4). The vertical axis shows the concentration in the medium (pg/ml).

[Fig. 7] In FIG. Figure 7 shows the results of the reaction of a mixed culture of lymphocytes (incorporation of ³ H-thymidine) with the addition of cells obtained in the presence of anti-CD80 and anti-CD86 antibodies (IgG1, IgG2, IgG4). The vertical axis represents the amount of ³ H-thymidine incorporation based on the absence of added antibodies (Allo). 1/2, 1/4 and 1/8 represent the ratios of a mixture of cells obtained in the presence of anti-CD80 and anti-CD86 antibodies (IgG1, IgG2, IgG4) to naïve responder cells.

[Fig. 8] In FIG. 8 shows the results of a mixed lymphocyte culture reaction (IFNγ production) in the presence of chimeric anti-CD80 antibodies and chimeric anti-CD86 antibodies. The vertical axis shows the concentration in the medium (pg/ml).

[Fig. 9] In FIG. 9 shows the results of a mixed culture reaction of lymphocytes (incorporation of ³ H-thymidine) with the addition of a cell obtained in the presence of chimeric anti-CD80 and chimeric anti-CD86 antibodies. The vertical axis represents the amount of ³ H-thymidine incorporation based on the absence of added antibodies (Allo). 1/2, 1/4, 1/8 and 1/16 represent the ratios of the mixture of cells obtained in the presence of chimeric anti-CD80 and chimeric anti-CD86 antibodies to naïve responder cells.

DESCRIPTION OF IMPLEMENTATION OPTIONS

[0014]

Terms used in this specification are to be understood in the meaning in which they are commonly used in the art unless specifically stated otherwise. Therefore, unless otherwise indicated, all terms and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention relates. In the event of any conflict, this specification (including definitions) shall control.

[0015]

Definition of terms

As used herein, the term “about” refers to a range of ±10% of the next numerical value.

[0016]

As used herein, “immune tolerance” refers to a condition in which a specific immune response to a particular antigen is not demonstrated or the specific immune response is suppressed. Immune tolerance can also refer to either or both of: a state in which an immune cell (particularly a T cell) does not exhibit a specific immune response to a particular antigen or the specific immune response is suppressed, and a state in which a person does not exhibit a specific immune response to a particular antigen. the immune response to a particular antigen or specific immune response is suppressed. Immune tolerance has attracted attention because the creation of immune tolerance allows the treatment of immune rejection or allergies. As used herein, the term “anergy” refers to a condition in which costimulation does not occur upon presentation of an antigen by an antigen-presenting cell, resulting in the cell being unable to respond to stimulation under costimulation conditions at a subsequent time. As used herein, the term “immune tolerance-induced PBMC (or T cell)” is synonymous with the term “anergic PBMC (or T cell).”

[0017]

As used herein, “individual” includes domestic animals (eg, cows, sheep, cats, dogs and horses), primates (eg, humans and non-human primates such as monkeys), rabbits and rodents (eg, mice and rats). In a specific embodiment, the individual is a human.

[0018]

As used herein, “immunostimulation” refers to stimulation applied to cells of the immune system (eg, macrophages, neutrophils, natural killer (NK) cells, T cells, B cells, etc.) or proliferation of cells in the immune system. The production of cytokines (eg, INF) or cytotoxic activity is induced by stimulation of cells of the immune system.

[0019]

As used herein, the term “substantially does not induce cytokine production by immunostimulation” refers to the induction of cytokine production that is approximately 20% or less of the amount of cytokine produced by cells (e.g., macrophages, neutrophils, natural killer (NK) cells, T cells , B cells, etc.) obtained from an individual in the presence of an antigen and in the absence of an antibody of the present invention.

[0020]

As used herein, the term “antibody” broadly refers to a molecule or group of molecules that can specifically bind to a specific epitope on an antigen. As used herein, an “antibody” may be a full-length antibody (ie, an antibody with an Fc moiety) or an antibody that does not contain an Fc moiety. An antibody lacking the Fc moiety should only be able to bind to the antigen of interest. Examples of such an antibody include, but are not limited to, Fab antibodies, F(ab') ₂ antibodies, Fab' antibodies, Fv antibodies and scFv antibodies, and the like. Antibodies also include modified and unmodified antibodies. For modified antibodies, the antibody can be linked to various molecules such as polyethylene glycol. The modified antibody can be obtained by chemically modifying the antibody using a known method.

[0021]

As used herein, the term “antibody” in the narrow sense refers to an immunoglobulin or group thereof that can specifically bind to a specific epitope of an antigen. Its variant form is called a “variant antibody.” As used herein, the term “antibody”, in the narrow sense, can be a full-length antibody (ie, an antibody with an Fc fragment). As used herein, an “antibody variant” may be a variant that lacks the Fc portion of the antibody described above. Therefore, as used herein, an antibody in the narrow sense may also be referred to as a full-length antibody, and a variant antibody may also be referred to as a variant of a full-length antibody. The variant lacking the Fc fragment should only be able to bind to the antigen of interest. Examples of such an option include, but are not limited to, Fab antibodies, F(ab') ₂ antibodies, Fab' antibodies, Fv antibodies and scFv antibodies, and the like. Antibody variants also include modified antibodies and unmodified antibodies. For modified antibodies, the antibody can be linked to various molecules such as polyethylene glycol. The modified antibody can be obtained by chemically modifying the antibody using a known method.

[0022]

In one embodiment, a "polyclonal antibody" may be produced, for example, by administering an immunogen containing the antigen of interest to a mammal (eg, rat, mouse, rabbit, cow, monkey, or the like), birds, or the like. . to induce the production of a polyclonal antibody specific for the antigen. The immunogen can be administered using one or more immunological agents and adjuvant infusion, if necessary. The adjuvant may be used to enhance immune responses and may include Freund's adjuvant (complete or incomplete), a mineral gel (aluminum hydroxide or the like), a surfactant (lysolecithin or the like), or the like. The immunization protocol is known in the art and can be performed by any method that induces an immune response in accordance with the selected host organism (Tanpakushitsu Jikken Handobukku [Protein experiment handbook], Yodosha (2003): 86-91).

[0023]

In one embodiment of the present invention, a “monoclonal antibody” includes individual antibodies constituting a population that are identical antibodies corresponding to substantially the same epitope, except antibodies containing a mutation that may occur naturally in small quantities. In addition, the individual antibodies that make up a population may be antibodies that are essentially the same, except for antibodies that have a mutation that may occur naturally in small quantities. Monoclonal antibodies are highly specific and differ from conventional polyclonal antibodies, which typically include different antibodies corresponding to different epitopes and/or different antibodies corresponding to the same epitope. In addition to specificity, monoclonal antibodies are useful in that they can be synthesized from a hybridoma culture that is not contaminated with other immunoglobulins. The description “monoclonal” may indicate that the antibody is derived from a substantially homogeneous population of antibodies. However, such a description does not mean that the antibodies must be produced by a particular method. For example, monoclonal antibodies can be produced by a method similar to the hybridoma method described in “Kohler G, Milstein C., Nature. 1975 Aug 7; 256(5517): 495-497.” Alternatively, monoclonal antibodies can be produced by a method similar to the recombinant method described in US patent No. 4816567. Monoclonal antibodies can also be isolated from a phage antibody library using a method similar to that described in Clackson et al., Nature. 1991 Aug 15; 352(6336): 624-628" or "Marks et al., J Mol Biol. 1991 Dec 5; 222(3): 581-597.” Monoclonal antibodies can also be produced by the method described in "Tanpakushitsu Jikken Handobukku [Protein experiment handbook], Yodosha (2003): 92-96".

[0024]

In one embodiment of the present invention, a "chimeric antibody" is, for example, an antibody variable region linked to an antibody constant region between xenogeneic organisms, and can be constructed using genetic engineering techniques. A mouse-human chimeric antibody can be produced, for example, by the method described in "Roguska et al., Proc Natl Acad Sci USA. 1994 Feb 1; 91(3): 969-973". For example, in a basic method for producing a mouse-human chimeric antibody, a mouse leader and variable region sequence in a cloned cDNA is linked to a sequence encoding a human antibody constant region already present in a mammalian cell expression vector. After linking the murine leader and variable region sequences in the cloned cDNA to the human antibody constant region coding sequence, the resulting sequence can be linked to a mammalian cell expression vector. A human antibody constant region fragment may be derived from any human antibody H chain constant region and a human antibody L chain constant region. Examples of a human H chain fragment include Cγ1, Cγ2, Cγ3 or Cγ4, and examples of an L chain fragment include Cλ or Cκ.

[0025]

In one embodiment, a “humanized antibody” is, for example, an antibody that has one or more CDRs derived from a non-human species, a framework region (FR) derived from a human immunoglobulin, and a constant region derived from a human immunoglobulin, wherein said antibody binds to the desired antigen. Antibodies can be humanized using various approaches known in the art (Almagro et al., Front Biosci. 2008 Jan 1; 13: 1619-1633). Examples include CDR grafting (Ozaki et al., Blood. 1999 Jun 1; 93(11): 3922-3930), recoating (Roguska et al., Proc Natl Acad Sci US A. 1994 Feb 1; 91(3): 969-973.), FR shuffling (Damschroder et al., Mol Immunol. 2007 Apr; 44(11): 3049-3060. Epub 2007 Jan 22.), etc. The amino acid residue of the human FR region can be replaced by the corresponding residue from the CDR donor antibody to alter (preferably improve) antigen binding. The FR replacement can be accomplished in a manner well known in the art (Riechmann et al., Nature. 1988 Mar 24; 332 (6162): 323-327). For example, a FR residue that is important for antigen binding can be identified by modeling the interaction between the CDR and the FR residue. In addition, an abnormal FR residue at a specific position can be identified by sequence comparison.

[0026]

In one embodiment, a “human antibody” is, for example, an antibody in which the region comprising the heavy chain variable region and constant region and the light chain variable region and constant region constituting the antibody is derived from a gene encoding a human immunoglobulin. Examples of basic production methods include a method in which transgenic mice are used to produce human antibodies, a phage display method, and the like. A method in which transgenic mice are used to produce human antibodies produces instead of mouse antibodies human antibodies that differ in antigen-binding ability if a functional human Ig gene is introduced into a mouse with a knockout of endogenous Ig. In addition, this mouse can be immunized to produce human monoclonal antibodies using the conventional hybridoma method. They can be obtained, for example, by the method described in Lonberg et al., Int Rev Immunol. 1995; 13(1): 65-93.” Phage display is a system that typically expresses an exogenous gene as a fusion protein without losing phage infectivity at the N-terminus of the coat protein (g3p, g10p, etc.) of a filament phage such as M13 or T7 E coli virus . Antibodies can be prepared, for example, by the method described in Vaughan et al., Nat Biotechnol. Mar 1996; 14(3): 309-314.”

[0027]

As used herein, a "variant sequence", in the case of an amino acid sequence, refers to a sequence with a change (substitution, addition or deletion) of at least one amino acid residue relative to the original sequence and, in the case of a nucleic acid sequence, refers to a change (substitution, addition or deletion) of at least one base that will not result in a frameshift. The variant sequence has a function similar to the original sequence, which is, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the original sequence. If the parent sequence is an antibody, the variant sequence preferably has no changes within the CDR, but may have one or more changes, preferably 3 or fewer changes, more preferably 2 or fewer changes, and most preferably one change, as long as binding capacity or function is retained original antibody. When a change is introduced into the CDR, those skilled in the art can select an appropriate change that preserves the binding ability or function of the original antibody.

[0028]

As used herein, the term “cell obtained from an individual” refers to a cell obtained from an individual to whom a composition of the present invention is administered, or a cell obtained from a cell which is obtained from an individual. As used herein, “subject-derived antigen” refers to an antigen produced in the individual's own body that induces an immune response, such as an antigen produced in the individual's body that causes an autoimmune disease in the individual with the autoimmune disease. In the context of the present invention, the term “non-subject antigen” refers to an exogenous antigen that can induce an immune response. As used herein, the term “antigen-containing material that is not derived from an individual” refers to any substance or set of substances containing an antigen that is not derived from an individual. Examples of this include a cell, population of cells, tissue, etc. expressing an antigen not derived from an individual.

[0029]

As used herein, the term “graft rejection” refers to an individual's immune system attacking, damaging, or destroying a transplanted organ, tissue, or cell in an individual receiving a transplanted organ, tissue, or cell.

[0030]

As used herein, the term “allergy” refers to a hyperactive immune response to an antigen not originating from the individual. An antigen that does not originate from an individual that causes an allergy is also called an allergen. Examples thereof include, but are not limited to, tick antigen, egg white antigen, milk antigen, wheat antigen, peanut antigen, soy antigen, buckwheat antigen, sesame antigen, rice antigen, crustacean antigen, kiwi antigen, apple antigen, banana antigen, peach antigen, tomato antigen, tuna antigen, salmon antigen, mackerel antigen, beef antigen, chicken antigen, pork antigen, cat dander antigen, insect antigen, pollen antigen, dog dander antigen, fungal antigen, bacterial antigen, latex, hapten, metal, etc. P.

[0031]

As used herein, the term "autoimmune disease" refers to any disease in which the immune system produces an unwanted immune response against its own cell, tissue, or organ. Examples of an autoimmune disease include, but are not limited to, rheumatoid arthritis, multiple sclerosis, type 1 diabetes, inflammatory bowel disease (eg, Crohn's disease or ulcerative colitis), systemic lupus erythematosus, psoriasis, scleroderma, autoimmune thyroid disease, alopecia areata, Graves' disease, Guillain-Barré syndrome, celiac disease, Sjögren's syndrome, rheumatic fever, gastritis, autoimmune atrophic gastritis, autoimmune hepatitis, pancreatitis, otitis media, orchitis, uveitis, lens-induced uveitis, myasthenia gravis, primary hemolytic myxedema, pernicious anemia, autoimmune hemolytic anemia, Addison's disease , scleroderma, Goodpasture's syndrome, nephritis (eg, glomerulonephritis), psoriasis, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, Wegener's granulomatosis, granuloma and polymyositis/dermatomyulositis.

[0032]

As used herein, the term graft-versus-host refers to a transplanted organ, tissue, or cell that attacks, damages, or destroys, through an immune response, the cell, tissue, or organ of the individual receiving the transplant.

[0033]

As used herein, the term “immune rejection caused by transplantation of an iPS cell or ES cell, or a cell, tissue, or organ derived from said cells” refers to immune rejection caused by an iPS cell or ES cell antigen, or a cell antigen, tissue or organ derived from an iPS cell or ES cell.

[0034]

(Preferred options)

Preferred embodiments are described below. It is understood that the embodiments are examples of the present invention, therefore, the scope of the present invention is not limited to such preferred embodiments. It will be understood that those skilled in the art may refer to the following preferred embodiments to make modifications or changes within the scope of the present invention. Any of these embodiments can be combined by those skilled in the art as appropriate.

[0035]

By attempting to induce a non-immune response (anergy) with an antibody having a human Fc region that inhibits the interaction between CD80 and/or CD86 and CD28, the inventors have discovered that such attempts result in activation of a cell that is a component of the immune system, such as macrophage, neutrophil or natural killer (NK) cell, and cause the release of humoral factors such as interleukin or interferon (IFN), which are responsible for immune responses. The inventors also discovered that the induction of a non-immune response (anergy) by an antibody having a human Fc region is associated with the problem of weakening the effect of immune tolerance, since the release of these humoral factors causes unwanted activation of the immune system, which is the opposite of immune tolerance, and it was found that the effect of immune tolerance can be improved by selecting a human Fc region.

[0036]

Specifically, a chimeric antibody was produced that inhibits the interaction between CD80 and/or CD86 and CD28 and induces immune tolerance by immunizing Balb/c mice with a recombinant/human CD80/CD86-Fc fusion protein, followed by isolating mRNA from mouse spleens and cDNA synthesis. , selecting a high-affinity CD80/CD86 Fab antibody identified using a phage display library generated using an RT-PCR-amplified gene, followed by genetically engineered introduction of the human Fc region.

[0037]

To minimize the likelihood of rejection of the antibody used to induce immune tolerance, the chimeric antibody was humanized using a CDR grafting method developed by Xoma to produce a humanized antibody (subclass; IgG1). The resulting humanized anti-CD80 antibodies and anti-CD86 antibodies (subclass; IgG1) were added to a mixed culture reaction of lymphocytes with PBMCs (peripheral blood mononuclear cells) obtained from two volunteers, as donor or recipient, to evaluate the suppressor function when induced cells were stimulated the same antigen. Specifically, PBMCs from one volunteer were collected, irradiated with 30 Gy of radiation, and cocultured for 7 days with PBMCs obtained from another volunteer in the presence of a humanized anti-CD80/CD86 antibody to induce an anergic cell specific for the donor antigen. Induced cells were added in a certain ratio to a culture of mixed lymphocytes obtained from the same recipient and donor, and the ability to suppress cell division and proliferation by antigen stimulation was measured by the level of tritium-labeled thymidine incorporation. As a result, it was confirmed that humanized anti-CD80 antibodies and anti-CD86 antibodies significantly suppressed thymidine incorporation, and cells induced by such antibodies regained their suppressor function.

[0038]

However, when the amount of IFNγ in the supernatant was measured after 7 days of culture, IFNγ was produced upon addition of antibody, even without antigen stimulation. It was also found that IFNγ, normally produced upon alloantigen stimulation by the addition of radiation-irradiated PBMCs, was present in essentially the same amount in the supernatant after the addition of antibody.

[0039]

In this regard, as a result of extensive research aimed at obtaining an antibody that does not induce IFNγ production but still retains the ability to induce immune tolerance, the inventors have discovered that immune tolerance inducing cells without IFNγ production can be obtained using humanized anti-CD80/CD86 antibodies of the IgG2 or IgG4 subclass.

[0040]

(Antibodies)

In one embodiment, the present invention provides an antibody that inhibits the interaction between CD80 and/or CD86 expressed on the surface of a cell and CD28 expressed on the surface of another cell, wherein the antibody does not substantially induce cytokine production by immunostimulation. The antibody of the present invention may be an anti-CD80 antibody and/or an anti-CD86 antibody, a bispecific antibody to CD80 and CD86, an anti-CD28 antibody, or a mixture thereof. In some embodiments, the Fc fragment of an antibody may be a fragment that does not substantially induce cytokine production by immunostimulation. In a specific embodiment, the antibody of the present invention is of the IgG2 or IgG4 subclass. Surprisingly, it has been found that the antibody of the present invention, which belongs to the IgG2 or IgG4 subclass, does not induce the production of cytokines by a cell of the immune system. Since the antibody used in the present invention may cause an undesirable effect by activating the receptor, an antagonistic antibody is preferred.

[0041]

In some embodiments, an antibody that does not substantially induce cytokine production through immunostimulation may induce cytokine production of about 20% or less, preferably about 10% or less, more preferably about 5% or less, and most preferably about 0% of the amount cytokines produced by a cell derived from an individual (eg, macrophage, neutrophil, natural killer (NK) cell, T cell, B cell, etc.) in the absence of an antibody of the present invention.

[0042]

As used herein, the term "antagonistic (antibody)" is synonymous with antagonist (antibody), inhibit (antibody), block (antibody), or the like, which (represents an antibody in the case of antibodies) has the function of inhibiting a target function or signaling function with the purpose of weakening or inactivating the action of a living organism.

[0043]

It has been surprisingly discovered that the induction of cytokine production can be suppressed by changing the antibody subclass from IgG1 to IgG2 or IgG4, as described above. Without wishing to be bound by theory, the induction of cytokine production is mediated by the Fc portion of the IgG1 antibody, since the function mediated by the Fc portion varies among IgG subclasses. Thus, in another embodiment, the antibody of the present invention may be an antibody lacking an Fc moiety. Examples of such an antibody include, but are not limited to, a Fab antibody, an F(ab') ₂ antibody, a Fab' antibody, an Fv antibody, a scFv antibody, and the like.

[0044]

In some embodiments, the cell expressing CD80 and/or CD86 may be an antigen presenting cell, and the other cell expressing CD28 may be a T cell.

[0045]

In another aspect of the present invention, an antibody of the invention can inhibit the interaction between CD80 and/or CD86 expressed on the surface of a cell and CD28 expressed on the surface of another cell. The ability to inhibit the interaction between CD80 and/or CD86 expressed on the surface of one cell with CD28 expressed on the surface of another cell can be determined, for example, using coimmunoprecipitation. Specifically, these proteins are bound and then immunoprecipitated using an antibody that binds to one of the proteins (bait) and the other immunoprecipitated protein (prey) is measured. If the prey contained in the immunoprecipitation reaction is reduced with the addition of a candidate antibody compared to a control without the candidate antibody, the candidate antibody is determined to inhibit protein-protein binding. The prey contained in the immunoprecipitation reaction can be measured by labeling it or the like as needed. The amount of prey bound to a vehicle can be compared by binding one of the proteins (bait) to the vehicle using a push-down assay and contacting the other protein (prey) in the presence/absence of a candidate antibody.

[0046]

Preferably, the antibody of the present invention can inhibit the interaction between CD80 and/or CD86 expressed on the surface of an antigen presenting cell and CD28 expressed on the surface of a T cell. The ability of a candidate antibody to inhibit the interaction between CD80 and/or CD86 expressed on the surface of an antigen presenting cell and CD28 expressed on the surface of a T cell can be determined by contacting an antigen presenting cell expressing CD80 and/or CD86 with the T cell, expressing CD28, in the presence of a candidate antibody and determining that the interaction between CD80 and/or CD86 expressed on the surface of the antigen presenting cell and CD28 expressed on the surface of the T cell is inhibited if the T cell is not activated.

[0047]

In addition, the antibody of the present invention is characterized in that it does not induce the production of cytokines by human PBMC. The ability of a candidate antibody to induce the production of cytokines by human PBMCs can be determined by contacting the candidate antibody with human PBMCs in a medium and measuring the amount of cytokines released into the medium. A candidate antibody resulting in less/more cytokines released in the presence of the candidate antibody compared to the amount of cytokines released by human PBMCs in the absence of the antibody is defined as non-stimulatory/stimulatory of cytokine production by human PBMCs. Examples of cytokines include interleukins such as IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-14, IL-15, IL-17 and IL-18 ; chemokines such as CC, CXC, CX3C and C; interferons such as IFNα, IFNβ and IFNγ; and cytotoxic agents such as TNFα. The cytokines are preferably inflammatory cytokines and more preferably IFNγ. Cytokines can be measured using a commercially available kit. For example, IFNγ can be measured using Biolegend's Human IFNγ ELISA MAX TM Deluxe.

[0048]

Preferably, the antibody of the present invention can contact a PBMC along with an antigen or a cell bearing the antigen on its surface to induce immune tolerance to the antigen. The ability of a candidate antibody to induce immune tolerance to an antigen by contacting the antibody with a PBMC along with the antigen or a cell bearing the antigen on its surface can be determined by contacting the antigen or a cell bearing the antigen on its surface with the PBMC for several to 7 days in the presence of the candidate antibody, then adding ³ H-thymidine and removing ³ H-thymidine from the culture 16-20 hours after its addition, and then measuring the level of ³ H-thymidine incorporation into PBMC. If the level of ³ H-thymidine incorporation into PBMC is low upon addition of a candidate antibody compared to a control without application of the candidate antibody, the antibody may be determined to be capable of inducing immune tolerance to the antibody by contacting the antibody to the PBMC together with the antigen or a cell bearing the antigen on its surface.

[0049]

As used herein, the term “immune tolerance” means a condition in which a specific immune response to a particular antigen is not demonstrated or the specific immune response is suppressed. Immune tolerance can mean one or both of: a state in which an immune cell (particularly a T cell) does not mount a specific immune response to a particular antigen or the specific immune response is suppressed, and a state in which a person does not mount a specific immune response to a specific antigen or specific immune response is suppressed. As used herein, the term “anergy” refers to a T cell that will no longer be able to respond even when subsequently stimulated under costimulation conditions due to the lack of costimulation upon presentation of antigen by the antigen presenting cell. As used herein, the terms “immune tolerance-induced PBMC (or T cell)” and “anergic PBMC (or T cell)” are synonymous.

[0050]

Preferably, the present disclosure relates to an anti-CD80 antibody and an anti-CD86 antibody. According to the present description, the anti-CD80 antibody and the anti-CD86 antibody specifically bind to CD80 and CD86, respectively. As used herein, the term “specific” binding of an antibody refers to the binding of an antibody to a protein of interest (CD80 or CD86) with substantially higher affinity than for other proteins or peptides. In this regard, “substantially higher affinity binding” refers to a higher affinity that allows a particular protein or peptide of interest to be clearly identified from other proteins or peptides using an appropriate measuring device or measurement method. For example, substantially higher affinity may mean about 3-fold or greater, about 4-fold or greater, about 5-fold or greater, about 6-fold or greater, about 7-fold or greater, about 8-fold or greater. about 9 times or more, about 10 times or more, about 20 times or more, about 30 times or more, about 40 times or more, about 50 times or more, or about 100 times or more affinity, according to intensity (eg, fluorescence intensity) determined by ELISA or EIA.

[0051]

Examples of the association rate constant (Ka1) for binding of an antibody of the present invention to CD80, CD86 or CD28 include about 1×10 ⁴ MS ^-1 or more, about 1×10 ⁵ MS ^-1 or more, and about 5×10 ⁵ MS ^-1 or more. Examples of the dissociation rate constant (Kd1) for binding of an antibody of the present invention to CD80, CD86 or CD28 include about 1×10 ^-3 MS ^-1 or less and about 1×10 ^-4 MS ^-1 or less. Examples of the association constant (KD) for binding of an antibody of the present invention to CD80, CD86 or CD28 include about 1x10 ^-8 (M) or less, about 5x10 ^-8 (M) or less, about 1x10 ^-9 ( M) or less and about 5×10 ^-9 (M) or less. To measure the association rate constant (Ka1), dissociation rate constant (Kd1) and association constant (KD) of the antibodies herein, BIACORE (GE Healthcare Bio-Sciences KK, BIACORE-X100) can be used according to the guidance provided by the manufacturer by immobilization biotinylated CD80, CD86 or CD28 or a cell expressing them on the SA chip, then providing a flow of test antibody to measure the Ka1 association rate constant and the Kd1 dissociation rate constant, and the KD association constant, using a two-valued function fit for the measurement.

[0052]

The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. As used herein, “monoclonal antibodies” are antibodies with a homogeneous structure that react against a single antigenic determinant. In addition, the antibody of the present invention includes an antibody having the amino acid sequence of a non-human animal antibody and the amino acid sequence of an antibody derived from a human and a human antibody. Examples of non-human animal antibodies include antibodies from mouse, rat, hamster, guinea pig, rabbit, dog, monkey, sheep, goat, camel, chicken, duck and the like, preferably antibodies from an animal that can be used to produce a hybridoma , and more preferably mouse, rat or rabbit antibodies. Examples of antibodies with the amino acid sequence of a non-human animal antibody and the amino acid sequence of an antibody derived from a human include human-type chimeric antibodies and humanized antibodies. A "chimeric antibody" as described above is a non-human animal antibody modified by genetic engineering such that the constant region of the antibody that specifically binds to the antigen of interest (CD80, CD86, or CD28) is the same constant region , like a human antibody. The chimeric antibody is preferably a human/mouse chimeric antibody (see patent application publication EP No. 0125023). A “humanized antibody” is a non-human animal antibody modified by genetic engineering such that the primary structure is different from the complementarity determining regions (CDRs) of the H chain and L chain of the antibody that specifically binds to CD80, CD86, or CD28 , is the primary structure corresponding to a human antibody. In this case, CDR can be defined according to one of the following definitions: Kabat et al. (“Sequences of Proteins of Immunological Interest”, Kabat, E. et al., U.S. Department of Health and Human Services, 1983), or Chothia et al. (Chothia & Lesk (1987) J. Mol. Biol., 196: 901-917). "Human antibody" is a human antibody that is the product of expression of a fully human antibody gene. Examples thereof include monoclonal antibodies produced using a transgenic animal into which the gene used to produce a human antibody is introduced (EP Patent Application Publication No. 0546073), and the like. When, for example, an antibody of the present invention is used for treatment, prophylaxis, or diagnosis by administering the antibody to the body, the antibody of the present invention is preferably a human/non-human animal chimeric antibody, a humanized antibody, or a human antibody.

[0053]

Preferably, the immunoglobulin class of the antibody of the present invention is IgG. The antibody subclass of the present invention is preferably IgG2 or IgG4. More preferably, the antibody subclass of the present invention is IgG4. Alternatively, the antibody subclass of the present invention is preferably IgG2. The antibody of the present invention may be monospecific, bispecific (bispecific antibody), trispecific (trispecific antibody) (eg WO 1991/003493), tetraspecific (tetraspecific antibody) or polyspecific (polyantigen specific).

[0054]

In one embodiment, the antibody of the present invention has CDRH3 with the amino acid sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 33. Preferably, the antibody of the present invention has CDRH1 with the amino acid sequence set forth in SEQ ID NO: 25, CDRH2 with the amino acid sequence shown in SEQ ID NO: 26, and CDRH3 with the amino acid sequence shown in SEQ ID NO: 27, or CDRH1 with the amino acid sequence shown in SEQ ID NO: 31, CDRH2 with the amino acid sequence shown in SEQ ID NO: 32, and CDRH3 with the amino acid sequence shown in SEQ ID NO: 33. More preferably, the antibody of the present invention has CDRH1 with the amino acid sequence shown in SEQ ID NO: 25, CDRH2 with the amino acid sequence shown in SEQ ID NO: 26, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 27, and CDRL1 having the amino acid sequence set forth in SEQ ID NO: 28, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 29, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 30, or CDRH1 with the amino acid sequence given in SEQ ID NO: 31, CDRH2 with the amino acid sequence given in SEQ ID NO: 32, and CDRH3 with the amino acid sequence given in SEQ ID NO: 33, and CDRL1 with the amino acid sequence the sequence shown in SEQ ID NO: 34, CDRL2 with the amino acid sequence shown in SEQ ID NO: 35, and CDRL3 with the amino acid sequence shown in SEQ ID NO: 36.

[0055]

In one embodiment, an antibody of the present invention may be an antibody or variant thereof having a heavy chain having the amino acid sequence set forth in SEQ ID NO: 38 or 42, or a variant sequence thereof, and a light chain having the amino acid sequence set forth in SEQ ID NO: 40 or 44, or a variant sequence thereof. Preferably, the antibody of the present invention may be an antibody or a variant thereof comprising (a) a VH comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 53, or a variant sequence thereof, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 54, or a variant sequence thereof, and CDRH3 with the amino acid sequence given in SEQ ID NO: 55, or a variant sequence thereof, and VL containing CDRL1 with the amino acid sequence given in SEQ ID NO: 56, or a variant sequence thereof, CDRL2 with the amino acid sequence shown in SEQ ID NO: 57, or a variant sequence thereof, and CDRL3 with the amino acid sequence shown in SEQ ID NO: 58, or a variant sequence thereof; or (b) a VH comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 59, or a variant sequence thereof, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 60, or a variant sequence thereof, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 61, or a variant sequence thereof, and VL comprising CDRL1 having the amino acid sequence set forth in SEQ ID NO: 62, or a variant sequence thereof, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 63, or a variant sequence thereof , and CDRL3 with the amino acid sequence shown in SEQ ID NO: 64, or a variant sequence thereof. In a specific embodiment, the antibody may be an antibody or variant thereof comprising (a) a VH having the amino acid sequence set forth in SEQ ID NO: 46, or a variant sequence thereof, and a VL having the amino acid sequence set forth in SEQ ID NO: 48, or a variant sequence thereof, or (b) a VH having the amino acid sequence set forth in SEQ ID NO: 50 or a variant sequence thereof, and a VL having the amino acid sequence set forth in SEQ ID NO: 52 or a variant sequence thereof. The variant sequence preferably does not contain a change (amino acid substitution, addition or deletion) in the CDR, but may contain several changes (3 or less, 2 or less, preferably 1 or less) in each CDR, provided the ability of the antibody to bind and/or function is retained in the same way as before the change. Preferably, the variant sequence may have at least 90% identity with the original sequence.

[0056]

In another aspect, an antibody of the present invention refers to an antibody having a VH with the amino acid sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 22. Preferably, the antibody of the present invention has a VH with the amino acid sequence set forth in SEQ ID NO: 18 , and VL having the amino acid sequence set forth in SEQ ID NO: 20, or VH having the amino acid sequence set forth in SEQ ID NO: 22, and VL having the amino acid sequence set forth in SEQ ID NO: 24.

[0057]

Furthermore, the present invention provides an antibody having a VH with an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 22. Preferably, the present invention relates to to an antibody having a VH with an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18, and a VL with an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 20, or an antibody having a VH with an amino acid sequence encoded by a nucleic acid sequence that hybridizes, under stringent conditions, to a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 22, and a VL with an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24. As used herein, stringent hybridization means hybridization under hybridization conditions commonly used by those skilled in the art. Sequence hybridization can be determined using a method described, for example, in Molecular Cloning, a Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012), Current Protocols in Molecular Biology, Wiley Online Library, etc. For example, the hybridization condition could be a hybridization condition at 42°C in 6×SSC (0.9M NaCl, 0.09M trisodium citrate) or 6×SSPE (3M NaCl, 0.2M _{NaH2PO4 ,} 20mM EDTA⋅2Na, pH 7.4), and then washed at 42°C in 0.5xSSC.

[0058]

The present invention also provides an antibody having a VH with an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 22. Preferably, the antibody of the present invention has a VH with an amino acid sequence having 80% or more more identical to the amino acid sequence of SEQ ID NO: 18, and a VL having an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 20, or a VH having 80% or more identity to the amino acid sequence of SEQ ID NO: 22, and VL with an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 24. Amino acid sequence identity refers to the ratio (%) of the number of amino acids of the same type between two types of proteins within the amino acid sequences intended for comparison, and can be determined using a well-known program such as BLAST or FASTA. The identity described above may be an identity that exceeds 80% or more, such as an identity of 85% or more, 90% or more, 95% or more, 98% or more, 99% or more.

[0059]

An antibody having the above-described VH with an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions, or an antibody having an amino acid sequence with 80% or more identity, preferably has the CDR sequence described above.

[0060]

As a more specific example, the present invention provides an antibody having a heavy chain with the amino acid sequence shown in one of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14. Preferred is an antibody of the present invention having a heavy chain with the amino acid sequence shown in SEQ ID NO: 2, and a light chain with the amino acid sequence shown in SEQ ID NO: 4, a heavy chain with the amino acid sequence shown in SEQ ID NO: : 6, and a light chain having the amino acid sequence given in SEQ ID NO: 8, a heavy chain having the amino acid sequence given in SEQ ID NO: 10, and a light chain having the amino acid sequence given in SEQ ID NO: 12, or a heavy chain with the amino acid sequence shown in SEQ ID NO: 14, and a light chain with the amino acid sequence shown in SEQ ID NO: 16.

[0061]

In a preferred embodiment, the antibody may comprise (a) a VH comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 25, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 26, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: : 27, and VL containing CDRL1 with the amino acid sequence given in SEQ ID NO: 28, CDRL2 with the amino acid sequence given in SEQ ID NO: 29, and CDRL3 given in SEQ ID NO: 30, or (b) VH, containing CDRH1 with the amino acid sequence given in SEQ ID NO: 31, CDRH2 with the amino acid sequence given in SEQ ID NO: 32, and CDRH3 with the amino acid sequence given in SEQ ID NO: 33, and VL containing CDRL1 with the amino acid sequence, shown in SEQ ID NO: 34, CDRL2 with the amino acid sequence shown in SEQ ID NO: 35, and CDRL3 shown in SEQ ID. NO: 36. In another preferred embodiment, the antibody may comprise (a) a VH having the amino acid sequence set forth in SEQ ID NO: 18, or a variant sequence thereof, and a VL having the amino acid sequence set forth in SEQ ID NO: 20, or a variant thereof sequence, or (b) VH having the amino acid sequence set forth in SEQ ID NO: 22, or a variant sequence thereof, and VL having the amino acid sequence set forth in SEQ ID NO: 24, or a variant sequence thereof. The variant sequence preferably contains no change (amino acid substitution, addition or deletion) in the CDR, but may contain several changes (3 or less, 2 or less, preferably 1 or less) in each CDR, provided it retains the ability to bind and/or function with antibody in the same way as before the change. Preferably, the variant sequence may have at least 90% identity with the original sequence.

[0062]

The antibody of the present invention can be obtained by immunizing a non-human animal with an immune response, preferably immunizing a non-human animal (eg, MRL/lpr mouse) with an autoimmune response (autoantibody) due to hyperimmunization, with CD80, CD86 or CD28 or an extracellular domain thereof or a cell expressing CD80, CD86 or CD28 as an immunogen, together with an immunostimulant (for example, mineral oil or aluminum precipitate and heated dead bacteria or lipopolysaccharide, Freund's complete adjuvant, Freund's incomplete adjuvant, or the like) , if necessary. There are no special restrictions on the animal to be immunized, provided that it is an animal that can be used to produce a hybrid, such as a mouse, rat, hamster, guinea pig, rabbit, dog, monkey, sheep, goat, chicken or duck, but is preferably is a mouse or rat, more preferably a mouse and most preferably an MRL/lpr mouse. The immunogen can be administered to the animal by subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection or injection into the sole of the foot in an amount of 1 x 10 ⁶ cells one or more times at a suitable interval (usually one immunization every 1-6 weeks in total from 2 to 10 times). 1-2 weeks after the last immunization, blood samples are taken from the eye socket or tail vein of the immunized animal to measure the antibody titer using its serum. The antibody of the present invention can be obtained by purification from the serum of an animal with sufficient antibody titer.

[0063]

A monoclonal antibody can be produced by culturing a hybridoma obtained by fusing an antibody-producing cell obtained from an immunized animal that has been immunized in the manner described above with a myeloma cell. Examples of the fusion method include the method described by Milstein et al. (Galfre, G. & Milstein, C. (1981) Methods Enzymol., 73: 3-46). The antibody-producing cells to be used can be obtained from the spleen, pancreas, lymph node or peripheral blood of a mouse or rat immunized as described above and having serum of sufficient antibody titer. There are no special restrictions on the myeloma cell to be used, provided that the cell is derived from a mammal such as a mouse, rat, guinea pig, hamster, rabbit or human, and can proliferate in vitro . Examples of such a cell include P3-X63Ag8 (X63) (Nature, 256, 495, 1975), P3/NS1/1-Ag4-1 (NS1) (Eur. J. Immunol., 6, 292, 1976), P3X63Ag8U1 (P3U1 ) (Curr. Top. Microbiol. Immunol., 81, 1, 1978), P3X63Ag8.653 (653) (J. Immunol., 123, 1548, 1979), Sp2/0-Ag14 (Sp2/O) (Nature, 276, 269, 1978), Sp2/O/FO-2 (FO-2) (J. Immunol. Methods, 35, 1, 1980), SP2ab, etc. The cell is preferably a cell derived from an animal of the same species as the antibody producing cell, and more preferably a cell derived from an animal of the same phylogeny as the antibody producing cell.

[0064]

After culture, the culture supernatant is collected and clones that bind to CD80, CD86 or CD28 are selected by ELISA using the CD80, CD86 or CD28 protein or its extracellular domain, or a cell expressing CD80, CD86 or CD28. Monoclonal antibody producing cells can be prepared by repeating serial dilutions of 1 to 5 times in a selected clone to obtain a single cell.

[0065]

Alternatively, an antibody that binds to CD80, CD86 or CD28 can be produced, for example, using a phage antibody library (Tomizuka et al., Nature Genet., 15, 146-156 (1997)). When using a phage antibody library, the desired clone can be obtained, for example, by immobilizing the CD80, CD86 or CD28 protein or its extracellular domain or a cell expressing CD80, CD86 or CD28 on a solid phase, reacting it with the phage library, washing and removing unbound phage , and then collecting the bound phage (panning).

[0066]

Alternatively, it can be obtained by screening an antibody with high specificity for CD80, CD86 or CD28 in accordance with the method described above from antibodies obtained by engineering the amino acid sequence of the antibody of interest or an immunoresponsive fragment thereof, with reference to the amino acid sequence presented in the present description , obtaining DNA encoding the designed amino acid sequence, incorporating the DNA into an expression vector, and introducing and expressing said vector into a suitable host cell.

[0067]

When the antibody of the present invention is a human-type chimeric antibody, the antibody can be produced by obtaining DNA encoding the VH and VL of a non-human animal monoclonal antibody, linking it to a human immunoglobulin constant region cDNA and integrating it into an expression vector, and introducing and expression of the vector in a suitable host cell (Morrison, SL et al., Proc. Natl. Acad. Sci. USA, 81, 6851-6855, 1984).

[0068]

When the antibody of the present invention is a humanized antibody, the antibody can be produced by constructing DNA encoding a V region obtained by transplanting the amino acid sequence encoding the CDRs VH and VL of a non-human animal monoclonal antibody into the framework region (FR) of VH and VL human antibody, linking the engineered DNA to the human immunoglobulin constant region cDNA and integrating it into an expression vector, and introducing and expressing the vector into a suitable host cell (see L. Rieohmann et al., Nature, 332, 323, 1988: Kettleborough, CA et al., Protein Eng., 4, 773-783, 1991; Clark M., Immunol. Today., 21, 397-402, 2000). The CDR of a non-human animal monoclonal antibody can be obtained by comparing the amino acid sequence predicted from the DNA sequence encoding the VH and VL of the non-human animal monoclonal antibody obtained by the method described above with the full-length VH and VL amino acid sequences of a known antibody. The amino acid sequences of a known antibody can be obtained from the amino acid sequences of antibodies that are registered in a database, such as a protein data bank. The FR of the humanized antibody is not particularly limited, provided that the grafted antibody must provide the effect disclosed herein, and is preferably the FR of a human antibody with a three-dimensional structure, wherein the variable region (hereinafter referred to as the "V region") of the humanized antibody is similar The V region of a non-human animal monoclonal antibody from which the CDR or FR of a human antibody is derived with high homology to the amino acid sequence of the FR of the non-human animal monoclonal antibody intended for use. Some of the amino acids constituting the FR derived from a human antibody (in particular, the amino acids located adjacent to the CDR in three-dimensional space) may be replaced by the FR sequence of the non-human animal monoclonal antibody from which the humanized antibody CDR is, if desired, derived ( see Queen et al., US patent No. 5585089). The DNA sequence encoding the V region of a humanized antibody to be used is designed as a DNA sequence corresponding to the amino acid sequence obtained by linking the CDR amino acid sequence of a non-human animal monoclonal antibody to the FR amino acid sequence of a human antibody. DNA encoding the V region of a humanized antibody can be obtained in a manner well known to those skilled in the art based on an engineered DNA sequence.

[0069]

For example, a human antibody can be produced using a human antibody phage library or a transgenic mouse producing human antibodies (Tomizuka et al., Nature Genet., 15, 146-156 (1997)). When using a phage library of human antibodies, the desired clone can be obtained, for example, by binding the CD80, CD86 or CD28 protein or its extracellular domain, or a cell expressing CD80, CD86 or CD28, to the solid phase, followed by interaction with the phage library, washing and by removing the unbound phage and then collecting the bound phage (panning). A human antibody transgenic mouse is a mouse that is produced by introducing a human antibody Ig gene into an endogenous immunoglobulin (Ig) gene knockout mouse. A human antibody that specifically recognizes CD80, CD86 or CD28 can be produced by immunization with an antigenic protein in accordance with the method for producing antibodies of the present invention described above, using a transgenic mouse producing a human antibody as the immunized animal.

[0070]

(Nucleic acid molecule/vector/host cell)

In another aspect, the present invention relates to a nucleic acid molecule containing a polynucleotide encoding an antibody of the present invention described above. For example, the nucleic acid molecule of the present invention includes the nucleotide sequence shown in SEQ ID NO: 17 or SEQ ID NO: 21 as VH. In another aspect, the nucleic acid molecule of the present invention contains the nucleotide sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 23 as VL. As a more specific example, the nucleic acid molecule of the present invention contains a polynucleotide encoding VH, with the nucleotide sequence shown in any of SEQ ID NO: 1, 5, 9 and 13, and a polynucleotide encoding VL, with the nucleotide sequence shown in any of SEQ ID NOs: 3, 7, 11 and 15. The nucleic acid molecule of the present invention may have a nucleic acid sequence that hybridizes to the SEQ ID NO described above under stringent conditions. In addition, the present invention includes a vector containing the specified nucleic acid molecule. There are no special restrictions on such a vector as long as it is a vector that can be used to express an antibody. Suitable viral vector, plasmid vector, etc. can be selected according to the host used. In another aspect, the present invention relates to a host cell containing the specified vector. There is no particular limitation on the host cell as long as it is a host cell that can be used to express the antibody. Examples of such cells include mammalian cells (mouse cells, rat cells, rabbit cells, human cells, etc.), yeast and microorganisms ( E. coli , etc.). The present invention relates to a method for producing the antibody of the present invention, comprising culturing a host cell as described above. Those skilled in the art can determine a culture medium appropriate for the host cell being used using well known means.

[0071]

The nucleic acid of the present invention can be obtained by cloning from a hybridoma producing the antibody obtained above, or by designing a corresponding nucleic acid sequence based on the amino acid sequence of the antibody obtained above or an immunosensitive fragment thereof. The vector of the present invention can be obtained by incorporating a suitably prepared nucleic acid into a suitable expression vector. The vector of the present invention may contain, in addition to the nucleic acid of the present invention, a region that is necessary for expression (promoter, enhancer, terminator, etc.). The host cell of the present invention can be obtained by introducing a vector of the present invention into a suitable cell line (eg, an animal cell, an insect cell, a plant cell, a yeast cell, or a microorganism such as E. coli ).

[0072]

(Composition)

In one embodiment, the present invention relates to a composition for producing a cell with induced immune tolerance, which contains at least one of the antibodies presented in the present description. In one embodiment of the present invention, the composition may comprise an anti-CD80 antibody, an anti-CD86 antibody, an anti-CD28 antibody, a bispecific antibody to CD80 and CD86, or any combination thereof. Rejection due to organ transplantation can be reduced, for example, by administering an immune tolerance-induced cell to the individual receiving the organ transplant. The induced immune tolerance cell may be a cell obtained from an individual (recipient) or a donor, or a mixture of such cells. Surprisingly, it has been found that the composition of the present invention does not substantially cause the release of cytokines by cytokine-producing cells (eg, macrophages, neutrophils or natural killer (NK) cells). Since cytokines weaken immune tolerance, it is preferable that cytokine release is not induced.

[0073]

In another aspect, the present invention relates to a medical composition containing an antibody as described herein. In one example, the medical composition of the present invention contains an anti-CD80 antibody, an anti-CD86 antibody, an anti-CD28 antibody, or a bispecific antibody to CD80 and CD86. The medical composition of the present invention can be used to induce immune tolerance. Throughout the specification, the term “inducing immune tolerance” includes applications such as for inducing immune tolerance to a transplanted organ, i.e. to suppress excessive immune responses to a pathogen that becomes an immunogen in a patient with an autoimmune disease or allergic disease, to relieve or treat the symptoms of such diseases for the purpose of suppressing rejection in organ transplantation. In another embodiment, the pharmaceutical composition of the present invention is used to obtain a cell with induced immune tolerance.

[0074]

In the context of the present description, the term “organ” to be transplanted in “organ transplantation” is not particularly limited. Examples of such an organ include the heart, kidneys, lungs, pancreas, esophagus, stomach, small intestine, large intestine, skin, nerve, blood, blood cells including immune system cells, bone, cartilage, blood vessel, cornea, eyeball, bone brain and liver. As used herein, the term "organ" includes a portion of an organ and the cells composing that organ, in addition to the whole or mass of the organ. For example, transplanted organs also include myocardial cells, corneal cells, and the like. Organ transplantation also includes transplantation into a patient of an organ obtained using heterologous stem cells in regenerative medicine and the like, in addition to transplantation of another person's organ into the patient.

[0075]

The antibody of the present invention can be used as a medical composition by purification and then, if necessary, formulation according to a conventional method. The present invention also includes the use of an antibody of the present invention for the production of a medical composition for inducing immune tolerance. Alternatively, the present invention relates to the use of an antibody of the present invention to induce immune tolerance. In addition, the present invention relates to a method for inducing immune tolerance, comprising adding or administering an antibody of the present invention. For example, the present invention relates to a method for suppressing rejection in organ transplantation, comprising administering an antibody of the present invention to a patient receiving an organ transplant; a method for treating an autoimmune disease or an allergic disease, comprising administering an antibody of the present invention to a patient with an autoimmune disease or an allergic disease; and a method for suppressing rejection in transplantation of an iPS cell or ES cell or a cell, tissue or organ derived (differentiated) from such a cell, comprising administering an antibody of the present invention to a patient receiving a transplant of an iPS cell or ES cell or cell, tissue or an organ obtained (differentiated) from such a cell.

[0076]

Examples of cells, tissue or organ derived (differentiated) from iPS cells or ES cells include, but are not limited to, nerve cells or tissue, corneal cells or tissue, myocardial cells or tissue, liver cells or tissue, cartilage cells or tissue, skin cells or tissue, kidney cells or tissue, etc. In a preferred embodiment, the cells, tissue or organ derived from iPS cells or ES cells include nerve cells or tissue, myocardial cells or tissue, cartilage cells or tissue, and skin cells or tissue.

[0077]

The medical composition of the present invention can be used by direct administration to a patient or by contacting the composition with a PBMC obtained from a patient in which immune tolerance is to be induced, together with an ex vivo immune tolerance-inducing antigen of interest to obtain an antigen-anergic immune cell .

[0078]

When the medical composition of the present invention is administered directly to a patient, any oral or parenteral formulation may be used. Examples of the composition for parenteral administration include eye drops, injections, nasal drops, suppositories, patches, ointments and the like. The composition is preferably an injection. Examples of dosage forms of the medical composition of the present invention include a liquid agent and a lyophilized formulation. When the medicinal composition of the present invention is used as an injection, an additional agent such as a solubilizing agent such as propylene glycol or ethylenediamine, a buffer such as phosphate, a tonicity adjusting agent such as sodium chloride or glycerol, a stabilizer such as as a sulfite, a preservative such as phenol, or an analgesic such as lidocaine (see Japanese Pharmaceutical Excipients Directory, Yakuji Nippo, Limited, Handbook of Pharmaceutical Excipients Fifth Edition, APhA). When the medical composition of the present invention is used as an injection, examples of the storage container include an ampoule, a vial, a pre-filled syringe, a pen-shaped injection cartridge, an intravenous infusion bag and the like.

[0079]

For example, the medical composition of the present invention (therapeutic drug or prophylactic drug) can be used in the form of an injection. Its dosage form includes intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intravitreal injection, intravenous drip injection and the like. Such injections can be prepared in accordance with a known method, for example, by dissolving, suspending or emulsifying the antibody described above in an aseptic aqueous or oily solution commonly used for injection. For example, a tonic solution containing saline, glucose, sucrose, mannitol or other adjuvant or the like may be used as an aqueous injection solution, or such an aqueous solution may be used in combination with a suitable solubilizing agent such as an alcohol (eg, ethanol), polyhydric alcohol (eg, propylene glycol or polyethylene glycol), anionic surfactant [eg, polysorbate 80, polysorbate 20, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] or the like. For example, sesame oil, soybean oil, etc. can be used as an oil solution, which can be used simultaneously with benzyl benzoate, benzyl alcohol or the like. as a solubilizing agent. The prepared injection is usually poured into a suitable ampoule, vial or syringe. Optionally, a suitable excipient may be added to the antibody of the present invention or an immunosensing fragment thereof to form a lyophilized injection formulation that is dissolved in water for injection, saline or the like. Although it is generally believed that oral injection of a protein, such as an antibody, involves difficulties associated with the breakdown of the protein in the digestive system, oral administration is also possible through creativity and ingenuity in the manufacture of a dosage form containing such an antibody fragment or a modified antibody fragment . Examples of formulations for oral administration include capsules, tablets, syrup, granules and the like.

[0080]

The medicinal composition of the present invention is suitably formulated in a dosage form with a dosage unit that corresponds to the dose of the active ingredient. A typical dosage unit dosage form includes an injection (ampoule, vial, or prefilled syringe). A dosage unit dosage form may typically contain from 5 to 500 mg, from 5 to 100 mg, or from 10 to 250 mg of an antibody of the present invention or an immunoresponsive fragment thereof.

[0081]

Administration of the medical composition of the present invention may be local or systemic. There are no special restrictions on the route of administration. The composition is administered parenterally or orally as described above. Examples of parenteral routes of administration include intraocular, subcutaneous, intraperitoneal, blood (intravenous or intra-arterial) or by injection into cerebrospinal fluid, in the form of drops and the like, and are preferably administered into the blood. The medical composition of the present invention (therapeutic drug or prophylactic drug) may be administered temporarily, continuously or periodically. For example, administration may be continuous and last from 1 minute to 2 weeks.

[0082]

The dosage of the medical composition of the present invention is not particularly limited as long as it is a dosage that provides the desired therapeutic or prophylactic effect. The dose can be determined accordingly depending on the symptom, gender, age and the like. The dose of the medical composition of the present invention can be determined, for example, by the degree of induction of immune tolerance used as an indicator. For example, as a single dose of the active ingredient of the medicinal composition of the present invention, it is generally preferable to administer from about 0.01 to 20 mg/kg body weight, preferably from about 0.1 to 10 mg/kg body weight, and more preferably from about 0.1 up to 5 mg/kg body weight about 1 to 10 times per month, and preferably about 1 to 5 times per month by intravenous injection. A corresponding amount can be administered by another parenteral or oral route of administration. If the symptom is very severe, the dose or number of administrations may be increased depending on the severity of the symptom.

[0083]

When the medical composition of the present invention is contacted with PBMC obtained from a patient in whom immune tolerance is to be induced, the composition together with the ex vivo immune tolerance-inducing antigen of interest to produce an immune cell anergic to the antigen can be formulated in such a form as as a liquid, powder, tablet, gel or granules, in accordance with the above-described composition for administration.

[0084]

(Cells with induced immune tolerance)

In another aspect, the present invention relates to a cell with immune tolerance induced by an antibody as provided herein. Immune tolerance can be induced by mixing a cell derived from an individual with an antigen that does not originate from the individual or with material containing that antigen. In another aspect, the present invention relates to a method of producing a cell for the treatment or prevention of a disease, disorder or condition caused by an antigen foreign to an individual, comprising mixing an antibody as provided herein, a cell obtained from the individual, and a non-individual antigen or material containing this antigen (for example, cells). The disease, disorder, or condition may be selected from the group consisting of graft rejection, allergy, autoimmune disease, graft-versus-host disease, and immune rejection caused by transplantation of an iPS cell or ES cell and a cell, tissue, or organ derived from said cells.

[0085]

In an embodiment in which the disease or the like addressed by the present invention is graft rejection, an anergic cell can be induced by mixing the antibody described above with a cell obtained from the recipient (PBMC or splenocyte) and the antigen obtained from a donor, or material containing an antigen obtained from a donor. The material containing the donor-derived antigen may be a PBMC, a splenocyte, or a cell obtained from the organ to be transplanted.

[0086]

In an embodiment in which the disease addressed by the present invention or the like is an allergy, an anergic cell can be induced by mixing the antibody described above with a cell obtained from an individual (PBMC or splenocyte) and an antigen causing allergies foreign to the individual.

[0087]

In an embodiment in which the disease addressed by the present invention or the like is an autoimmune disease, an anergic cell can be induced by mixing the antibody described above with a cell obtained from an individual (PBMC or splenocyte) and obtained from an individual with an antigen that is the cause of an autoimmune disease.

[0088]

In an embodiment in which the disease targeted by the present disclosure or the like is graft-versus-host disease, an anergic cell can be induced by mixing the antibody described above with a PBMC or splenocyte from a graft donor and an antigen obtained from the recipient, or material containing this antigen. The antigen-containing material obtained from the recipient may be a PBMC, a splenocyte, a cell from a site adjacent to the organ transplant site, or a cell derived therefrom.

[0089]

In an embodiment in which the disease targeted by the present disclosure or the like is an immune rejection caused by transplantation of an iPS cell or ES cell and a cell, tissue, or organ derived from said cells, the anergic cell may be induced by mixing the antibody described above with a cell derived from an individual (PBMC or splenocyte) and a cell differentiated from an iPS cell or ES cell used in the transplantation.

[0090]

Therapeutic examples of disease or the like. in accordance with the present disclosure are given below, but the invention is not limited to them.

[0091]

(Allergies and autoimmune diseases)

For allergies and autoimmune diseases, a macrophage obtained from the patient's peripheral blood is differentiated into a dendritic cell (dendritic cell derived from macrophages) with high antigen-presenting capacity in a conventional manner. After exposure to radiation (γ-rays), the cell is injected with an antigen that causes hyperreaction in allergies or autoimmune disease and co-cultured for 1-2 weeks with a group of T cells obtained from the peripheral blood of the same patient, in the presence of anti- CD80 antibodies and/or anti-CD86 antibodies to produce an anergic cell specific for an antigen that causes an allergy or autoimmune disease. An anergic cell is administered to a patient to induce immune tolerance specific to an antigen causing an allergy or autoimmune disease for use in the prevention or treatment of allergies and autoimmune disease. Multiple doses may be administered depending on whether the dose is for prophylactic treatment or therapeutic treatment, and depending on various conditions such as the severity of symptoms.

[0092]

(Graft versus host disease)

In graft-versus-host disease, as opposed to the treatment of graft rejection, a cell that may be causing graft-versus-host disease, such as a PBMC or a T cell from the graft donor, is cultured along with the host-derived PBMC irradiated with radiation (γ- rays), or other cells in the presence of anti-CD80 antibody and/or anti-CD86 antibody for 1 to 2 weeks to obtain a host-specific anergic cell. Administration of such an anergic cell to a host for the prevention or treatment of graft-versus-host disease suppresses the graft-versus-host disease responses in the host to the graft (and induces immune tolerance). Multiple doses may be administered depending on whether the dose is being administered for prophylactic treatment or therapeutic treatment, and depending on various conditions such as the tissue to be transplanted, its size, or the severity of symptoms.

[0093]

(Immune rejection caused by transplantation of an iPS cell or ES cell and a cell, tissue or organ derived from said cells)

In the case of a treatment application where an iPS cell or an ES cell is used, a dendritic cell or a transplant cell differentiated from an iPS cell or an ES cell is irradiated with radiation (γ-rays), and the cell is co-cultured with a group PBMC or T cells from the transplant patient in the presence of anti-CD80 antibody and/or anti-CD86 antibody for 1-2 weeks to produce an anergic cell that is specific to a cell differentiated from an iPS cell or ES cell. Administration of such an anergic cell to the host induces immune tolerance specific to the iPS cell or transplanted ES cell-derived cells, tissues and organs and provides prevention and treatment of rejection of these cells. Multiple doses may be administered depending on whether the dose is being administered for prophylactic treatment or therapeutic treatment, as well as various conditions, such as the tissue to be transplanted, its size, or the severity of symptoms.

[0094]

In a specific embodiment, the present invention relates to a method of inducing anergy in a patient's PBMC to an antigen, comprising contacting an antibody described above (including a composition containing the antibody described above; the same applies hereinafter) with an antigen to which immune tolerance is to be induced, or a cell bearing the specified antigen on its surface, and PBMC. The immune tolerance induction method of the present invention is carried out ex vivo . If, for example, an organ transplant is intended to suppress rejection of the transplanted organ of the transplant recipient, the PBMC is a cell obtained from the transplant recipient. If the PBMC is intended to treat or improve an autoimmune disease or allergic disease, the PBMC is a patient-derived cell.

[0095]

For example, immune tolerance for suppressing organ transplant rejection can be induced by the following method. PBMC of the patient (recipient) receiving an organ transplant and PBMC of the individual (donor) providing the organ are prepared, and the donor PBMC are irradiated with 30 Gy of radiation. Donor PBMCs are mixed with recipient PBMCs in a 1:1 ratio and an antibody of the present invention (preferably a combination of human anti-CD80 antibody and human anti-CD86 antibody) is added. Recipient PBMCs, anergic to the donor, can be obtained by culturing for 3-7 days in an incubator at 37°C and 5% CO ₂ in PBMC medium.

[0096]

For example, immune tolerance for treating or improving an autoimmune disease or an allergic disease can also be induced by the following method. The patient's PBMC is prepared and mixed with an antigen that causes an autoimmune disease or an allergic disease, and an antibody of the present invention (preferably a combination of human anti-CD80 antibody and human anti-CD86 antibody) is added. Recipient PBMCs, anergic to the antigen, can be obtained by culturing for 3-7 days in an incubator at 37°C and 5% CO ₂ in PBMC medium.

[0097]

The present invention relates to a cell (eg, PBMC) with anergy induced by the method described above. The anergy-induced cell (eg, PBMC) can induce immune tolerance in a patient by administration to the patient so that the anergy-induced cell (eg, PBMC) can be used as a cell therapy agent. Accordingly, the present invention relates to a cell therapy agent containing as an active ingredient a cell (for example, PBMC) with anergy induced by the above method, preferably the invention relates to a cell therapy agent for suppressing rejection in organ transplantation containing as an active ingredient a cell ( e.g., PBMC) of a transplant patient with induced anergy to the transplanted organ, or to a cell therapy agent for the treatment of an autoimmune disease or allergic disease containing as an active ingredient a cell (e.g., PBMC) with induced anergy to an antigen causing the autoimmune disease or an allergic disease caused by an overactive immune response, or to a cell therapy agent to suppress rejection upon transplantation of an iPS cell or ES cell and a cell, tissue or organ derived from said cells. An example of rejection includes graft-versus-host disease.

[0098]

The cell therapy agent of the present invention can induce immune tolerance in a patient by administration to the patient. For example, the present invention includes a method for suppressing organ transplant rejection, comprising administering to a patient receiving an organ transplant a cell therapy agent for suppressing organ transplant rejection containing as an active ingredient a cell (eg, PBMC) from the transplant recipient with induced anergy to transplanted organ. The present invention also includes a method of treating an autoimmune disease or allergic disease, comprising administering to a patient with an autoimmune disease or allergic disease a cell therapy agent for treating the autoimmune disease or allergic disease containing as an active ingredient a cell (eg, PBMC) with induced anergy to an antigen that causes an autoimmune disease or allergic disease caused by an overactive immune response. The present disclosure also includes a method of suppressing rejection in transplantation of an iPS cell or ES cell or a cell, tissue or organ derived from such cells, comprising administering to a patient receiving an organ transplant a cell therapy agent for suppressing rejection in transplantation of an iPS cell or ES cell. cell or cell, tissue or organ derived from said cells, comprising as an active ingredient a cell (for example, PBMC) of a transplant recipient with anergy to the iPS cell or ES cell, or cell, tissue or organ to be transplanted, obtained from these cells.

[0099]

The cell therapy agent of the present invention contains PBMC in serum containing/not containing medium or saline that can be administered to a human. Once the cells are adsorbed, the cells can be contained and stored on a removable support and used by detaching the cells before use. For example, a substrate from which cells can be separated by changing temperature (RepCell, CellSeed Inc., Japan) or the like can be used.

[0100]

The present invention may be a method of inducing immune tolerance in a patient, comprising contacting an antibody as described above with an antigen to which immune tolerance is to be induced, or a cell bearing the antigen on its surface, and a cell (eg, PBMC) to induce anergy to the cell's antigen (eg, PBMC) of the patient and administering the anergy-induced cell (eg, PBMC) to the patient. For example, the present invention relates to a method of suppressing rejection in organ transplantation, comprising preparing a cell (eg, PBMC) obtained from a patient with immune tolerance to the transplanted organ induced by inducing anergy to the transplanted organ, in a cell (eg, PBMC) of the patient by contacting a cell obtained from a donor with a cell (eg, PBMC) from a patient and administering the resulting cell (eg, PBMC) to a patient receiving an organ transplant. The present invention may also be a method of treating or ameliorating an autoimmune disease or allergic disease, comprising preparing a cell (eg, PBMC) obtained from a patient with immune tolerance to an antigen causing the autoimmune disease or allergic disease, induced by inducing anergy to the antigen, in cell (eg, PBMC) of a patient by contacting an antigen that causes an autoimmune disease or allergic disease with a cell (eg, PBMC) of the patient and administering the resulting cell (eg, PBMC) to a patient with an autoimmune disease or allergic disease. The present invention also includes a method for suppressing rejection in transplantation of an iPS cell or an ES cell, or a cell, tissue or organ derived from such cells, comprising preparing a patient-derived cell (eg, PBMC) that is immune tolerant to the iPS to be transplanted. -a cell or ES cell, or a cell, tissue or organ derived from said cells, induced by inducing anergy to an iPS cell or ES cell, or a cell, tissue or organ derived from said cells, in a cell (eg, PBMC ) of a patient by contacting an iPS cell or ES cell or a cell, tissue or organ derived from said cells with a cell (eg, PBMC) of the patient and administering the resulting cell (eg, PBMC) to the patient receiving an iPS cell or ES cell transplant cells, or cells, tissue or organ derived from said cells.

[0101]

Administration of a cell therapy agent (anergy-induced cells such as PBMC) is not particularly limited in the present specification as long as the dosage provides the desired therapeutic or prophylactic effect and can be appropriately determined depending on the symptom, gender, age or the like. . The dose of the cell therapy agent of the present invention can be determined, for example, by the degree of induction of immune tolerance used as an indicator. For example, a single dose of PBMC with anergy-induced cell therapy agent of the present invention is preferably administered by intravenous injection about 1 to 10 times per month, and preferably 1 to 5 times per month, and is about 1.0 to 3.7× 10 ⁷ cells/kg body weight, preferably from about 2.0 to 5.0 x 10 ⁷ cells/kg body weight, and more preferably from about 3.0 to 5.0 x 10 ⁷ cells/kg body weight. If no immunosuppressive effect is observed, the dose or number of administrations can be increased depending on the symptom.

[0102]

(Note)

As used herein, "or" is used when "at least one or more" of the following can be used in a sentence. When a range is explicitly stated herein, such as “within a range limited to two values,” the specified range also includes those two values.

[0103]

Documents cited by reference, such as scientific literature, patents and patent applications cited herein, are incorporated herein by reference as if each of these documents were specifically described.

[0104]

To facilitate understanding of the present invention, preferred embodiments have been demonstrated herein. Although the present description is disclosed below on the basis of examples, the above description and examples below are provided for purposes of illustration and not limitation of the present invention only. Thus, the scope of the present invention is not limited to the embodiments and examples that are particularly presented in the present description, and is limited only by the scope of the claims.

[Examples]

[0105]

The present invention is more specifically described below based on examples. However, the present invention is not limited to the examples. Throughout the application, all references cited are expressly incorporated herein by reference.

[0106]

Examples are described below. The microorganisms used in the examples below were used in accordance with the guidelines established by Juntendo University and regulatory authorities. Animal experiments were performed in accordance with the Regulations for the Control of Animal Experiments of Juntendo University, which are adopted based on the Act on Welfare and Management of Animals, Guideline for Raising and Keeping of Laboratory Animals (2006 Ministry of the Environment Notification No. 88 ) and “Fundamental guidelines for proper conduct of animal experiment and related activities in Academic research institutions” (2006 Ministry of Education, Culture, Sports, Science and Technology Notification No. 71). The experiments were planned on the basis of the so-called 3R. The plan was approved and accepted after presentation and review by the Animal Experimentation Committee of Juntendo University. The use of genetically engineered mice was approved after a university safety committee reviewed the experimental design for the use of recombinant DNA producing animals. For recombinant DNA experiments, the plan was approved and accepted after its presentation and review by the DNA Safety Committee at Juntendo University; The research was carried out in accordance with relevant regulations such as "Law Concerning the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms". The experiment using human peripheral blood (PBMC) is based on the principle of objective review and disclosure and approved as appropriate by the ethics committee of Juntendo University. The studies were performed under strict protection of personal information and maintaining anonymity after obtaining informed consent regarding the objectives/design/method of the studies from the sample providers. The experiments were carried out in accordance with the ethical principles established by the ethics committees of Juntendo University School of Medicine and Sagamihara National Hospital and the “guidelines for clinical research” established by the Ministry of Health, Labor and Welfare. The specific products described in the examples were used as reagents. However, the reagents may be replaced by an equivalent product from another manufacturer (Sigma-Aldrich, Wako Pure Chemical, Nacalai Tesque, R&D Systems, USCN Life Science INC, BD bioscience, BioLegend, etc.).

[0107]

(Example 1) Mixed lymphocyte culture reaction (MLR) in the presence of human anti-CD80 antibodies and anti-CD86 antibodies (IgG1 subclass)

(Materials and methods)

Mononuclear cells (PBMCs) were isolated from the peripheral blood of three human volunteers (one designated as donor (Donor C) and two designated as recipients (Recipient A and Recipient B)) using Promo cell lymphocyte separation medium (cat. no. . C 44010) and adjusted the concentration to 4×10 ⁶ cells/ml in ALyS505N-0 medium containing 2% AB type human serum (pool), Biowest, (Cell Science & Technology Institute (CSTI) 1020P10). Donor PBMCs were irradiated with radiation (γ-rays) at a dose of 30 Gy.

[0108]

Donor PBMCs and recipient PBMCs were distributed into 4 wells of a 96-well plate (Corning, Cat. No. 3799) at 100 μl/well in a 1:1 ratio (for a total of 200 μl/well). To these were added a combination of humanized antibodies to human CD80 (v2 (IgG1 subclass)) and human CD86 (v5 (IgG1 subclass)) or a combination of eBioscience mouse anti-human CD80 (cat. no. 16-0809-85) (IgG1 subclass) and mouse antibodies to human CD86 (Cat. No. 16-0869-85) (IgG2 subclass) in an amount of 10 μl so that the concentration of each antibody after addition is 0.1, 1 or 10 μg/ml, and begin cultivation in an incubator at 5% CO ₂ at 37°C. On the 4th day from the start of cultivation, ³ H-thymidine (10 μl) was added. On day 5 from the start of cultivation (16-20 hours after the addition of ³ H-thymidine), the cultured cells were collected using a cell harvester (Molecular Devices). The amount of ³ H-thymidine incorporation was measured with a scintillation counter.

[0109]

To produce IFNγ, the PBMC mixture, which was mixed as described above with the addition of each antibody as above, was added to 3 wells of a 48-well plate (Corning, Cat. No. 3548) at 500 μl/well, and culture was started in an incubator at 5 % CO ₂ at 37°C. On day 6 of cultivation, the supernatant was collected. The collected sample was diluted 10-25 times and then measured using Biolegend's Human IFNγ ELISA MAX TM Deluxe, Cat. 430104.

[0110]

(Results)

As a result, as shown in FIG. 1, the combination of humanized anti-human CD80 (v2 (IgG1 subclass)) and humanized anti-human CD86 (v5 (IgG1 subclass)) had a low level of ^3H -thymidine incorporation, almost at the same level as the combination of mouse anti-human CD80 from eBioscience and mouse anti-human CD86, i.e. suppressed activation of immune cells was observed. Meanwhile, as shown in FIG. 2, assessment of IFNγ production showed a low level of production in the presence of eBioscience murine antibodies to human CD80 and CD86, while in the presence of humanized antibodies to human CD80 and CD86 (IgG1 subclass), IFNγ production was observed, which was dependent on the amount of added antibodies. At 10 μg/ml, the observed level of production was equal to or greater than the level of production in the absence of antibodies (Figure 2: Allo). Without wishing to be bound by any theory, it is understood that the low level of IFNγ production in the presence of eBioscience antibodies is due to the fact that these antibodies are murine and have low reactivity.

[0111]

(Example 2) Mixed lymphocyte culture reaction (MLR) in the presence of antibodies to human CD80 and CD86 (IgG1 subclass)

(Materials and methods)

³ H-thymidine was added to a combination of humanized anti-human CD80 (v2 (IgG1 subclass)) and human CD86 (v5 or v9 (IgG1 subclass)) or eBioscience mouse anti-human CD80 combination (cat. no. 16-0809-85) and mouse anti-human CD86 antibody (cat. no. 16-0869-85) until the concentration of each antibody was reached after addition of 10 μg/ml as in Example 1. Of the three volunteers, one was designated as a donor (donor F), and two were designated as recipients (recipient D and recipient E).

[0112]

In addition, IFNγ production was assessed for the combination of a humanized anti-human CD80 antibody (v2 (IgG1 subclass)) and a humanized anti-human CD86 antibody (v9 (IgG1 subclass)) and for the combination of an eBioscience mouse anti-human CD80 antibody and an anti-human CD86 antibody. In addition to the method described in Example 1, IFNγ production was also assessed for the combination of a humanized anti-human CD80 antibody (v2 (IgG1 subclass)) and an anti-human CD86 antibody (v9 (IgG1 subclass)) without the addition of radiation-irradiated donor PBMCs.

[0113]

(Results)

As a result, as shown in FIG. 3, the combination of humanized anti-human CD80 and human CD86 antibodies showed greater inhibition of ³ H-thymidine incorporation than the combination of mouse anti-human CD80 and anti-human CD86. Meanwhile, as shown in FIG. 4, IFNγ production in the presence of eBioscience murine anti-human CD80 and CD86 antibodies was as low as in Example 1. For the humanized anti-human CD80 and CD86 antibodies, production levels were the same or higher than when the donor was simulated in the absence of antibody. In addition, the same IFNγ production was observed with the addition of humanized antibodies to human CD80 and human CD86, even without the addition of radiation-irradiated donor PBMCs.

[0114]

(Example 3) Mixed lymphocyte culture reaction (MLR) in the presence of anti-human CD80 antibodies and anti-CD86 antibodies (IgG1, IgG2 and IgG4 subclasses)

In Example 2, IFNγ production was observed even without the addition of radiation-irradiated donor PBMCs. Thus, the classes of humanized antibodies to human CD80 and human CD86 were changed, and the incorporation of ³ H-thymidine by adding a combination of humanized antibodies to human CD80 (v2 (IgG2 or IgG4 subclass)) and human CD86 (v9 (IgG2 or IgG4 subclass)) was assessed. of the same subclass, as well as a combination of eBioscience mouse anti-human CD80 antibody (cat. no. 16-0809-85) and anti-human CD86 antibody (cat. no. 16-0869-85) until the concentration of each antibody after addition is equal to 1.3 or 10 μg/ml, and the level of IFNγ production upon addition of antibodies was assessed with a final concentration of each antibody after addition of 10 μg/ml. Of the three volunteers, one was designated as a donor (Donor I) and two were designated as recipients (Recipient G and Recipient H). Below is a detailed description of the experiment.

[0115]

[ ³ H-thymidine inclusion (concentration of each antibody: 1, 3 and 10 μg/ml) (Fig. 5)

- without the addition of antibody (Allo), with the addition of radiation-irradiated donor PBMCs

- eBioscience's mouse anti-human CD80 antibodies and mouse anti-human CD86 antibodies

- humanized antibody to human CD80 (v2 (IgG2)) and humanized antibody to human CD86 (v9 (IgG2))

- humanized antibody to human CD80 (v2 (IgG4)) and humanized antibody to human CD86 (v9 (IgG4))

[0116]

[IFNγ production (concentration of each antibody: 10 μg/ml) (Fig. 6)

With the addition of radiation-irradiated donor PBMCs (Allo)

- without added antibodies

- eBioscience's mouse anti-human CD80 and anti-human CD86

- humanized antibody to human CD80 (v2 (IgG2)) and humanized antibody to human CD86 (v9 (IgG2))

- humanized antibody to human CD80 (v2 (IgG4)) and humanized antibody to human CD86 (v9 (IgG4))

Without the addition of radiation-irradiated donor PBMCs (Naïve)

- without adding antibodies (Allo)

- eBioscience anti-mouse CD80 antibodies and anti-CD86 antibodies

- humanized anti-CD80 antibody (v2 (IgG1)) and humanized anti-human CD86 antibody (v9 (IgG1))

- humanized anti-CD80 antibody (v2 (IgG2)) and humanized anti-human CD86 antibody (v9 (IgG2))

- humanized anti-CD80 antibody (v2 (IgG4)) and humanized anti-human CD86 antibody (v9 (IgG4))

[0117]

(Results)

As a result, as shown in FIG. 5, the IgG2 and IgG4 subclasses showed suppression ^{of 3H} -thymidine incorporation similar to that observed in Examples 1 and 2. Meanwhile, as shown in FIG. 6, IFNγ production was observed in the IgG1 subclass, but surprisingly it was found that in the IgG2 and IgG4 subclasses there was no IFNγ production at all.

[0118]

(Example 4) Mixed lymphocyte culture reaction (MLR) with the addition of cells obtained from culture in the presence of anti-human CD80 antibodies and anti-human CD86 antibodies (IgG1, IgG2 or IgG4 subclass)

(Materials and methods)

Cultivation was continued for 14 days in the manner described in Example 1, in the presence of a combination of humanized anti-human CD80 (v2) and anti-human CD86 (v9) IgG1, IgG2 and IgG4 subclasses. The humanized anti-human CD80 (v2) antibody of the IgG2 subclass consisted of a heavy chain with the amino acid sequence SEQ ID NO: 2 and a light chain with the amino acid sequence SEQ ID NO: 4. The humanized anti-human CD80 (v2) antibody of the IgG4 subclass consisted of a heavy chain with the amino acid sequence SEQ ID NO: 6 and a light chain with the amino acid sequence SEQ ID NO: 8. The anti-human CD86 (v9) antibody of the IgG2 subclass consisted of a heavy chain with the amino acid sequence SEQ ID NO: 10 and a light chain with the amino acid sequence SEQ ID NO: 12 The antibody to human CD86 (v9) of the IgG4 subclass consisted of a heavy chain with the amino acid sequence SEQ ID NO: 14 and a light chain with the amino acid sequence SEQ ID NO: 16. After culturing, the resulting anergic cells, recipient PBMCs (R-PBMCs) and irradiated with radiation Donor PBMCs (D-PBMCs) were mixed according to the table below, and then ³ H-thymidine incorporation was assessed after mixing by the method described in Example 1.

[0119]

[Table 1]

[0120]

(Results)

As a result, as shown in FIG. 7, cells obtained in the presence of antibodies of any subclass equally suppressed the incorporation of ³ H-thymidine.

[0121]

(Example 5) Induction of immune tolerance in a patient receiving an organ transplant.

Tests were performed using a method similar to that described in Satoru Todo et al. Hepatorogy, 64 (vol. 2), 632-643 (2016). Briefly, anergic donor antigen-specific T cells were induced by co-culturing lymphocytes collected from the recipient's spleen or peripheral blood, donor-derived lymphocytes irradiated with 30 Gy of radiation, and humanized anti-CD80 and anti-CD86 IgG4 antibodies used in the example. 4. Anergic T cells were administered to a liver transplant recipient on day 13 after surgery. The status was then monitored.

[0122]

(Example 6) Chimeric antibodies to human CD80 and chimeric antibodies to human CD86 (IgG1 subclass)

Antibody drug

We have attempted to improve anti-human CD80 antibodies and anti-human CD86 antibodies for use in cell therapy. In particular, chimeric antibodies were prepared based on eBioscience's mouse anti-human CD80 antibody and mouse anti-human CD86 antibody. The resulting chimeric anti-human CD80 antibody (IgG1 subclass) consisted of a heavy chain with the amino acid sequence SEQ ID NO: 38 and a light chain with the amino acid sequence SEQ ID NO: 40. The resulting chimeric anti-human CD86 antibody (IgG1 subclass) consisted of a heavy chain with the amino acid sequence SEQ ID NO: 42 and a light chain with the amino acid sequence SEQ ID NO: 44. The plasmid encoding them was ordered from a third party (TPG Biologics, Inc. Taiwan) and expressed in CHO cells, and antibodies were produced/purified.

[0123]

Mixed lymphocyte culture reaction (MLR) in the presence of chimeric antibodies to human CD80 and chimeric antibodies to CD86 (IgG1 subclass)

IFNγ production

In essentially the same manner as the method described in Example 1, mononuclear cells (PBMCs) were isolated from human peripheral blood of two volunteers (one designated as donor and the other as recipient) using Promo cell lymphocyte medium (cat. No. C - 44010), and the cell concentration was adjusted to 2×10 ⁶ cells/ml in ALyS505N-0 medium containing 2% human serum type AB (pool), Biowest, (Cell Science & Technology Institute (CSTI) 1020P10). Donor PBMCs were irradiated with radiation (γ-rays) at a dose of 30 Gy.

[0124]

The experimental system was as follows.

1. “naïve”: recipient PBMC, 1×10 ⁶ cells.

2. “allo”: recipient PBMC, 1×10 ⁶ cells, and donor PBMC, 1×10 ⁶ cells.

3. “Chimeric anti-CD80”: recipient PBMC, 1x10 ⁶ cells, and chimeric anti-human CD80 antibody, 4 μg.

4. "Chimeric anti-CD86": recipient PBMC, 1x10 ⁶ cells, and chimeric anti-human CD86 antibody, 4 μg.

Systems 1-4 were placed in each well of a 96-well plate (Corning, cat. no. 3799) with a final reaction volume of 200 μl (final antibody concentration was approximately 20 μg/ml), and cultured in a 5% CO ₂ incubator at 37°C C. On days 5 and 6 from the start of cultivation, the supernatant was collected. The collected sample was diluted 20-25 times and then measured using Biolegend's Human IFNγ ELISA MAX TM Deluxe, cat. No. 430104.

[0125]

Results

As shown in FIG. 8, neither the chimeric anti-human CD80 antibody nor the chimeric anti-human CD86 antibody demonstrated detectable IFNγ production.

[0126]

Measuring the amount of ^3H -thymidine incorporated

Experiments were generally performed in accordance with the methods described in Examples 1 and 4. Recipient PBMC and donor PBMC were used, freshly isolated from human peripheral blood or frozen at -80°C, which were quickly thawed and used. These cells were brought to a concentration of 4×10 ⁶ cells/ml in ALyS505N-0 medium containing 2% human serum type AB (pool), Biowest, (Cell Science & Technology Institute (CSTI) 1020P10). Donor PBMCs were irradiated at a dose of 20 Gy. Donor PBMC and recipient PBMC were mixed in a 1:1 ratio, and anti-human CD80 chimeric antibody and anti-human CD86 chimeric antibody were added to the mixture to a final concentration of each antibody of 10 μg/ml. Cells were cultured for 7 days in an incubator at 5% CO ₂ at 37°C in a Petri dish with a diameter of 6 to 10 cm (culture volume from 6 to 18 ml).

[0127]

On day 7 from the start of culture, cultured recipient PBMCs were collected by centrifugation and adjusted to a concentration of 4×10 ⁶ cells/ml in the medium described above. Freshly prepared and irradiated donor PBMCs were added to cultured recipient PBMCs so that the cell number ratio was 2:1, and anti-human CD80 chimeric antibody and anti-human CD86 chimeric antibody were added to a final concentration of each antibody of 5 μg/ml. Cells were cultured for 7 days under the same conditions as described above (cell density: 4×10 ⁶ cells/ml).

[0128]

On day 14 after the start of culture, the induced cells were collected by centrifugation. Each cell was mixed in the following ratio:

1. “naïve”: recipient PBMC, 4×10 ⁵ cells

2. “allo”: recipient PBMC, 4x10 ⁵ cells, and donor PBMC, 4x10 ⁵ cells.

3. “1:1/2”: recipient PBMC, 4x10 ⁵ cells, donor PBMC, 4x10 ⁵ cells, and induced cells, 2x10 ⁵ cells.

4. “1:1/4”: recipient PBMC, 4x10 ⁵ cells, donor PBMC, 4x10 ⁵ cells, and induced cells, 1x10 ⁵ cells.

5. “1:1/8”: recipient PBMC, 4x10 ⁵ cells, donor PBMC, 4x10 ⁵ cells, and induced cells, 5x10 ⁴ cells.

6. “1:1/16”: recipient PBMC, 4x10 ⁵ cells, donor PBMC, 4x10 ⁵ cells, and induced cells, 2.5x10 ⁴ cells.

Each of systems 1-6 was added to 4 wells of a 96-well plate (Corning, cat. no. 3799) and co-cultured in a 5% CO ₂ incubator at 37°C. On day 4 after the start of co-culture, ³ H-thymidine (10 μl) was added. On day 5 from the start of co-culture (16-20 hours after the addition of ³ H-thymidine), ³ H-thymidine was removed from the culture to measure the amount of ³ H-thymidine incorporated.

[0129]

results

As shown in FIG. 9, induced cells reduced the amount of incorporated ³ H-thymidine in a dose-dependent manner (ie immune cell activation was suppressed). Thus, it has been demonstrated that anti-human CD80 chimeric antibodies and anti-human CD86 chimeric antibodies are antibodies suitable for cell therapy and can induce anergic T cells without causing deleterious IFNγ production.

[0130]

[Note]

As described above, the present invention is illustrated using its preferred embodiments. However, it should be understood that the scope of the present disclosure should be interpreted solely on the basis of the claims. It is also understood that any patent, any patent application and any other references cited herein are incorporated herein by reference in the same manner as if their contents were disclosed herein. The present application claims priority to Japanese Patent Application No. 2018-118996 (filed June 22, 2018). It is understood that its contents (maybe the entire document) are incorporated herein by reference. In addition, part or all of the contents of Japanese Patent Application No. 2018-119001 and Japanese Patent Application No. 2018-1190031 (both filed on June 22, 2018) and international applications claiming priority are incorporated herein by reference.

[Industrial applicability]

[0131]

The present description provides an antibody with a structure capable of improving the effect of immune tolerance or not weakening the effect of immune tolerance. A technology is proposed that can be used in industries (pharmaceuticals) related to the preparation of compounds, etc., based on such technology.

[Free text list of sequences]

[0132]

SEQ ID NO: 1: Anti-CD80 antibody heavy chain nucleic acid sequence (IgG2).

SEQ ID NO: 2: Amino acid sequence of anti-CD80 antibody heavy chain (IgG2).

SEQ ID NO: 3: Anti-CD80 antibody light chain nucleic acid sequence (IgG2).

SEQ ID NO: 4: Amino acid sequence of anti-CD80 antibody light chain (IgG2).

SEQ ID NO: 5: Anti-CD80 antibody heavy chain nucleic acid sequence (IgG4).

SEQ ID NO: 6: Amino acid sequence of anti-CD80 antibody heavy chain (IgG4).

SEQ ID NO: 7: Anti-CD80 light chain nucleic acid sequence (IgG4).

SEQ ID NO: 8: Amino acid sequence of anti-CD80 antibody light chain (IgG4).

SEQ ID NO: 9: Anti-CD86 antibody heavy chain nucleic acid sequence (IgG2).

SEQ ID NO: 10: Amino acid sequence of anti-CD86 antibody heavy chain (IgG2).

SEQ ID NO: 11: Anti-CD86 antibody light chain nucleic acid sequence (IgG2).

SEQ ID NO: 12: Amino acid sequence of anti-CD86 antibody light chain (IgG2).

SEQ ID NO: 13: Anti-CD86 antibody heavy chain nucleic acid sequence (IgG4).

SEQ ID NO: 14: Amino acid sequence of anti-CD86 antibody heavy chain (IgG4).

SEQ ID NO: 15: Anti-CD86 antibody light chain nucleic acid sequence (IgG4).

SEQ ID NO: 16: Amino acid sequence of anti-CD86 antibody light chain (IgG4).

SEQ ID NO: 17: Nucleic acid sequence of the anti-CD80 antibody heavy chain variable region (IgG2).

SEQ ID NO: 18: Amino acid sequence of the anti-CD80 antibody heavy chain variable region (IgG2).

SEQ ID NO: 19: Anti-CD80 antibody light chain variable region nucleic acid sequence (IgG2).

SEQ ID NO: 20: Amino acid sequence of the anti-CD80 antibody light chain variable region (IgG2).

SEQ ID NO: 21: Anti-CD86 antibody heavy chain variable region nucleic acid sequence (IgG2).

SEQ ID NO: 22: Amino acid sequence of the anti-CD86 antibody heavy chain variable region (IgG2).

SEQ ID NO: 23: Anti-CD86 antibody light chain variable region nucleic acid sequence (IgG2).

SEQ ID NO: 24: Amino acid sequence of the anti-CD86 antibody light chain variable region (IgG2).

SEQ ID NO: 25: Amino acid sequence of anti-CD80 anti-(IgG2) antibody CDRH1.

SEQ ID NO: 26: CDRH2 amino acid sequence of anti-CD80 antibody (IgG2).

SEQ ID NO: 27: CDRH3 amino acid sequence of anti-CD80 antibody (IgG2).

SEQ ID NO: 28: Amino acid sequence of anti-CD80 antibody CDRL1 (IgG2).

SEQ ID NO: 29: amino acid sequence of anti-CD80 antibody CDRL2 (IgG2).

SEQ ID NO: 30: amino acid sequence of anti-CD80 antibody CDRL3 (IgG2).

SEQ ID NO: 31: CDRH1 amino acid sequence of anti-CD86 antibody (IgG2).

SEQ ID NO: 32: CDRH2 amino acid sequence of anti-CD86 antibody (IgG2).

SEQ ID NO: 33: CDRH3 amino acid sequence of anti-CD86 antibody (IgG2).

SEQ ID NO: 34: amino acid sequence of anti-CD86 antibody CDRL1 (IgG2).

SEQ ID NO: 35: Amino acid sequence of anti-CD86 antibody CDRL2 (IgG2).

SEQ ID NO: 36: Amino acid sequence of anti-CD86 antibody CDRL3 (IgG2).

SEQ ID NO: 37: Nucleic acid sequence of the chimeric anti-CD80 antibody heavy chain.

SEQ ID NO: 38: Amino acid sequence of the chimeric anti-CD80 antibody heavy chain.

SEQ ID NO: 39: Nucleic acid sequence of the chimeric anti-CD80 antibody light chain.

SEQ ID NO: 40: Amino acid sequence of the chimeric anti-CD80 antibody light chain.

SEQ ID NO: 41: Nucleic acid sequence of the chimeric anti-CD86 antibody heavy chain.

SEQ ID NO: 42: Amino acid sequence of the chimeric anti-CD86 antibody heavy chain.

SEQ ID NO: 43: Nucleic acid sequence of the chimeric anti-CD86 antibody light chain.

SEQ ID NO: 44: Amino acid sequence of the chimeric anti-CD86 antibody light chain.

SEQ ID NO: 45: Nucleic acid sequence of the heavy chain variable region of the chimeric anti-CD80 antibody.

SEQ ID NO: 46: Amino acid sequence of the heavy chain variable region of the chimeric anti-CD80 antibody.

SEQ ID NO: 47: Nucleic acid sequence of the light chain variable region of the chimeric anti-CD80 antibody.

SEQ ID NO: 48: Amino acid sequence of the light chain variable region of the chimeric anti-CD80 antibody.

SEQ ID NO: 49: Nucleic acid sequence of the heavy chain variable region of the chimeric anti-CD86 antibody.

SEQ ID NO: 50: Amino acid sequence of the heavy chain variable region of the chimeric anti-CD86 antibody.

SEQ ID NO: 51: Nucleic acid sequence of the light chain variable region of the chimeric anti-CD86 antibody.

SEQ ID NO: 52: Amino acid sequence of the light chain variable region of the chimeric anti-CD86 antibody.

SEQ ID NO: 53: Amino acid sequence of CDRH1 chimeric anti-CD80 antibody.

SEQ ID NO: 54: CDRH2 amino acid sequence of chimeric anti-CD80 antibody.

SEQ ID NO: 55: CDRH3 amino acid sequence of chimeric anti-CD80 antibody.

SEQ ID NO: 56: Amino acid sequence of CDRL1 chimeric anti-CD80 antibody.

SEQ ID NO: 57: Amino acid sequence of CDRL2 chimeric anti-CD80 antibody.

SEQ ID NO: 58: Amino acid sequence of CDRL3 chimeric anti-CD80 antibody.

SEQ ID NO: 59: Amino acid sequence of CDRH1 chimeric anti-CD86 antibody.

SEQ ID NO: 60: CDRH2 amino acid sequence of chimeric anti-CD86 antibody.

SEQ ID NO: 61: CDRH3 amino acid sequence of chimeric anti-CD86 antibody.

SEQ ID NO: 62: Amino acid sequence of CDRL1 chimeric anti-CD86 antibody.

SEQ ID NO: 63: Amino acid sequence of CDRL2 chimeric anti-CD86 antibody.

SEQ ID NO: 64: Amino acid sequence of CDRL3 chimeric anti-CD86 antibody.

--->

LIST OF SEQUENCES

<110> DZUNTEN BIO CO., LTD.

<120> Antibodies inducing immune tolerance, induced

lymphocytes, and a therapeutic agent/method of cell therapy with

using induced lymphocytes

<130>JB002PCT

<150> JP 2018-118996

<151> 2018-06-22

<160> 64

<170> Patent In version 3.5

<210> 1

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<223> antibody

<220>

<221>CDS

<222> (1)..(1347)

<400> 1

gag gtg cag ttg gtt gaa tct ggc gga gga ctg gtt cag cct ggc gga

48

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

1 5 10 15

tct ctg aga ctg tct tgt gcc gcc tct ggc ttc gtg ttc agc tcc gac

96

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Asp

20 25 30

gat atg tcc tgg gtc cga cag gct cct ggc aaa gga ttg gag tgg gtc

144

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

gcc tac atc tct tct ggc ggc gac tac acc tac tat ccc gac acc gtg

192

Ala Tyr Ile Ser Ser Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

aag ggc aga ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tac

240

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

65 70 75 80

ctg cag atg aac agc ctg aga 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

gcc aga cac ctg tac ggc tcc tcc agc tac tac gtg atg gac tat tgg

336

Ala Arg His Leu Tyr Gly Ser Ser Ser Tyr Tyr Val Met Asp Tyr Trp

100 105 110

ggc cag ggc acc ctg gtc aca gtg tcc tct gct tcc acc aag gga ccc

384

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

115 120 125

agc gtt ttc cct ctg gct cct tgc tcc aga tcc acc tcc gag tct aca

432

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

gct gct ctg ggc tgc ctg gtc aag gac tac ttt cct gag cct gtg acc

480

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

gtg tcc tgg aac tct ggc gct ctg aca tct ggc gtg cac acc ttt cca

528

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

gct gtg ctg cag tcc tct ggc ctg tac tct ctg tcc tcc gtc gtg acc

576

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

gtg cct tcc tct aac ttt ggc acc cag acc tac acc tgt aat gtg gac

624

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

cac aag cct tcc aac acc aag gtg gac aag acc gtg gaa cgg aag tgc

672

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

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720

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

gtg ttt ctg ttc cct cca aag cct aag gac acc ctg atg atc tct cgg

768

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

acc cct gaa gtg acc tgc gtg gtg gtg gat gtg tct cac gag gat ccc

816

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

gaa gtg cag ttc aat tgg tac gtg gac ggc gtg gaa gtg cac aac gcc

864

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

aag acc aag cct aga gag gaa cag ttc aac tcc acc ttc aga gtg gtg

912

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val

290 295 300

tcc gtg ctg acc gtg gtg cat cag gat tgg ctg aac ggc aaa gag tac

960

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

305 310 315 320

aag tgc aag gtg tcc aac aag ggc ctg cct gct cct atc gaa aag acc

1008

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

atc tct aag acc aag ggg cag ccc cgg gaa cct cag gtt tac aca ctg

1056

Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

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cct cca agc cgg gaa gag atg acc aag aac cag gtg tcc ctg acc tgc

1104

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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1152

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

aat ggc cag cct gag aac aac tac aag acc aca cct cct atg ctg gac

1200

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp

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1248

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

405 410 415

aga tgg cag cag ggc aac gtg ttc tcc tgc tcc gtg atg cac gag gcc

1296

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

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1344

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

tga

1347

<210> 2

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Asp

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Ala Tyr Ile Ser Ser Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Thr Val

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

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

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Ala Arg His Leu Tyr Gly Ser Ser Ser Tyr Tyr Val Met Asp Tyr Trp

100 105 110

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

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val

290 295 300

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

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

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Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

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Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp

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

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Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

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Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

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

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gac aga atc acc atc acc tgt cac gcc agc cag aac atc aac gtg tgg

96

Asp Arg Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp

20 25 30

ctg tcc tgg tat cag cag aag ccc ggc aag atc ccc aag ctg ctg gtg

144

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ile Pro Lys Leu Leu Val

35 40 45

tac aag gcc tcc aat ctg cac acc ggc gtg ccc tct aga ttc tcc gga

192

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

tct ggc tct ggc acc gac ttt acc ctg aca atc tcc agc ctg cag cct

240

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

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gag gac ttc gcc acc tac tat tgt cag cag ggc cag agc tac cct ctg

288

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

85 90 95

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336

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

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cct tcc gtg ttc atc ttc cca cct tcc gac gag cag ctg aag tcc ggc

384

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

aca gct tct gtc gtg tgc ctg ctg aac aac ttc tac cct cgg gaa gcc

432

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

aag gtg cag tgg aag gtg gac aat gcc ctg cag tcc ggc aac tcc caa

480

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

gag tct gtg acc gag cag gac tcc aag gac agc acc tac agc ctg tcc

528

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

tcc aca ctg acc ctg tcc aag gcc gac tac gag aag cac aag gtg tac

576

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

180 185 190

gcc tgc gaa gtg acc cat cag ggc ctg tct agc cct gtg acc aag tct

624

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

195 200 205

ttc aac cgg ggc gag tgc tga

645

Phe Asn Arg Gly Glu Cys

210

<210> 4

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

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

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Asp Arg Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp

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Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ile Pro Lys Leu Leu Val

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 5

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> antibody

<220>

<221>CDS

<222> (1)..(1350)

<400> 5

gag gtg cag ttg gtt gaa tct ggc gga gga ctg gtt cag cct ggc gga

48

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

1 5 10 15

tct ctg aga ctg tct tgt gcc gcc tct ggc ttc gtg ttc agc tcc gac

96

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Asp

20 25 30

gat atg tcc tgg gtc cga cag gct cct ggc aaa gga ttg gag tgg gtc

144

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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192

Ala Tyr Ile Ser Ser Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

aag ggc aga ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tac

240

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

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288

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

85 90 95

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336

Ala Arg His Leu Tyr Gly Ser Ser Ser Tyr Tyr Val Met Asp Tyr Trp

100 105 110

ggc cag ggc acc ctg gtc aca gtg tcc tct gct tct acc aag ggc ccc

384

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

115 120 125

tcc gtg ttc cct ctg gcc cct tgc tcc aga tcc acc tcc gag tct acc

432

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

gcc gct ctg ggc tgc ctc gtg aag gac tac ttc ccc gag ccc gtg aca

480

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

gtg tcc tgg aac tct ggc gct ctg acc tcc ggc gtg cac acc ttt cca

528

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

gca gtg ctg cag tcc tcc ggc ctg tac tcc ctg tcc tcc gtc gtg act

576

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

gtg ccc tcc agc tct ctg ggc acc aag acc tac acc tgt aac gtg gac

624

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

195 200 205

cac aag ccc tcc aac acc aag gtg gac aag aga gtg gaa tct aag tac

672

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

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720

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

225 230 235 240

agc gtg ttc ctg ttc ccc cca aag ccc aag gac acc ctg atg atc tcc

768

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

cgg acc ccc gaa gtg acc tgc gtg gtg gtg gat gtg tcc cag gaa gat

816

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

260 265 270

ccc gag gtg cag ttc aat tgg tac gtg gac ggc gtg gaa gtg cac aac

864

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

gcc aag acc aag cct aga gag gaa cag ttc aac tcc acc tac cgg gtg

912

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

290 295 300

gtg tcc gtg ctg acc gtg ctg cac cag gat tgg ctg aac ggc aaa gag

960

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

tac aag tgc aag gtg tcc aac aag ggc ctg ccc agc tcc atc gaa aag

1008

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

325 330 335

acc atc tcc aag gcc aag ggc cag ccc cgg gaa ccc cag gtg tac aca

1056

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

ctg cct cca agc cag gaa gag atg acc aag aac cag gtg tcc ctg acc

1104

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

tgt ctc gtg aaa ggc ttc tac ccc tcc gat atc gcc gtg gaa tgg gag

1152

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

tcc aac ggc cag cct gag aac aac tac aag acc acc ccc cct gtg ctg

1200

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

gac tcc gac ggc tcc ttc ttc ctg tac tct cgc ctg acc gtg gac aag

1248

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

405 410 415

tcc cgg tgg cag gaa ggc aac gtg ttc tcc tgc tcc gtg atg cac gag

1296

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

gcc ctg cac aac cac tac acc cag aag tcc ctg tct ctg tcc ctg ggc

1344

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

aag tga

1350

Lys

<210> 6

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 6

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Asp

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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

Ala Arg His Leu Tyr Gly Ser Ser Ser Tyr Tyr Val Met Asp Tyr Trp

100 105 110

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

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

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

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

Lys

<210> 7

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<223> antibody

<220>

<221>CDS

<222> (1)..(645)

<400> 7

gac atc cag atg acc cag tct cca tcc tct ctg tct gcc agc ctg ggc

48

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

1 5 10 15

gac aga atc acc atc acc tgt cac gcc agc cag aac atc aac gtg tgg

96

Asp Arg Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp

20 25 30

ctg tcc tgg tat cag cag aag ccc ggc aag atc ccc aag ctg ctg gtg

144

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ile Pro Lys Leu Leu Val

35 40 45

tac aag gcc tcc aat ctg cac acc ggc gtg ccc tct aga ttc tcc gga

192

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

tct ggc tct ggc acc gac ttt acc ctg aca atc tcc agc ctg cag cct

240

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

65 70 75 80

gag gac ttc gcc acc tac tat tgt cag cag ggc cag agc tac cct ctg

288

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

85 90 95

acc ttt ggc gct ggc acc aag ctg gaa atc aag cgg aca gtg gcc gct

336

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

cct tcc gtg ttc atc ttc cca cct tcc gac gag cag ctg aag tcc ggc

384

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

aca gct tct gtc gtg tgc ctg ctg aac aac ttc tac cct cgg gaa gcc

432

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

aag gtg cag tgg aag gtg gac aat gcc ctg cag tcc ggc aac tcc caa

480

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

gag tct gtg acc gag cag gac tcc aag gac agc acc tac agc ctg tcc

528

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

tcc aca ctg acc ctg tcc aag gcc gac tac gag aag cac aag gtg tac

576

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

180 185 190

gcc tgc gaa gtg acc cat cag ggc ctg tct agc cct gtg acc aag tct

624

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

195 200 205

ttc aac cgg ggc gag tgc tga

645

Phe Asn Arg Gly Glu Cys

210

<210> 8

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 8

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

1 5 10 15

Asp Arg Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ile Pro Lys Leu Leu Val

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 9

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<223> antibody

<220>

<221>CDS

<222> (1)..(1347)

<400> 9

cag gtg cag ttg gtt cag tct ggc gcc gaa gtg aag aaa cct ggc gcc

48

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

1 5 10 15

tct gtg aag gtg tcc tgc aag gct tcc ggc tac acc ttt acc agc tac

96

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

20 25 30

gtg atc cac tgg gtc cga cag gct cca gga caa ggc ttg gag tgg atg

144

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

35 40 45

ggc tac atc aac ccc tac cac gac gtg acc aag tac aac gag aag ttc

192

Gly Tyr Ile Asn Pro Tyr His Asp Val Thr Lys Tyr Asn Glu Lys Phe

50 55 60

cag ggc aga gtg acc atg acc aga gac acc tcc acc agc acc gtg tac

240

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

atg gaa ctg tcc agc ctg aga tcc gag gac acc gcc gtg tac tac tgc

288

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

85 90 95

gcc aga cag ggc gac cct tac tcc ggc aac tac cag ttt acc tac tgg

336

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

100 105 110

ggc cag ggc acc ctg gtc aca gtt tct tcc gct tcc acc aag gga ccc

384

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

115 120 125

agc gtt ttc cct ctg gct cct tgc tcc aga tcc acc tcc gag tct aca

432

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

gct gct ctg ggc tgc ctg gtc aag gac tac ttt cct gag cct gtg acc

480

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

gtg tcc tgg aac tct ggc gct ctg aca tct ggc gtg cac acc ttt cca

528

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

gct gtg ctg cag tcc tct ggc ctg tac tct ctg tcc tcc gtc gtg acc

576

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

gtg cct tcc tct aac ttt ggc acc cag acc tac acc tgt aat gtg gac

624

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

cac aag cct tcc aac acc aag gtg gac aag acc gtg gaa cgg aag tgc

672

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

tgc gtg gaa tgc cct cct tgt cct gct cct cct gtg gct ggc cct tcc

720

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

gtg ttt ctg ttc cct cca aag cct aag gac acc ctg atg atc tct cgg

768

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

acc cct gaa gtg acc tgc gtg gtg gtg gat gtg tct cac gag gat ccc

816

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

gaa gtg cag ttc aat tgg tac gtg gac ggc gtg gaa gtg cac aac gcc

864

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

aag acc aag cct aga gag gaa cag ttc aac tcc acc ttc aga gtg gtg

912

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val

290 295 300

tcc gtg ctg acc gtg gtg cat cag gat tgg ctg aac ggc aaa gag tac

960

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

305 310 315 320

aag tgc aag gtg tcc aac aag ggc ctg cct gct cct atc gaa aag acc

1008

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

atc tct aag acc aag ggg cag ccc cgg gaa cct cag gtt tac aca ctg

1056

Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

cct cca agc cgg gaa gag atg acc aag aac cag gtg tcc ctg acc tgc

1104

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

ctc gtg aag ggc ttc tac cct tcc gat atc gcc gtg gaa tgg gag agc

1152

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

aat ggc cag cct gag aac aac tac aag acc aca cct cct atg ctg gac

1200

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp

385 390 395 400

tcc gac ggc tca ttc ttc ctg tac tcc aag ctg aca gtg gac aag tcc

1248

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

405 410 415

aga tgg cag cag ggc aac gtg ttc tcc tgc tcc gtg atg cac gag gcc

1296

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

ctg cac aat cac tac acc cag aag tcc ctg tct ctg tcc cct ggc aaa

1344

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

tga

1347

<210> 10

<211> 448

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Tyr Ile Asn Pro Tyr His Asp Val Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val

290 295 300

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

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp

385 390 395 400

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

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 11

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<223> antibody

<220>

<221>CDS

<222> (1)..(645)

<400> 11

gac atc cag atg acc cag tct cca tcc tct ctg tcc gcc tct gtg ggc

48

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

1 5 10 15

gac aga gtg acc atc acc tgt aga gcc tct cag gac gtg tcc acc gag

96

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

20 25 30

gtc gtg tgg tat cag cag aag cct ggc aag gcc cct aag ctg ctg atc

144

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

35 40 45

tac tcc gcc tcc tac cgg tac tct ggc gtg ccc tct aga ttc tcc ggc

192

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

50 55 60

tct ggc tct ggc acc gac ttt acc ctg aca atc tcc agc ctg cag cct

240

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

65 70 75 80

gag gac ttc gcc acc tac tac tgc cag cag cac tac agc acc cct tac

288

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

85 90 95

acc ttt ggc cag ggc acc aag ctg gaa atc aag cgg aca gtg gcc gct

336

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

cct tcc gtg ttc atc ttc cca cct tcc gac gag cag ctg aag tcc ggc

384

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

aca gct tct gtc gtg tgc ctg ctg aac aac ttc tac cct cgg gaa gcc

432

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

aag gtg cag tgg aag gtg gac aat gcc ctg cag tcc ggc aac tcc caa

480

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

gag tct gtg acc gag cag gac tcc aag gac agc acc tac agc ctg tcc

528

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

tcc aca ctg acc ctg tcc aag gcc gac tac gag aag cac aag gtg tac

576

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

180 185 190

gcc tgc gaa gtg acc cat cag ggc ctg tct agc cct gtg acc aag tct

624

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

195 200 205

ttc aac cgg ggc gag tgc tga

645

Phe Asn Arg Gly Glu Cys

210

<210> 12

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 12

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 Asp Val Ser Thr Glu

20 25 30

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

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr 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 His Tyr Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 13

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> antibody

<220>

<221>CDS

<222> (1)..(1350)

<400> 13

cag gtg cag ttg gtt cag tct ggc gcc gaa gtg aag aaa cct ggc gcc

48

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

1 5 10 15

tct gtg aag gtg tcc tgc aag gct tcc ggc tac acc ttt acc agc tac

96

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

20 25 30

gtg atc cac tgg gtc cga cag gct cca gga caa ggc ttg gag tgg atg

144

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

35 40 45

ggc tac atc aac ccc tac cac gac gtg acc aag tac aac gag aag ttc

192

Gly Tyr Ile Asn Pro Tyr His Asp Val Thr Lys Tyr Asn Glu Lys Phe

50 55 60

cag ggc aga gtg acc atg acc aga gac acc tcc acc agc acc gtg tac

240

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

atg gaa ctg tcc agc ctg aga tcc gag gac acc gcc gtg tac tac tgc

288

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

85 90 95

gcc aga cag ggc gac cct tac tcc ggc aac tac cag ttt acc tac tgg

336

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

100 105 110

ggc cag ggc acc ctg gtc aca gtt tct tcc gct tct acc aag ggc ccc

384

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

115 120 125

tcc gtg ttc cct ctg gcc cct tgc tcc aga tcc acc tcc gag tct acc

432

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

gcc gct ctg ggc tgc ctc gtg aag gac tac ttc ccc gag ccc gtg aca

480

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

gtg tcc tgg aac tct ggc gct ctg acc tcc ggc gtg cac acc ttt cca

528

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

gca gtg ctg cag tcc tcc ggc ctg tac tcc ctg tcc tcc gtc gtg act

576

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

gtg ccc tcc agc tct ctg ggc acc aag acc tac acc tgt aac gtg gac

624

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

195 200 205

cac aag ccc tcc aac acc aag gtg gac aag aga gtg gaa tct aag tac

672

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

ggc cct ccc tgc ccc cct tgt cct gcc cct gaa ttt ctg ggc gga ccc

720

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

225 230 235 240

agc gtg ttc ctg ttc ccc cca aag ccc aag gac acc ctg atg atc tcc

768

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

cgg acc ccc gaa gtg acc tgc gtg gtg gtg gat gtg tcc cag gaa gat

816

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

260 265 270

ccc gag gtg cag ttc aat tgg tac gtg gac ggc gtg gaa gtg cac aac

864

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

gcc aag acc aag cct aga gag gaa cag ttc aac tcc acc tac cgg gtg

912

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

290 295 300

gtg tcc gtg ctg acc gtg ctg cac cag gat tgg ctg aac ggc aaa gag

960

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

tac aag tgc aag gtg tcc aac aag ggc ctg ccc agc tcc atc gaa aag

1008

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

325 330 335

acc atc tcc aag gcc aag ggc cag ccc cgg gaa ccc cag gtg tac aca

1056

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

ctg cct cca agc cag gaa gag atg acc aag aac cag gtg tcc ctg acc

1104

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

tgt ctc gtg aaa ggc ttc tac ccc tcc gat atc gcc gtg gaa tgg gag

1152

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

tcc aac ggc cag cct gag aac aac tac aag acc acc ccc cct gtg ctg

1200

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

gac tcc gac ggc tcc ttc ttc ctg tac tct cgc ctg acc gtg gac aag

1248

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

405 410 415

tcc cgg tgg cag gaa ggc aac gtg ttc tcc tgc tcc gtg atg cac gag

1296

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

gcc ctg cac aac cac tac acc cag aag tcc ctg tct ctg tcc ctg ggc

1344

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

aag tga

1350

Lys

<210> 14

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Tyr Ile Asn Pro Tyr His Asp Val Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

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

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

Lys

<210> 15

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<223> antibody

<220>

<221>CDS

<222> (1)..(645)

<400> 15

gac atc cag atg acc cag tct cca tcc tct ctg tcc gcc tct gtg ggc

48

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

1 5 10 15

gac aga gtg acc atc acc tgt aga gcc tct cag gac gtg tcc acc gag

96

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

20 25 30

gtc gtg tgg tat cag cag aag cct ggc aag gcc cct aag ctg ctg atc

144

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

35 40 45

tac tcc gcc tcc tac cgg tac tct ggc gtg ccc tct aga ttc tcc ggc

192

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

50 55 60

tct ggc tct ggc acc gac ttt acc ctg aca atc tcc agc ctg cag cct

240

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

65 70 75 80

gag gac ttc gcc acc tac tac tgc cag cag cac tac agc acc cct tac

288

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

85 90 95

acc ttt ggc cag ggc acc aag ctg gaa atc aag cgg aca gtg gcc gct

336

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

cct tcc gtg ttc atc ttc cca cct tcc gac gag cag ctg aag tcc ggc

384

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

aca gct tct gtc gtg tgc ctg ctg aac aac ttc tac cct cgg gaa gcc

432

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

aag gtg cag tgg aag gtg gac aat gcc ctg cag tcc ggc aac tcc caa

480

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

gag tct gtg acc gag cag gac tcc aag gac agc acc tac agc ctg tcc

528

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

tcc aca ctg acc ctg tcc aag gcc gac tac gag aag cac aag gtg tac

576

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

180 185 190

gcc tgc gaa gtg acc cat cag ggc ctg tct agc cct gtg acc aag tct

624

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

195 200 205

ttc aac cgg ggc gag tgc tga

645

Phe Asn Arg Gly Glu Cys

210

<210> 16

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 16

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 Asp Val Ser Thr Glu

20 25 30

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

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr 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 His Tyr Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 17

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> antibody variable region

<220>

<221>CDS

<222> (1)..(366)

<400> 17

gag gtg cag ttg gtt gaa tct ggc gga gga ctg gtt cag cct ggc gga

48

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

1 5 10 15

tct ctg aga ctg tct tgt gcc gcc tct ggc ttc gtg ttc agc tcc gac

96

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Asp

20 25 30

gat atg tcc tgg gtc cga cag gct cct ggc aaa gga ttg gag tgg gtc

144

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

gcc tac atc tct tct ggc ggc gac tac acc tac tat ccc gac acc gtg

192

Ala Tyr Ile Ser Ser Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

aag ggc aga ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tac

240

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

65 70 75 80

ctg cag atg aac agc ctg aga 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

gcc aga cac ctg tac ggc tcc tcc agc tac tac gtg atg gac tat tgg

336

Ala Arg His Leu Tyr Gly Ser Ser Ser Tyr Tyr Val Met Asp Tyr Trp

100 105 110

ggc cag ggc acc ctg gtc aca gtg tcc tct

366

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 18

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Asp

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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

Ala Arg His Leu Tyr Gly Ser Ser Ser Tyr Tyr Val Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> antibody variable region

<220>

<221>CDS

<222> (1)..(321)

<400> 19

gac atc cag atg acc cag tct cca tcc tct ctg tct gcc agc ctg ggc

48

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

1 5 10 15

gac aga atc acc atc acc tgt cac gcc agc cag aac atc aac gtg tgg

96

Asp Arg Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp

20 25 30

ctg tcc tgg tat cag cag aag ccc ggc aag atc ccc aag ctg ctg gtg

144

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ile Pro Lys Leu Leu Val

35 40 45

tac aag gcc tcc aat ctg cac acc ggc gtg ccc tct aga ttc tcc gga

192

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

tct ggc tct ggc acc gac ttt acc ctg aca atc tcc agc ctg cag cct

240

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

65 70 75 80

gag gac ttc gcc acc tac tat tgt cag cag ggc cag agc tac cct ctg

288

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

85 90 95

acc ttt ggc gct ggc acc aag ctg gaa atc aag

321

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 20

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 20

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

1 5 10 15

Asp Arg Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ile Pro Lys Leu Leu Val

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 21

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> antibody variable region

<220>

<221>CDS

<222> (1)..(366)

<400> 21

cag gtg cag ttg gtt cag tct ggc gcc gaa gtg aag aaa cct ggc gcc

48

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

1 5 10 15

tct gtg aag gtg tcc tgc aag gct tcc ggc tac acc ttt acc agc tac

96

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

20 25 30

gtg atc cac tgg gtc cga cag gct cca gga caa ggc ttg gag tgg atg

144

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

35 40 45

ggc tac atc aac ccc tac cac gac gtg acc aag tac aac gag aag ttc

192

Gly Tyr Ile Asn Pro Tyr His Asp Val Thr Lys Tyr Asn Glu Lys Phe

50 55 60

cag ggc aga gtg acc atg acc aga gac acc tcc acc agc acc gtg tac

240

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

atg gaa ctg tcc agc ctg aga tcc gag gac acc gcc gtg tac tac tgc

288

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

85 90 95

gcc aga cag ggc gac cct tac tcc ggc aac tac cag ttt acc tac tgg

336

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

100 105 110

ggc cag ggc acc ctg gtc aca gtt tct tcc

366

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 22

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Tyr Ile Asn Pro Tyr His Asp Val Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 23

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> antibody variable region

<220>

<221>CDS

<222> (1)..(321)

<400> 23

gac atc cag atg acc cag tct cca tcc tct ctg tcc gcc tct gtg ggc

48

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

1 5 10 15

gac aga gtg acc atc acc tgt aga gcc tct cag gac gtg tcc acc gag

96

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

20 25 30

gtc gtg tgg tat cag cag aag cct ggc aag gcc cct aag ctg ctg atc

144

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

35 40 45

tac tcc gcc tcc tac cgg tac tct ggc gtg ccc tct aga ttc tcc ggc

192

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

50 55 60

tct ggc tct ggc acc gac ttt acc ctg aca atc tcc agc ctg cag cct

240

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

65 70 75 80

gag gac ttc gcc acc tac tac tgc cag cag cac tac agc acc cct tac

288

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

85 90 95

acc ttt ggc cag ggc acc aag ctg gaa atc aag

321

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 24

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 24

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 Asp Val Ser Thr Glu

20 25 30

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

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr 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 His Tyr Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 25

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CDR

<400> 25

Ser Asp Asp Met Ser

15

<210> 26

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR

<400> 26

Tyr Ile Ser Ser Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Thr Val Lys

1 5 10 15

Gly

<210> 27

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> CDR

<400> 27

His Leu Tyr Gly Ser Ser Ser Tyr Tyr Val Met Asp Tyr

1 5 10

<210> 28

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> CDR

<400> 28

His Ala Ser Gln Asn Ile Asn Val Trp Leu Ser

1 5 10

<210> 29

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> CDR

<400> 29

Lys Ala Ser Asn Leu His Thr

15

<210> 30

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR

<400> 30

Gln Gln Gly Gln Ser Tyr Pro Leu Thr

15

<210> 31

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CDR

<400> 31

Ser Tyr Val Ile His

15

<210> 32

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR

<400> 32

Tyr Ile Asn Pro Tyr His Asp Val Thr Lys Tyr Asn Glu Lys Phe Gln

1 5 10 15

Gly

<210> 33

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> CDR

<400> 33

Gln Gly Asp Pro Tyr Ser Gly Asn Tyr Gln Phe Thr Tyr

1 5 10

<210> 34

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> CDR

<400> 34

Arg Ala Ser Gln Asp Val Ser Thr Glu Val Val

1 5 10

<210> 35

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> CDR

<400> 35

Ser Ala Ser Tyr Arg Tyr Ser

15

<210> 36

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR

<400> 36

Gln Gln His Tyr Ser Thr Pro Tyr Thr

15

<210> 37

<211> 1359

<212> DNA

<213> Artificial sequence

<220>

<223> chimeric anti-CD80 antibody heavy chain

<400> 37

gaagtgaagc tgttggagtc tggcggcgga ctggttaagc ctggcggatc tctgaagctg

60

tcttgtgccg cctctggctt cgtgttcagc tccgacgata tgtcctgggt ccgacagtct

120

cctgagaagc ggcttgagtg ggtcgcctac atctcttccg gcggagacta cacctactat

180

cccgacaccg tgaagggcag attcaccatc tccagagaca acgccaagaa caccctgtac

240

ctgcagatgt ccagcctgaa gtctgaggac accgccatgt actactgcgc cagacacctg

300

tacggctcct ccagctacta cgtgatggac tattggggcc agggcacctc tgtcacagtg

360

tcctctgctt ccaccaaggg acccagcgtg ttccctctgg ctccttccag caagtctacc

420

tctggcggaa cagctgctct gggctgcctg gtcaaggact actttcctga gcctgtgacc

480

gtgtcctgga actctggcgc tctgacatct ggcgtgcaca cattccctgc tgtgctgcag

540

tcctccggcc tgtactctct gtcctctgtc gtgaccgtgc cttccagctc tctgggaacc

600

cagacctaca tctgcaatgt gaaccacaag ccttccaaca ccaaggtgga caagaaggtg

660

gaacccaagt cctgcgacaa gacccacacc tgtcctccat gtcctgctcc agaactgctc

720

ggcggacctt ccgtgttcct gtttcctcca aagcctaagg acaccctgat gatctctcgg

780

acccctgaag tgacctgcgt ggtggtggat gtgtcccacg aggacccaga agtgaagttc

840

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcctag agaggaacag

900

tacaactcca cctacagagt ggtgtccgtg ctgaccgtgc tgcaccagga ttggctgaac

960

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgctcctat cgaaaagacc

1020

atctccaagg ccaagggcca gcctcgagaa ccccaggttt acaccttgcc tccatctcgg

1080

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc

1140

gacattgccg tggaatggga gtctaatggc cagccagaga acaactacaa gacaacccct

1200

cctgtgctgg actccgacgg ctcattcttc ctgtactcca agctgacagt ggacaagtcc

1260

agatggcagc agggcaacgt gttctcctgc tccgtgatgc acgaggccct gcacaatcac

1320

tacacacaga agtccctgtc tctgagcccc ggcaaatga

1359

<210> 38

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<223> chimeric anti-CD80 antibody heavy chain

<400> 38

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Asp

20 25 30

Asp Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

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

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Leu Tyr Gly Ser Ser Ser Tyr Tyr Val Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 39

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<223> chimeric anti-CD80 antibody light chain

<400> 39

gacatccaga tgacccagtc tccatcctct ctgtctgcca gcctgggcga caccatcacc

60

atcacatgtc acgccagcca gaacatcaac gtgtggctgt cctggtatca gcagaagccc

120

ggcaacatcc ccaagctgct ggtgtacaag gcctccaatc tgcacaccgg cgtgccctct

180

agattctccg gatctggctc tggcaccggc tttaccctga caatcagctc cttgcagccc

240

gaggacattg ccacctacta ctgtcagcag ggccagagct accctctgac ctttggcgct

300

ggcaccaagc tggaactgaa gcggacagtg gccgctcctt ccgtgttcat cttcccacct

360

tccgacgagc agctgaagtc cggcacagct tctgtcgtgt gcctgctgaa caacttctac

420

cctcgggaag ccaaggtgca gtggaaggtg gacaatgccc tgcagtccgg caactcccaa

480

gagtctgtga ccgagcagga ctccaaggac agcacctaca gcctgtcctc cacactgacc

540

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccatcagggc

600

ctgtctagcc ctgtgaccaa gtctttcaac cggggcgagt gctga

645

<210> 40

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> chimeric anti-CD80 antibody light chain

<400> 40

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

1 5 10 15

Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Val

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 41

<211> 1359

<212> DNA

<213> Artificial sequence

<220>

<223> chimeric anti-CD86 antibody heavy chain

<400> 41

gaggttcagt tgcagcagtc tggacccgag ctggtcaagc ctggcacctc cgtgaagatg

60

tcctgcaagg cctccggcta caccttcacc agctacgtga tccactgggt caaagagaag

120

ccaggccagg gccttgagtg gatcggctac atcaacccct accacgacgt gaccaagtac

180

aacgagaagt tcaagggcag agctaccctg acctccgaca agtcctcttc caccgcctac

240

atggaactgt ccagcctgac ctctgaggac tccgccgtgt actactgctc tagacagggc

300

gacccttact ccggcaacta ccagtttacc tactggggcc agggcaccct ggtcacagtt

360

tctgccgctt ccaccaaggg acccagcgtg ttccctctgg ctccttccag caagtctacc

420

tctggcggaa cagctgctct gggctgcctg gtcaaggact actttcctga gcctgtgacc

480

gtgtcctgga actctggcgc tctgacatct ggcgtgcaca cattccctgc tgtgctgcag

540

tcctccggcc tgtactctct gtcctctgtc gtgaccgtgc cttccagctc tctgggaacc

600

cagacctaca tctgcaatgt gaaccacaag ccttccaaca ccaaggtgga caagaaggtg

660

gaacccaagt cctgcgacaa gacccacacc tgtcctccat gtcctgctcc agaactgctc

720

ggcggacctt ccgtgttcct gtttcctcca aagcctaagg acaccctgat gatctctcgg

780

acccctgaag tgacctgcgt ggtggtggat gtgtcccacg aggacccaga agtgaagttc

840

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcctag agaggaacag

900

tacaactcca cctacagagt ggtgtccgtg ctgaccgtgc tgcaccagga ttggctgaac

960

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgctcctat cgaaaagacc

1020

atctccaagg ccaagggcca gcctcgagaa ccccaggttt acaccttgcc tccatctcgg

1080

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc

1140

gacattgccg tggaatggga gtctaatggc cagccagaga acaactacaa gacaacccct

1200

cctgtgctgg actccgacgg ctcattcttc ctgtactcca agctgacagt ggacaagtcc

1260

agatggcagc agggcaacgt gttctcctgc tccgtgatgc acgaggccct gcacaatcac

1320

tacacacaga agtccctgtc tctgagcccc ggcaaatga

1359

<210> 42

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<223> chimeric anti-CD86 antibody heavy chain

<400> 42

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

1 5 10 15

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

20 25 30

Val Ile His Trp Val Lys Glu Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr His Asp Val Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

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

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 43

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<223> chimeric anti-CD86 antibody light chain

<400> 43

gacatcgtga tgtcccagag ccacaagttc atgtccacct ccgtgggcga cagagtgtct

60

atcgcctgca aggcctctca ggacgtgtcc acagaggtcg tgtggttcca gcagaagcct

120

ggccagtctc ctaagctgct gatctactcc gcctcctaca gatacaccgg cgtgcccgat

180

agattcaccg gctctggctc tggcaccgac tttaccttca ccatcagctc cgtgcaggcc

240

gaggatctgg ccgtgtacta ctgtcagcag cactacagca ccccttacac ctttggcgga

300

ggcaccaagc tggaaatcaa gcggacagtg gccgctcctt ccgtgttcat cttcccacct

360

tccgacgagc agctgaagtc cggcacagct tctgtcgtgt gcctgctgaa caacttctac

420

cctcgggaag ccaaggtgca gtggaaggtg gacaatgccc tgcagtccgg caactcccaa

480

gagtctgtga ccgagcagga ctccaaggac agcacctaca gcctgtcctc cacactgacc

540

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccatcagggc

600

ctgtctagcc ctgtgaccaa gtctttcaac cggggcgagt gctga

645

<210> 44

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> chimeric anti-CD86 antibody light chain

<400> 44

Asp Ile Val Met Ser Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Ala Cys Lys Ala Ser Gln Asp Val Ser Thr Glu

20 25 30

Val Val Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 45

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> heavy chain variable region of chimeric anti-CD80 antibody

<400> 45

gaagtgaagc tgttggagtc tggcggcgga ctggttaagc ctggcggatc tctgaagctg

60

tcttgtgccg cctctggctt cgtgttcagc tccgacgata tgtcctgggt ccgacagtct

120

cctgagaagc ggcttgagtg ggtcgcctac atctcttccg gcggagacta cacctactat

180

cccgacaccg tgaagggcag attcaccatc tccagagaca acgccaagaa caccctgtac

240

ctgcagatgt ccagcctgaa gtctgaggac accgccatgt actactgcgc cagacacctg

300

tacggctcct ccagctacta cgtgatggac tattggggcc agggcacctc tgtcacagtg

360

tccctct

366

<210> 46

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> heavy chain variable region of chimeric anti-CD80 antibody

<400> 46

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Asp

20 25 30

Asp Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

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

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Leu Tyr Gly Ser Ser Ser Tyr Tyr Val Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 47

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> chimeric anti-CD80 antibody light chain variable region

<400> 47

gacatccaga tgacccagtc tccatcctct ctgtctgcca gcctgggcga caccatcacc

60

atcacatgtc acgccagcca gaacatcaac gtgtggctgt cctggtatca gcagaagccc

120

ggcaacatcc ccaagctgct ggtgtacaag gcctccaatc tgcacaccgg cgtgccctct

180

agattctccg gatctggctc tggcaccggc tttaccctga caatcagctc cttgcagccc

240

gaggacattg ccacctacta ctgtcagcag ggccagagct accctctgac ctttggcgct

300

ggcaccaagc tggaactgaa g

321

<210> 48

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> chimeric anti-CD80 antibody light chain variable region

<400> 48

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

1 5 10 15

Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Val

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 49

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> heavy chain variable region of chimeric anti-CD86 antibody

<400> 49

gaggttcagt tgcagcagtc tggacccgag ctggtcaagc ctggcacctc cgtgaagatg

60

tcctgcaagg cctccggcta caccttcacc agctacgtga tccactgggt caaagagaag

120

ccaggccagg gccttgagtg gatcggctac atcaacccct accacgacgt gaccaagtac

180

aacgagaagt tcaagggcag agctaccctg acctccgaca agtcctcttc caccgcctac

240

atggaactgt ccagcctgac ctctgaggac tccgccgtgt actactgctc tagacagggc

300

gacccttact ccggcaacta ccagtttacc tactggggcc agggcaccct ggtcacagtt

360

tctgcc

366

<210> 50

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> heavy chain variable region of chimeric anti-CD86 antibody

<400> 50

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

1 5 10 15

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

20 25 30

Val Ile His Trp Val Lys Glu Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr His Asp Val Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 51

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> chimeric anti-CD86 antibody light chain variable region

<400> 51

gacatcgtga tgtcccagag ccacaagttc atgtccacct ccgtgggcga cagagtgtct

60

atcgcctgca aggcctctca ggacgtgtcc acagaggtcg tgtggttcca gcagaagcct

120

ggccagtctc ctaagctgct gatctactcc gcctcctaca gatacaccgg cgtgcccgat

180

agattcaccg gctctggctc tggcaccgac tttaccttca ccatcagctc cgtgcaggcc

240

gaggatctgg ccgtgtacta ctgtcagcag cactacagca ccccttacac ctttggcgga

300

ggcaccaagc tggaaatcaa g

321

<210> 52

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> chimeric anti-CD86 antibody light chain variable region

<400> 52

Asp Ile Val Met Ser Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Ala Cys Lys Ala Ser Gln Asp Val Ser Thr Glu

20 25 30

Val Val Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 53

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CDRH1 chimeric anti-CD80 antibody

<400> 53

Ser Asp Asp Met Ser

15

<210> 54

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDRH2 chimeric anti-CD80 antibody

<400> 54

Tyr Ile Ser Ser Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Thr Val Lys

1 5 10 15

Gly

<210> 55

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> CDRH3 chimeric anti-CD80 antibody

<400> 55

His Leu Tyr Gly Ser Ser Ser Tyr Tyr Val Met Asp Tyr

1 5 10

<210> 56

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> CDRL1 chimeric anti-CD80 antibody

<400> 56

His Ala Ser Gln Asn Ile Asn Val Trp Leu Ser

1 5 10

<210> 57

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> CDRL2 chimeric anti-CD80 antibody

<400> 57

Lys Ala Ser Asn Leu His Thr

15

<210> 58

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDRL3 chimeric anti-CD80 antibody

<400> 58

Gln Gln Gly Gln Ser Tyr Pro Leu Thr

15

<210> 59

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CDRH1 chimeric anti-CD86 antibody

<400> 59

Ser Tyr Val Ile His

15

<210> 60

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDRH2 chimeric anti-CD86 antibody

<400> 60

Tyr Ile Asn Pro Tyr His Asp Val Thr Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 61

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> CDRH3 chimeric anti-CD86 antibody

<400> 61

Gln Gly Asp Pro Tyr Ser Gly Asn Tyr Gln Phe Thr Tyr

1 5 10

<210> 62

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> CDRL1 chimeric anti-CD86 antibody

<400> 62

Lys Ala Ser Gln Asp Val Ser Thr Glu Val Val

1 5 10

<210> 63

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> CDRL2 chimeric anti-CD86 antibody

<400> 63

Ser Ala Ser Tyr Arg Tyr Thr

15

<210> 64

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDRL3 chimeric anti-CD86 antibody

<400> 64

Gln Gln His Tyr Ser Thr Pro Tyr Thr

15

<---

Claims (34)

1. An antibody combination for inducing immune tolerance to a cell obtained from an individual, wherein the antibody combination comprises an anti-CD86 antibody and an anti-CD80 antibody, or variants thereof, wherein the anti-CD86 antibody has a heavy chain with the amino acid sequence of SEQ ID NO:42 and a light chain with the amino acid sequence of SEQ ID NO:44, wherein the antibody option is a Fab antibody, an F(ab') ₂ antibody, a Fab' antibody, an Fv antibody, or a scFv antibody, wherein the antibody combination does not substantially induce cytokine production by immunostimulation and reduces 3H-thymidine reuptake in a cell obtained from an individual compared to a cell in which immune tolerance has not been induced. 2. The combination according to claim 1, where anti-CD86 antibody contains: VH containing CDRH1 with amino acid sequence SEQ ID NO:59, CDRH2 with amino acid sequence SEQ ID NO:60, and CDRH3 with amino acid sequence SEQ ID NO:61, and VL containing CDRL1 with amino acid sequence SEQ ID NO:62, CDRL2 with amino acid sequence SEQ ID NO:63, and CDRL3 with amino acid sequence SEQ ID NO:64 and anti-CD80 antibody contains: VH containing CDRH1 with amino acid sequence SEQ ID NO:53, CDRH2 with amino acid sequence SEQ ID NO:54, and CDRH3 with amino acid sequence SEQ ID NO:55, and VL containing CDRL1 with amino acid sequence SEQ ID NO:56, CDRL2 with amino acid sequence SEQ ID NO:57, and CDRL3 with amino acid sequence SEQ ID NO:58. 3. An antibody or variant thereof according to claim 1 or 2, wherein the chimeric antibody is a mouse-human chimeric antibody. 4. A nucleic acid molecule encoding an anti-CD86 antibody or a variant thereof according to claim 1 or 2. 5. A nucleic acid molecule encoding an anti-CD80 antibody or a variant thereof according to claim 1 or 2, comprising a polynucleotide encoding VH having the nucleotide sequence of SEQ ID NO:45, and a polynucleotide encoding VL having the nucleotide sequence of SEQ ID NO:47. 6. An expression vector containing a nucleic acid molecule according to claims 4 and 5. 7. A cell for obtaining a combination according to claim 1 or 2, containing a vector according to claim 6. 8. A method for obtaining a combination according to claim 1 or 2, including culturing the cell according to claim 7. 9. A method of obtaining a cell for treating or preventing a disease, disorder or condition, comprising mixing the combination according to claim 1 or 2, a T cell obtained from an individual, and CD80 and CD86 antigens foreign to the individual, or material containing these antigens, wherein the disease, disorder or condition is selected from the group consisting of graft rejection, allergy, autoimmune disease, graft-versus-host disease, and immune rejection caused by transplantation of an iPS cell or ES cell and a cell, tissue, or organ derived from said iPS cells or said ES cells.