antibodies directed against icos and their uses

专利摘要:
ANTIBODIES DIRECTED AGAINST ICOS AND USE OF THE SAME. The present invention provides antibodies directed against ICOS or a derivative thereof which neutralize ICOS coupling in Treg by inhibiting the fixation between ICOS and ICOS-L and nullify the proliferation of Treg induced by plasmacytoid dendritic cells. The present invention further provides antibodies directed against ICOS or a derivative thereof which induce the production of IL-10 and IFN (Gamma), induce the proliferation of CD4 + T cells, reduce the proliferation of Tconv and increase the immunosuppressive function of Treg.
公开号:BR112013025045B1
申请号:R112013025045-3
申请日:2012-03-29
公开日:2020-10-27
发明作者:Julien Faget；Christophe Caux；Christine Menetrier-Caux；Jacques Nunes；Daniel Olive
申请人:INSERM (Institut National de la Santé et de la Recherche Médicale)；Université D' Aixmarseille；Institut Jean Paoli & Irene Calmettes；Centre Leon Berard；Universite Claude Bernard - Lyon 1.；
IPC主号:

专利说明:

FIELD OF THE INVENTION
The invention relates to antibodies directed against ICOS and their uses. BACKGROUND OF THE INVENTION
In several types of cancer, the establishment of an immunosuppressive T-cell response is correlated with poor prognosis and disease progression.
Among the different cellular effectors involved in the establishment of immune tolerance, the subset of regulating CD4 + T lymphocytes (Treg) is specialized in the suppression of other T cells (Tconv), as well as in dendritic function. This suppression may be correlated with a low survival rate for cancer patients, especially breast cancer.
Large amounts of IL-10 and low amounts of IFNy produced by CD4 + T cells have been shown to be associated with reduced CD8 + T cell cytotoxicity, less T cell proliferation and participation in monocyte differentiation in M2c-type immunosuppressive macrophages, Macrophage Associated with Tumor (Tumor Associated Macrophage - TAM).
The inventors previously reported that memory CD3 + CD4 + T cells that cover large amounts of Treg (Ta-Treg) infiltrate primary breast tumors. Infiltrations of both Ta-Treg and plasmacytoid dendritic cell (pDC) in a primary breast tumor are associated with a poor prognosis and poor survival of the patient suffering from breast tumors.
The inventors further confirmed that immunosuppressive mechanisms involving Treg are seen in most cancers and chronic infections. These suppressive mechanisms prevent an efficient immune response against cancer and chronic viral infection.
Currently, Treg's are targeted at cancers and chronic infections using cell therapy, anti-CD25 mAbs or low-dose chemotherapy. However, these strategies did not provide acceptable results.
In addition, it has been reported that Tregs can play an important role in diseases associated with or caused by an excessive immune response.
However, there is currently no effective and available strategy for treating diseases associated with Treg. Thus, there is still a great need to provide efficient therapeutic strategies that target diseases involving Treg. SUMMARY OF THE INVENTION
Surprisingly, the inventors have shown that the interaction between ICOS and its ligand plays a central role in the functions of activation, proliferation and suppression of Treg in some cancers through interaction with plasmacytoid dendritic cells (pDC). So, they focused their efforts on generating specific antibodies with agonist and antagonist effects.
Antagonistic antibodies are effective for treating a disease or condition associated with suppression of Treg-mediated immune response. Agonist antibodies are effective for treating a disease or condition associated with or caused by an excessive immune response.
Thus, the present invention relates to an antibody directed against ICOS or a derivative thereof: - it neutralizes the coupling of ICOS on Treg by inhibiting the fixation between ICOS and ICOS-L; and - cancels pDC-induced Treg proliferation.
In the context of the present invention, said antibody can also be called "antagonist antibody".
The invention also relates to an antibody directed against ICOS, wherein said antibody is selected from the group consisting of Icos 145-1 and Icos 314-8, respectively obtainable from the hybridoma deposited in the "Collection Nationale de Cultures de Microorganismes" (CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France), in accordance with the terms of the Budapest Treaty, on 2 July 2009, under access numbers CNCM 1-4179 and CNCM 1- 4180, and derivatives thereof.
The invention also relates to an antagonist antibody directed against ICOS according to the invention or a derivative thereof for use as a medicament. The invention further relates to an antagonist antibody directed against ICOS according to the invention or a derivative thereof for use in the treatment of cancers or chronic infections.
The present invention also relates to an antibody directed against ICOS or a derivative thereof which: - induces the production of IL-10 and IFNy; - induces the proliferation of CD4 + T cells; - reduces the proliferation of Tconv; and - increases the immunosuppressive function of Treg.
In the context of the present invention, said antibody can also be called "agonist antibody".
The invention also relates to an antibody directed against ICOS, wherein said antibody is selected from the group consisting of Icos 53-3, Icos 88-2 and Icos 92-17, respectively obtainable from the hybridoma deposited in the "Collection Nationale de Cultures de Microorganismes "(CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France), according to the terms of the Budapest Treaty, on 2 July 2009, under access numbers CNCM 1- 4176, CNCM 1-4177, CNCM 1-4178, and derivatives thereof.
The invention relates to an agonist antibody according to the invention or a derivative thereof for use as a medicament. The invention also relates to an agonist antibody according to the invention or a derivative thereof for use in the treatment of autoimmune diseases, transplant rejection or a host versus graft disease. DETAILED DESCRIPTION OF THE INVENTION Definition
As used herein, the terms "ICOS" or "inducible T cell co-stimulator" refer to a 55 to 60 kDa homodimeric transmembrane glycoprotein THAT has an IgV-like domain in its extracellular portion and a tyrosine within a YMFM motif in its cytoplasmic portion. Coupling ICOS to its ligand has been shown to induce tyrosine phosphorylation in the cytoplasmic portion of ICOS. This phosphorylation is responsible for recruiting the regulatory subunit p85 PI3K, which activates the PI3K / AKT signaling pathway.
It is also described that ICOS coupling induces CD40L expression on the cell surface. CD40L is known to have an important effect on the cooperation between T lymphocytes and B lymphocytes.
ICOS was found to be expressed, after TCR activation, in conventional T cells (Tconv CD4 +, CD8 + subsets), as well as in Treg. The inventors showed that said activation was more important in patients suffering from melanoma or breast cancer.
As used herein, the terms "ICOSL", "ICOS-L" and "B7-H2" refer to an ICOS binder. Said ligand is present in lymphoid cells, such as B lymphocytes, macrophages, dendritic cells, as well as in non-lymphoid cells, such as endothelial or epithelial cells. ICOS coupling plays an important role in lymphocyte activation and induces T lymphocyte proliferation and survival, especially Treg.
As used herein, the term "JICOS 1" refers to a specific cell line that expresses ICOS.
As used herein, a "monoclonal antibody", in its various grammatical forms, refers to a population of antibodies that contains only one species of antibody combining site capable of immunoreacting with a particular epitope. Thus, a monoclonal antibody typically has a single binding affinity for any epitope with which it immunoreacts. A monoclonal antibody can therefore contain an antibody molecule that has a plurality of antibody combining sites, each immunospecific for a different epitope, for example, a bispecific monoclonal antibody. Although historically a monoclonal antibody has been produced by immortalizing a cell line that secretes clonally pure immunoglobulin, a monoclonally pure population of antibody molecules can also be prepared using the methods of the present invention. Laboratory methods for preparing monoclonal antibodies are well known in the art (see, for example, Harlow et al., 1988). Monoclonal antibodies (mAbs) can be prepared by immunizing purified TXAS with mutation in a mammal, for example, a mouse, rat, human and similar mammals. The antibody-producing cells in the immunized mammal are isolated and fused with myeloma or heteromyeloma cells to produce hybrid cells (hybridomas). The hybridoma cells that produce the monoclonal antibodies are used as a source of the desired monoclonal antibody. This standard method of hybridoma culture is described in Kohler and Milstein (1975). Although mAbs can be produced by hybridoma culture, the invention should not be so limited. Also contemplated is the use of mAbs produced by an expressed nucleic acid cloned from a hybridoma of the present invention. That is, the nucleic acid that expresses the molecules secreted by a hybridoma of the present invention can be transferred to another cell line to produce a transformant. The transformant is genotypically distinct from the original hybridoma, but is also capable of producing antibody molecules of the present invention, including immunologically active fragments of entire antibody molecules, corresponding to those secreted by the hybridoma. See, for example, U.S. Patent No. 4,642,334 to Reading; European Patent Publications No. 0239400 to Winter et al. and No. 0125023 to Cabilly et al. Antibody generation techniques that do not involve immunization are also considered, such as, for example, using phage display technology to analyze virgin libraries (from non-immunized animals); see Barbas et al. (1992) and Waterhouse et al. (1993).
As used herein, the term "anti-ICOS antibody" refers to a monoclonal antibody directed against ICOS, preferably obtained using recombinant ICOS-Fc as an immunogen.
As used herein, the term "derived from an antibody" refers to an antibody that comprises the 6 CDRs of said antibody.
As used herein, the term "mAb 53.3" or "Icos 53-3" refers to a monoclonal antibody directed against ICOS deposited at the CNCM on July 2, 2009 under accession number CNCN 1-4176. Said antibody is an ICOS agonist. The term "a derivative of mAb 53.3" refers to an anti-ICOS antibody that comprises the 6 CDRs of mAb 53.3.
As used herein, the term "mAb 88.2" or "Icos 88-2" refers to a monoclonal antibody directed against ICOS deposited at the CNCM on July 2, 2009 under accession number CNCN 1-4177. Said antibody is an ICOS agonist. The inventors have shown that the use of said antibody in the presence of IL-2 favors the proliferation of Treg and the secretion of IL-10. The term "a derivative of mAb 88.2" refers to an anti-ICOS antibody that comprises the 6 CDRs of mAb 88.2.
The 6 CDRs of mAb 88.2 are as listed in Table 1 below: Table 1


As used herein, the expression "mAb 92.17" or "Icos 92-17" refers to a monoclonal antibody directed against ICOS deposited at the CNCM on July 2, 5 2009 under accession number CNCN 1-4178. Said antibody is an ICOS agonist. The term "a derivative of mAb 92.17" refers to an anti-ICOS antibody that comprises the 6 CDRs of mAb 92.17.
As used herein, the term "mAb 145.1" or "Icos 145-1" refers to a monoclonal antibody directed against ICOS deposited at the CNCM on July 2, 2009 under accession number CNCN 1-4179. Said antibody is an ICOS antagonist. The term "a derivative of mAb 145.1" refers to an anti-ICOS antibody that comprises the 6 CDRs of mAb 145-1.
As used herein, the words "mAb 314.8" or "Icos 314-8" refer to a monoclonal antibody directed against ICOS deposited at the CNCM on July 2, 2009 under accession number CNCM 1-4180. The inventors have shown that the use of said antibody blocks the secretion of IL-10 by Tconv. Said antibody is an ICOS antagonist and is highly adapted to prevent dendritic cell-mediated expansion and suppressive function of regulatory T cells in cancer, such as breast cancer. The term "a derivative of mAb 314.8" refers to an anti-ICOS antibody comprising the 6 CDRs of mAb 15 314.8.
The 6 CDRs for mAb 314.8 are as shown in Table 2 below: Table 2


As used herein, the term "an antibody of the invention" refers to: an antibody directed against ICOS capable of neutralizing ICOS coupling in Treg by inhibiting the fixation between ICOS and ICOS-L and canceling Treg proliferation induced by plasmacytoid dendritic cells, that is, an antagonist antibody; as well as an antibody directed against ICOS capable of inducing the production of IL-10 and IFNy, inducing the proliferation of CD4 + T cells, reducing the proliferation of Tconv and increasing the immunosuppressive function of Treg, that is, an agonist antibody.
Said expression also covers any derivatives of said antibodies. chosen from mAb 53.3, mAb 88.2, mAb 92.17, mAb 145.1 and mAb 314.8 and the derivatives thereof.
As used herein, the term "antagonistic antibody directed against ICOS" refers to an antibody that is capable of binding to ICOS without triggering a cellular response similar to the naturally occurring ICOS-induced response. The term "the antagonistic antibodies of the invention" refers to mAb 145.1, mAb 314.8 and derivatives thereof.
As used herein, the term "agonist antibody directed against ICOS" refers to an antibody that is capable of binding to ICOS and eliciting a cellular response similar to the naturally occurring ICOS-induced response. The said antibody thus mimics the action of ICOS. The term "invention agonist antibody" refers to mAb 53.3, mAb 88.2, mAb 92.17 and derivatives thereof.
As used herein, the terms "antigen presenting cell" and "APC" refer to a class of cells in the immune system capable of internalizing and processing an antigen, so that antigenic determinants are presented on the cell surface as associated complexes to MHC, in a way capable of being recognized by the immune system (for example, MHC Class I restricted cytotoxic T lymphocytes and / or MHC Class II restricted helper T lymphocytes). The two required properties that allow a cell to function as an APC are the ability to process antigens that have undergone endocytosis and the expression of MHC gene products. Examples of APC include dendritic cells (DCs), mononuclear phagocytes (e.g., macrophages), B lymphocytes, skin Langerhans cells and, in humans, endothelial cells.
As used herein, the terms "Treg" and "regulatory T cells" refer to a specific population of T lymphocytes that have the ability to suppress the proliferation of responsive T cells in vitro and inhibit autoimmune diseases. Treg's were involved as the main contributors to the final failure of anti-tumor immune responses in humans. For example, in ovarian cancer, Treg suppresses tumor-specific T cells and high Treg numbers are associated with reduced survival time. The inventors have shown that Treg selectively inhibits the host's immune response and thus contributes to cancer progression, especially in breast cancer. Treg's were originally identified as a population of CD4 + CD25 + cells, but are also characterized by the expression of the Forkhead family transcription factor, FoxP3.
The inventors showed that Treg proliferates in situ in a patient's cancerous tissue and expresses the ICOS and CD39 cell surface markers, compared to Treg extracted from blood from the same patient.
In contrast, the term "Tconv" refers to T cells other than Treg. Thus, the term "Tconv" includes T cells that function to eliminate antigen (for example, through the production of cytokines that modulate the activation of other cells or by cytotoxic activity). This term includes helper T cells (for example, Th1 and Th2 cells) and cytotoxic T cells. In this regard, helper T cells preferably express CD4 and express low or undetectable levels of CD25. CTL cells preferably express CD8 and low or undetectable levels of CD4. Preferably, a non-Treg cell does not express both CD4 and CD25. Preferably, a non-Treg cell does not express FoxP3.
As used herein, the terms "tumor-associated regulatory T cells" and "Ta-Treg" refer to tumor-associated regulatory T cells, for example, breast tumors. The inventors have shown, in fact, that Ta-Treg are present in the lymphoid infiltrates of tissue surviving the breast cancer patient.
As used herein, the terms "plasmacytoid dendritic cells" and "pDC" refer to the innate immune cells that circulate in the blood and are found in peripheral lymphoid organs. They constitute a group of cells that belong to the group of peripheral blood mononuclear cells (PBMC).
As used herein, the terms "tumor-associated plasmacytoid dendritic cells" and "Ta-pDC" refer to tumor-associated plasmacytoid dendritic cells, for example, breast tumors. The inventors have shown that Ta-pDC are capable of inducing the proliferation of Ta-Treg under dependence on ICOS / ICOSL costimulation.
As used herein, the terms "IL-10" and "interleukin-10" refer to a human cytokine synthesis inhibiting factor (CSIF), which is an anti-inflammatory cytokine. This cytokine is produced primarily by monocytes and, to a lesser extent, by lymphocytes. This cytokine has pleiotropic effects on immunoregulation and inflammation. It down-regulates the expression of Thl cytokines, MHC Class II antigens. It also increases B cell survival, proliferation and antibody production. This cytokine can block NF-KB activity and is involved in the regulation of the JAK-STAT signaling pathway.
As used herein, the terms "IFNy" and "interferon gamma" refer to a dimeric protein with 146 amino acid subunits. The importance of IFN-y in the immune system stems, in part, from its ability to inhibit viral replication directly and, more importantly, from its immunostimulatory and immunomodulatory effects. IFNy is produced predominantly by natural killer cells (NK) and natural killer T cells (NKT) as part of the innate immune response, and by CD4 and CD8 cytotoxic T lymphocyte (CTL) effector T cells since antigen-specific immunity is develops.
As used herein, the terms "treat" or "treatment" mean reversal, relief, inhibition of progress or prevention of the disorder or condition to which that term applies, or one or more symptoms of said disorder or condition.
A "therapeutically effective amount" means a minimum amount of active agent that is necessary to confer therapeutic benefit to an individual. For example, a "therapeutically effective amount" is an amount that induces, relieves or otherwise causes an improvement in pathological symptoms, disease progression or physiological conditions associated with a disease or that improves resistance to a disorder.
As used herein, the term "prevention" refers to preventing the disease or condition from occurring in an individual who has not yet been diagnosed as having it. As used herein, the term "individual" means a mammal, such as a rodent, a feline, a canine and a primate. Preferably, an individual according to the invention is a human being.
The term "cancer" includes malignancies of the various organ systems, such as those that affect the lung, breast, thyroid, lymphoid system, gastrointestinal and genitourinary tract, as well as adenocarcinomas, which include malignancies, such as most colon cancers, carcinoma kidney cells, prostate cancer and / or testicular tumors, non-small cell lung cancer, cancer of the small intestine and cancer of the esophagus.
The term "disease associated with Treg" will be taken to encompass any disease or disorder or condition in which the modulation of Treg numbers and / or activity may confer a beneficial effect. This term covers: diseases and conditions associated with the suppression of an individual's immune response mediated by Treg, - diseases and conditions associated with or caused by an excessive immune response.
As used herein, the term "diseases and conditions associated with suppression of Treg-mediated immune response" refers to diseases and conditions caused by suppression of the proliferation of immunomodulatory cells by Treg, such as tumor-specific T cells. As mentioned earlier, the inventors showed that Treg's are associated with a poor diagnosis and survival rate in a cancer patient.
Non-limiting examples of diseases and conditions associated with the suppression of an individual's immune system mediated by Treg are cancer and chronic infections.
As used herein, "diseases and conditions associated with or caused by an excessive immune response" are, for example, autoimmune diseases, transplant rejection or host versus graft disease.
This term also covers inflammatory conditions, such as inflammatory nervous system disorder (eg multiple sclerosis), inflammatory mucosal disease (eg inflammatory bowel disease, asthma or tonsillitis), inflammatory skin disease (eg dermatitis, psoriasis or contact hypersensitivity), autoimmune arthritis (eg, rheumatoid arthritis).
As used herein, the term "immune response" refers to the action orchestrated by lymphocytes, antigen presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by the cells above or by the liver (including antibodies, cytokines and complement) that results in selective damage , destruction or elimination of cancer cells, metastatic tumor cells, malignant melanoma, invading pathogens, cells or tissues infected with pathogens in an individual's body or, in cases of autoimmunity or pathological inflammation, normal cells or tissues of an individual.
As used herein, an "autoimmune disease" is a disease or disorder arising from and directed against the individual's own tissues. Antagonistic Antibodies of the Invention
ICOS-L, which is an ICOS specific ligand, has been shown to be expressed in plasmacytoid dendritic cells. The inventors showed that tumor-associated Treg's were in close contact with tumor-associated plasmacytoid dendritic cells, indicating that such interaction allows the coupling of ICOS with ICOS-L in tumors.
They also showed that the interaction ICOS / ICOS-L in situ leads to the overloading of ICOS-L in the membrane of TDC. The inventors have developed an antagonist antibody directed against ICOS and have shown that the addition of said antibody completely abolishes the overloading of ICOS-L in pDC, which is responsible for the activation and proliferation of Ta-Treg.
The inventors have shown that the antagonist antibody according to the invention neutralizes ICOS coupling in Treg and cancels its pDC-induced expansion. More precisely, said antibody suppresses the proliferation of Treg and secretion of IL-10 induced by the ICOS / ICOSL interaction.
The antagonistic antibodies of the invention are therefore highly suitable for canceling the immunosuppressive response involved in the pathological mechanism. They are thus useful for treating diseases and conditions associated with suppression of Treg-mediated immune responses.
The invention thus relates to an antibody directed against ICOS and derivatives of the same as:
neutralize the ICOS coupling in Treg by inhibiting the fixation between ICOS and ICOS-L; and> cancel the Treg proliferation induced by plasmacytoid dendritic cells.
In one embodiment, said antibody is a monoclonal antibody.
In one embodiment, said antibody is a chimeric antibody.
In one embodiment, said antibody is a humanized antibody.
By "neutralization of ICOS coupling in Treg" is meant that the antibody interferes with the cooperation between ICOS and its ligand ICOS-L.
By "canceling Treg proliferation" is meant that a significant decrease, preferably a total interruption, of Treg proliferation is observed in a target tissue, preferably a tumor tissue, when compared to a control tissue, preferably a non-tumor tissue, more preferably blood.
The invention relates to an antibody directed against ICOS, in which said antibody is selected from the group consisting of Icos 145-1 and Icos 314-8, respectively obtainable from the hybridoma deposited at the CNCM on July 2, 2009 under the access numbers CNCM 1-4179 and CNCM 1-4180, and derivatives thereof.
The invention also relates to an antibody comprising the 6 CDRs of an antibody selected from the group consisting of Icos 145-1 and Icos 314-8, respectively obtainable from the hybridoma deposited at the CNCM on 2 July 2009 under the numbers CNCM 1-4179 and CNCM 1-4180 access codes, and derivatives thereof.
The invention also relates to an antibody that comprises the 6 CDRs in Table 2 above.
In another embodiment, the invention relates to an antibody derived from one of the antibodies selected from the group consisting of Icos 145-1 and Icos 314-8, respectively obtainable from the hybridoma deposited at the CNCM on July 2, 2009 under the numbers access codes CNCM 1-4179 and CNCM 1-4180. Therapeutic Use of Antagonistic Antibodies of the Invention
By neutralizing the coupling of ICOS to Treg and nullifying the proliferation of Treg, the antagonistic antibodies of the invention are highly suitable for use in the treatment of diseases and conditions associated with suppression of the Treg-mediated immune response, for example, cancers and chronic infections. Said antibodies can therefore be used to restore anti-tumor immunity.
Therefore, the invention relates to the antagonist antibody directed against ICOS according to the invention or a derivative thereof for use as a medicament.
The invention further relates to the antagonist antibody directed against ICOS according to the invention or a derivative thereof for use in the treatment of a disease or condition associated with the suppression of Treg-mediated immune response.
In a preferred embodiment, said disease or condition associated with suppression of immune response mediated by Treg is a disease selected from the group consisting of cancers and chronic infections.
In fact, the inventors have shown that the antagonistic antibodies of the invention are adapted for modulating Treg numbers and / or activity in order to nullify the Treg-related immunosuppressive effect. Therefore, said antagonistic antibodies represent a highly promising strategy for the treatment of diseases associated with suppression of the immune system, such as cancer and chronic infections.
Examples of cancers include, but are not limited to, human malignant lymphoma, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer, prostate cancer, head and neck cancer, pancreatic cancer, bladder cancer, cancer colon-rectal, bone cancer, cervical cancer, liver cancer, oral cancer, esophageal cancer, thyroid cancer, kidney cancer, stomach cancer, testicular cancer and skin cancer.
Examples of chronic infections include, but are not limited to, viral, bacterial, parasitic or fungal infections, such as chronic hepatitis, lung infections, lower respiratory tract infections, bronchitis, flu, pneumonia and sexually transmitted diseases.
Examples of viral infections include, but are not limited to, hepatitis (HAV, HBV, HCV), herpes simplex (HSV), herpes zoster, HPV, influenza (flu), AIDS and AIDS-related complex, chicken pox (chickenpox), common cold, cytomegalovirus (CMV) infection, smallpox, Colorado tick fever, dengue fever, Ebola hemorrhagic fever, foot and mouth disease, Lassa fever, measles, Marburg hemorrhagic fever, infectious mononucleosis, mumps, norovirus, polio, progressive multifocal leukoencephalopathy (PML ), rabies, rubella, SARS, viral encephalitis, viral gastroenteritis, viral meningitis, viral pneumonia, West Nile disease and yellow fever.
Examples of bacterial infections include, but are not limited to, pneumonia, bacterial meningitis, cholera, diphtheria, tuberculosis, anthrax, botulism, brucellosis, canphylobacteriosis, typhus, gonorrhea, listeriosis, Lyme disease, rheumatic fever, pertussis (pertussis), bubonic plague , salmonellosis, scarlet fever, shigellosis, syphilis, tetanus, trachoma, tularemia, typhoid fever and urinary tract infections.
Examples of parasitic infections include, but are not limited to, malaria, leishmaniasis, trypanosomiasis, Chagas' disease, cryptosporidiosis, fascioliasis, filariasis, amoebic infections, giardiase, oxymoron infection, schistosomiasis, teniasis, toxoplasmosis, trichinosis and trypanosomiasis. Examples of fungal infections include, but are not limited to, candidiasis, aspergillosis, coccidioidomycosis, cryptococcosis, histoplasmosis and tinea pedis.
In a preferred embodiment of the invention, the invention relates to antagonistic antibodies directed against ICOS according to the invention or a derivative thereof for use in treating cancer. Preferably, said cancer is selected from human malignant lymphoma, ovarian cancer, cervical cancer and breast cancer. Most preferably, said cancer is breast cancer.
The invention also relates to a method for treating a disease or condition associated with suppression of immune response mediated by Treg, in which the disease is selected from the group consisting of cancers and chronic infections, preferably cancers, in which said method it comprises the step of administering a therapeutically effective amount of an antagonist antibody directed against ICOS according to the invention or a derivative thereof to an individual in need thereof. Agonist antibodies directed against ICOS
ICOS coupling has been found to be associated with an immunosuppressive T cell response. In fact, it has been reported that said coupling reduces the production of IL-10 and IFNy and reduces the proliferation of CD4 + T cells.
Therefore, as evidenced by the inventors, an ICOS agonist antibody confers the opposite effect and is beneficial for the treatment of diseases associated with or caused by an excessive immune response. The invention thus relates to an antibody directed against ICOS or a derivative thereof which:> induces the production of IL-10 and IFNy; > induces proliferation of CD4 + T cells; > reduces the proliferation of Tconv; and> increases the immunosuppressive function of Treg.
By "inducing the production of IL-10 and IFNy" is meant that a significant increase in the production of IL-10 and IFNy is observed.
By "inducing CD4 + T cell proliferation" it is meant that a significant increase in CD4 + T cell proliferation is observed in a target tissue, preferably a tumor tissue, when compared to a control tissue, preferably a non-tumor tissue , more preferably blood.
By "reduction of Tconv proliferation" is meant that a significant reduction in Tconv proliferation is observed in a target tissue, preferably a tumor tissue, when compared to a control tissue, preferably a non-tumor tissue, more preferably blood .
By "increased Treg immunosuppressive function" is meant that a significant increase in Treg suppressive activity is observed.
In one embodiment, said antibody is a monoclonal antibody.
In one embodiment, said antibody is a chimeric antibody.
In one embodiment, said antibody is a humanized antibody.
The invention also relates to an antibody directed against ICOS, in which said antibody is selected from the group consisting of Icos 53-3, Icos 88-2 and Icos 92-17, respectively obtainable from the hybridoma deposited at the CNCM in 02 July 2009 under access numbers CNCM 1-4176, CNCM 1-4177, CNCM 1-4178, and derivatives thereof.
The invention also relates to an antibody that comprises the 6 CDRs of an antibody selected from the group consisting of Icos 53-3, Icos 88-2 and Icos 92-17, respectively obtainable from the hybridoma deposited at the CNCM on 2 July 2009 under access numbers CNCM 1-4176, CNCM 1-4177, CNCM 1-4178.
The invention also relates to an antibody comprising the 6 CDRs in Table 1 above.
In another embodiment, the invention relates to an antibody derived from one of the antibodies selected from the group consisting of Icos 53-3, Icos 88-2 and Icos 92-17, respectively obtainable from the hybridoma deposited at the CNCM on 2 July 2009 under access numbers CNCM 1-4176, CNCM 1-4177, CNCM 1-4178. Therapeutic use of the agonist antibodies of the invention
The invention also relates to the agonist antibody directed against ICOS according to the invention or a derivative thereof for use as a medicament.
The invention also relates to the agonist antibody directed against ICOS according to the invention or a derivative thereof for use in the treatment of a disease or condition associated with or caused by an excessive immune response.
The invention also relates to the agonist antibody directed against ICOS according to the invention or a derivative thereof for use in the treatment of an autoimmune disease, transplant rejection or a host versus graft disease.
In a particular embodiment, said autoimmune disease is selected from the group consisting of rheumatoid arthritis (RA), insulin-dependent diabetes mellitus (Type 1 diabetes), multiple sclerosis (MS), Crohn's disease, systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, pemphigus vulgaris, pemphigoid, Addison's disease, ankylosing spondylitis, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, celiac disease, dermatomyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barrash syndrome , idiopathic leukopenia, idiopathic thrombocytopenic purpura, male infertility, mixed connective tissue disease, myasthenia gravis, pernicious anemia, phageogenic uveitis, primary biliary cirrhosis, primary myxedema, Reiter's syndrome, rigid person's syndrome, thyrotoxicosis, granulomatosis and ulcerative colitis .
In another embodiment, the invention also relates to the agonist antibody directed against ICOS according to the invention or a derivative thereof for use in the treatment of an inflammatory disorder selected from the group consisting of an inflammatory disorder of the nervous system, such as multiple sclerosis, inflammatory mucosal disease, such as inflammatory bowel disease, asthma or tonsillitis, inflammatory skin disease, such as dermatitis, psoriasis or contact hypersensitivity, and autoimmune arthritis, such as rheumatoid arthritis.
The invention also relates to a method for treating a disease or condition associated with or caused by an excessive immune response, preferably an autoimmune disease, transplant rejection, host versus graft disease or an inflammatory disorder, wherein said method comprises the step of administering a therapeutically effective amount of an agonist antibody directed against ICOS according to the invention or a derivative thereof to an individual in need thereof. Nucleic acid sequence encoding an antibody of the invention
A further embodiment of the invention relates to a nucleic acid sequence that encodes an antibody to one of the antibodies selected from the group consisting of mAb 53.3, mAb88.2, mAb 92.17, mAb 145.1, mAb 314.8 and derivatives thereof.
In a particular embodiment, the invention relates to a nucleic acid sequence encoding the VH domain or the VL domain of one of the antibodies selected from the group consisting of mAb 53.3, mAb88.2, mAb 92.17, mAb 145.1, mAb 314.8 and derived from them.
Typically, said nucleic acid is a DNA or RNA molecule which can be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.
The terms "vector", "cloning vector" and "expression vector" mean the vehicle through which a DNA or RNA sequence (for example, a foreign gene) can be introduced into a host cell in order to transform the host and promote expression (for example, transcription and translation) of the introduced sequence. Thus, a further object of the invention relates to a vector that comprises a nucleic acid of the invention. Such vectors may comprise regulatory elements, such as a promoter, enhancer, finisher and the like, to cause or direct the expression of said antibody upon administration to an individual. Examples of promoters and enhancers used in the animal cell expression vector include the SV40 early promoter and enhancer, Moloney murine leukemia virus promoter and enhancer, immunoglobulin H chain promoter and enhancer and the like.
Any expression vector for animal cells can be used, as long as a gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSGl beta d2-4- and the like. Other examples of plasmids include replication plasmids comprising an origin of replication or integrative plasmids, such as, for example, pUC, pcDNA, pBR, etc. Other examples of viral vector include adenoviral, retroviral, herpesvirus and AAV vectors. Such recombinant viruses can be produced by methods known in the art, such as transfection of packaging cells or transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv + cells, 293 cells, etc. Detailed protocols for producing such defective recombinant viruses in replication can be found, for example, in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478.
An additional object of the present invention relates to a cell that has been transfected, infected or transformed by a nucleic acid and / or a vector according to the invention. The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence into a host cell, so that the host cell will express the introduced gene or sequence to produce a substance typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed". The nucleic acids of the invention can be used to produce an antibody of the invention in a suitable expression system. The term "expression system" means a host cell and compatible vector under suitable conditions, for example, for the expression of a protein encoded by the foreign DNA brought by the vector and introduced into the host cell.
Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and host cells and mammalian vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, the yeasts Saccharomyces or Kluyveromyces, mammalian cell lines (for example, Vero cells, CHO cells, 3T3 cells, COS cells, etc.), as well as primary or established mammalian cell cultures (for example, example, produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2 / 0- Agl4 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells in which a reductase dihydrofolate gene (hereinafter referred to as "DHFR gene ") is defective, rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as" YB2 / 0 cells ") and the like.
The present invention also relates to a method of producing a recombinant host cell that expresses an antibody according to the invention, said method comprising the steps of: (i) introducing, in vitro or ex vivo, a recombinant nucleic acid or a vector as described above in a competent host cell; (ii) culturing, in vitro or ex vivo, the obtained recombinant host cell; and (iii) optionally, selecting cells that express and / or secrete said antibody.
Such recombinant host cells can be used for the production of the antibodies of the invention. Pharmaceutical composition according to the invention
The invention also relates to pharmaceutical compositions that comprise an antibody of the invention.
Therefore, an antibody of the invention can be combined with pharmaceutically acceptable excipients and, optionally, sustained release matrices, such as biodegradable polymers, to form therapeutic compositions.
"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other unwanted reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a solid, semi-solid or non-toxic liquid filler, diluent, encapsulating material or formulation aid of any kind.
The form of the pharmaceutical compositions, the route of administration, the dosage and the regime will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc.
The pharmaceutical compositions of the invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration, and the like.
Preferably, the pharmaceutical compositions contain carriers that are pharmaceutically acceptable for a formulation capable of being injected. These can be, in particular, sterile isotonic saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like, or mixtures of such salts) or dry, especially lyophilized compositions that allow the constitution of injectable solutions through addition, depending on the case, of sterile water or saline.
The doses used for administration can be adapted according to different parameters and, in particular, depending on the mode of administration used, the relevant pathology or, alternatively, the desired duration of treatment. To prepare pharmaceutical compositions, an effective amount of the antibody can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the point that there is easy flow in a syringe. It must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi.
Solutions of the active compounds as a free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxy propylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
An antibody of the invention can be formulated into a composition in a neutral or saline form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acid, or organic acids, such as acetic, oxalic acid, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases, procaine and the like.
The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like), suitable mixtures thereof and vegetable oils.
Adequate fluidity can be maintained, for example, through the use of a coating, such as lecithin, through the maintenance of the required particle size in the case of dispersion and through the use of surfactants.
The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
Prolonged absorption of injectable compositions can be achieved by using, in the compositions, agents that delay absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients listed above, as required, followed by filtration sterilization.
Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle that contains the basic dispersion medium and the other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization techniques, which produce a powder of the active ingredient plus any additional desired ingredient from a previously filtered sterile solution of the same .
The preparation of more concentrated or highly concentrated solutions for direct injection is also considered, where the use of DMSO as a solvent is expected to result in extremely rapid penetration, providing high concentrations of the active agents to a small area of the tumor.
Upon formulation, the solutions will be administered in a manner compatible with the dosage formulation and in an amount such that they are therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above, but used capsules.
For parenteral administration in an aqueous solution, for example, the solution must be adequately buffered, if necessary, and the liquid diluent must first become isotonic with sufficient saline or glucose.
These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this context, sterile aqueous media that can be employed will be known to those skilled in the art in light of the present description. For example, a dosage can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected into the proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences", 15th Edition, pages 1035 -1038 and 1570-1580). Some variation in dosage will necessarily occur, depending on the condition of the individual being treated. The person responsible for administration, in any case, will determine the appropriate dose for the individual.
The antibodies of the invention can be formulated into a therapeutic mixture to comprise about 0.0001 to 1.0 milligram or about 0.001 to 0.1 milligram or about 0.1 to 1.0 or even about 10 milligrams per dose or more. Multiple doses can also be administered. In addition to compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration, prolonged release capsules and any other form currently used.
In certain embodiments, the use of liposomes and / or nanoparticles is considered for the introduction of antibodies into host cells. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art.
Nanocapsules can, in general, retain compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (about 0.1 µm in size) are, in general, designed using polymers capable of being degraded in vivo. Biodegradable polyalkyl cyanoacrylate nanoparticles that satisfy these requirements are contemplated for use in the present invention and such particles can be easily produced.
Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form vesicles in multilamellar concentric bilayers (also called multilamellar vesicles (MLV)). MLVs have, in general, diameters from 25 nm to 4 µm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 Ã…, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Method for producing the antibodies of the invention
Antibodies of the invention can be produced by any method known in the art such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.
Knowing the amino acid sequence of the desired sequence, those skilled in the art can easily produce said antibodies by means of standard techniques for producing polypeptides. For example, they can be synthesized using the well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California), following the manufacturer's instructions. Alternatively, the antibodies of the invention can be synthesized by means of recombinant DNA techniques well known in the art. For example, antibodies can be obtained as DNA expression products after incorporating DNA sequences that encode antibodies into expression vectors and introducing such vectors into suitable eukaryotic or prokaryotic hosts, which will express the desired antibodies, from from which they can be isolated in the future using well-known techniques.
In particular, the invention also relates to a method of producing an antibody of the invention, the method of which comprises the steps consisting of: (i) culturing a host cell transformed according to the invention under conditions suitable to allow expression of said antibody; and (ii) recovering the expressed antibody.
In another particular embodiment, the method comprises the steps of: (i) culturing the hybridomas deposited as CNCM I-4176, CNCM 1-4177, CNCM 1-4178, CNCM 1-4179 or CNCM 1-4180 under conditions suitable to allow antibody expression; and (ii) recovering the expressed antibody.
The antibodies of the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein, protein A-Sepharose hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
In a particular embodiment, the human chimeric antibody of the present invention can be produced by obtaining nucleic acid sequences that encode the VL and VH domains as described above, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cells that has human genes that encode antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. Regarding the CH domain of a human chimeric antibody, it can be any region that belongs to human immunoglobulin, but those of the IgG class are suitable and any of the subclasses that belong to the IgG class, such as IgGl, IgG2, IgG3 and TgG4, can also be used. In addition, in relation to the CL of a human chimeric antibody, it can be any region that belongs to Ig and those of the kappa class or lambda class can be used. Methods for producing chimeric antibodies involve conventional recombinant DNA techniques and transfection of genes well known in the art (see US Patent documents 5,202,238 and US 5,204,244).
The humanized antibody of the present invention can be produced by obtaining the nucleic acid sequences that encode the CDR domains, as previously described, construction of a humanized antibody expression vector by inserting them into an expression vector for cell animal that has genes that encode (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expression of the genes by introducing the expression vector into an animal cell.
The humanized antibody expression vector can be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exist in separate vectors or of a type in which both genes exist in the same vector (random type). Regarding the ease of construction of a humanized antibody expression vector, ease of introduction into animal cells and balance between the levels of antibody H and L chain expression in animal cells, the humanized antibody expression vector of the random type is preferred. Examples of humanized antibody expression vector of the random type include pKANTEX93 (WO 97/10354), pEE18 and the like.
Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art. Antibodies can be humanized using a variety of methods known in the art including, for example, insertion of CDR (EP 239,400; PCT Publication WO91 / 09967; United States Patent No = 5,225,539, 5,530,101 and 5,585,089), stratification or wear (documents EP 592,106, EP 519,596) and chain shuffling (United States Patent No. 5,565,332). The general recombinant DNA technology for the preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).
The Fab of the present invention can be obtained by treating an antibody that specifically reacts with ICOS with a protease, papain. In addition, Fab can be produced by inserting DNA encoding the antibody's Fab into a vector for a prokaryotic expression system or eukaryotic expression system, and introducing the vector into a prokaryote or eukaryote (as appropriate) to express the Fab.
The F (ab ') 2 of the present invention can be obtained by treating an antibody that specifically reacts with ICOS with a protease, pepsin.
In addition, F (ab ') 2 can be produced by binding the Fab' described below via a thioether bond or a disulfide bond.
The Fab 'of the present invention can be obtained by treating F (ab') 2, which specifically reacts with human ICOS with a reducing agent, dithiothreitol. In addition, Fab 'can be produced by inserting the DNA encoding the Fab' fragment of the antibody into an expression vector for prokaryotes or an expression vector for eukaryotes and introducing the vector into a prokaryote or eukaryote (as appropriate) for carry out your expression.
The scFv of the present invention can be produced by obtaining the cDNA encoding the VH and V2 domains, as described above, constructing the DNA encoding scFv, inserting the DNA into an expression vector for prokaryotes or an expression vector for eukaryotes and, then, introducing the expression vector into a prokaryote or eukaryote (as appropriate) to express scFv. To generate a humanized scFv fragment, a well-known technology called CDR insertion can be used, which involves selecting the complementarity determining regions (CDRs) of a donor scFv fragment and inserting them in a three-dimensional human scFv fragment support. known (see, for example, documents W098 / 45322, WO 87/02671; US 5,859,205, US 5,585,089; US 4,816,567; EP0173494).
Modification (s) is contemplated in the amino acid sequence of the antibodies described in this document. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. It is known that when a humanized antibody is produced simply by inserting only CDRs into VH and VL from an antibody derived from a non-human animal into VH and VL FRs from a human antibody, antigen-binding activity is reduced, in compared to that of the original antibody derived from a non-human animal. Various amino acid residues of VH and VL of the non-human antibody, not only in CDRs, but also in FRs, are considered to be directly or indirectly associated with antigen binding activity. Consequently, replacing these amino acid residues with different amino acid residues derived from human VH and VL FRs would reduce binding activity.
In order to solve the problem, in antibodies grafted with human CDR, attempts have been made to identify, among the amino acid sequences of the FR of VH and VL of human antibodies, an amino acid residue that is directly associated with binding to the antibody or that interact with a CDR amino acid residue, or that maintains the three-dimensional structure of the antibody and is directly associated with antigen binding. The reduced antigen binding activity can be increased by replacing the identified amino acids with amino acid residues from the original antibody derived from a non-human animal.
Modifications and alterations can be made in the structure of the antibodies of the present invention and in the DNA sequences that encode them, and also obtain a functional molecule that encodes an antibody with the desired characteristics. When making changes to the amino acid sequences, the hydropathic index of the amino acids can be considered. The importance of the hydropathic index of amino acids when conferring interactive biological function on a protein is, in general, understood in the technique. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resulting protein, which, in turn, defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens and the like .
Each amino acid was assigned a hydropathic index based on its hydrophobicity and charge characteristics; these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
A further embodiment of the present invention also encompasses conservative function variants of the antibodies of the present invention.
"Conservative function variants" are those in which a particular amino acid residue in a protein or enzyme has been altered without altering the overall conformation and function of the polypeptide, including, but not limited to, substituting an amino acid with one with similar properties (such as , for example, polarity, hydrogen bonding potential, acidity, basicity, hydrophobic, aromatic and the like).
Other amino acids other than those indicated as conserved may differ in one protein, so that the percent similarity of the amino acid sequence or protein between any two proteins of similar function may vary and may be, for example, 70% to 99%, as determined according to an alignment scheme, such as by the Cluster method, in which the similarity is based on the MEGALIGN algorithm.
A "conservative function variant" also includes a polypeptide that has at least 60% amino acid identity, as determined by the BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, even more preferably at least 90 % and, even more preferably, at least 95%, and which has properties or functions equal or substantially similar to the native or parent protein with which it is compared. Two amino acid sequences are "substantially homologous" or "substantially similar" when more than 80%, preferably more than 85%, preferably more than 90% of the amino acids are identical, or more than about 90%, preferably more than 95 % are similar (functionally identical) over the entire length of the shortest sequence. Preferably, similar or homologous sequences are identified by alignment using, for example, the GCG packaging program (Genetics Computer Group, Program Manual for the GCG package, Version 7, Madison, Wisconsin), or any of the matching algorithms sequence comparison, such as BLAST, FASTA, etc.
For example, certain amino acids can be replaced by other amino acids in the protein structure without appreciable loss of activity. Since the interactive ability and nature of a protein define the functional biological activity of the protein, certain amino acid substitutions can be made in a protein sequence and, of course, in its DNA coding sequence at the same time, however , a protein with similar properties is obtained. Thus, it is considered that several changes can be made to the antibody sequences of the invention, or corresponding DNA sequences that encode said antibodies without appreciable loss of their biological activity.
It is known in the art that certain amino acids can be replaced by other amino acids that have a similar hydropathic score or index and still result in a protein with similar biological activity, that is, still obtain a functionally equivalent biological protein. As outlined above, amino acid substitutions are therefore generally based on the relative similarity of amino acid side chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size and the like.
consideration of several of the foregoing characteristics are well known to those skilled in the art and include: arginine and lysine, glutamate and aspartate, serine and threonine, glutamine and asparagine, and valine, leucine and isoleucine. Another type of amino acid modification of the antibody of the invention may be useful for altering the original glycosylation pattern of the antibody.
By "alteration" is meant the deletion of one or more carbohydrate moieties found in the antibody and / or addition of one or more glycosylation sites that are not present in the antibody.
Antibody glycosylation is typically N-linked. "N-linked" refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The asparagine-X-serine and asparagine-X-threonine tripeptide sequences, where X is any amino acid except proline, are the recognition sequences for enzymatic binding of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. The addition of glycosylation sites to the antibody is conveniently effected by altering the amino acid sequence in such a way that it contains one or more of the tripeptide sequences described above (for N-linked glycosylation sites). Another type of covalent modification involves glycosides chemically or enzymatically coupled to the antibody. These procedures are advantageous in that they do not require the production of the antibody in a host cell that has glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling method used, the sugar (s) can be linked to the arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups, such as those of cysteine; (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline; (e) aromatic residues, such as those from phenylalanine, tyrosine or tryptophan; or (f) the amide group of glutamine. For example, such methods are described in document W087 / 05330.
The removal of any carbohydrate moieties present in the antibody can be carried out chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars, except the binding sugar (N-acetylglycosamine or N-acetylgalactosamine), leaving the antibody intact.
Enzymatic cleavage of carbohydrate moieties into antibodies can be achieved using a variety of endo- and exo-glycosidases.
Another type of covalent modification of the antibody comprises binding the antibody to one of a variety of non-proteinaceous polymers, for example, polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in US Patent No. 4,640,835;
4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337.
It may also be desirable to modify the antibody of the invention with respect to the effector function, for example, in order to improve the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions into an Fc region of the antibody. Alternatively or additionally, cysteine residue (s) can be introduced in the Fc region, thus allowing the formation of inter-chain disulfide bond in this region. The homodimeric antibody thus generated may have enhanced internalization capacity and / or complement-mediated cell death and / or increased antibody-dependent cell cytotoxicity (ADCC) (Caron PC et al., J Exp Med., October 1, 1992; 176 (4): 1191-5 and Shopes B., J
Immunol., May 1, 1992; 148 (9): 2918-22). Diagnostic Method
The present invention also relates to a diagnostic method of an increased risk of recurrence or early death in a patient with breast cancer. In fact, as shown in Example 3, the presence of a high number of ICOS + Treg cells is associated with lower Progression-free Survival or Global Survival for breast cancer patients.
Thus, the invention relates to a method for diagnosing an increased risk of recurrence or early death in a patient with breast cancer, comprising the step of quantifying ICOS positive Treg cells (ICOS +) in a sample of said patient. If said number is high, for example, greater than 1.7 ICOS + cells / dot when using the method of Example 3 and Figure 9, then there is an increased risk of recurrence or early death in said patient with breast cancer. .
The invention also relates to a method for selecting patients susceptible to be treated by anti-ICOS immunotherapy, comprising the step of quantifying positive ICOS Treg cells in a sample of said patient. Said immunotherapy can be with anti-ICOS antibodies of the invention.
Said sample may come from a biopsy. Said quantification of Treg ICOS + cells can be performed thanks to anti-ICOS antibodies, especially thanks to any of the antibodies described above. Treatment in the preclinical breast tumor model
As shown in Example 6, treatment of an established model in a murine breast tumor with a replacement anti-murine anti-ICOS neutralizing antibody (17G9, IgG2b) reduces tumor progression, reinforcing the potential of treatment with anti-neutralizing antibodies ICOS of the invention to favor tumor regression in the subpopulation of patients with high detection of Treg ICOS + in their primary breast tumors.
The invention will be further illustrated in view of the following Figures and Examples.
Figure 1: Ta-Treg strongly express ICOS, co-located with Ta-pDC and proliferate in situ, but do not proliferate in vitro.
A - Frozen sections of tumor were stained with anti-ICOS Ab (green) and Ab Ki67 (brown) and secondary anti-murine Ab conjugated with HRP and revealed, respectively, with Histogreen and DAB (10x and 40x magnification for the insertion).
B ~ Ki67 expression was analyzed using multicolored flow cytometry over Treg (CD4 + CD127 ~ CD25dlt0) and Tconv (CD4 + CD127 + CD25bdl '"' ° '“) in the primary tumor (Ta-Treg, Ta-Tconv) or paired blood (Treg, Tconv).
C - Treg and Tconv purified from either primary tumor or healthy blood were cultured in a 96-well U-bottom plate in the presence of 500 IU / ml IL-2. The number of cells was quantified every 4 days by enumeration.
D — F - Frozen tumor sections were stained with anti-CD3 Ab (brown) and counterstained with hematoxylin (blue) (10x and 40x in the insertion box) (D); Ab CD3 (green) and BDCA2 (brown) (20x and 40x in the insertion box) (E); Ab FoxP3 (brown) and BDCA2 (green) (20x and 40x in the insertion box) (F).
Figure 2: ICOS and ICOS-L blocking inhibits IL-10 secretion during pDC-mediated T cell activation without strongly interfering with MoDC / T co-culture. pDC or MoDC activated with R848 and purified were co-cultured for 5 days with allogeneic memory CD4 + T cells in the presence of Ab Ctrl, anti-ICOS (314.8) or anti-ICOS-L (MIH12). On day 5, IL-10 and IFNy were quantified by ELISA in supernatants from pDC / T (A) and MoDC / T (B) co-culture.
Figure 3: Co-stimulation with ICOS and CD3 favors the proliferation of Treg and Tconv, as well as IL-10, but does not favor the secretion of IFNy in the presence of exogenous IL-2.
A / B - Treg or Tconv chosen by FACS derived from amidala were cultured for 5 days alone or with granules coated with agonist mAb CD3 / IgG, CD3 / 88.2, CD3 / CD28 in the presence of IL-2 (n = 3). Proliferation was assessed by the incorporation of [3H] -thymidine (A). Levels of IFNy and IL-10 were measured by ELISA in the culture supernatant (B).
C - TaT CD4 + cells chosen from tumor were cultured for 5 days with granules coated with antiCD3 / IgG; antiCD3 / 88.2 or antiCD3 / antiCD28 in the presence of exogenous IL-2 (100 IU / ml). The concentrations of IL-10 and IFNy in the supernatant were quantified by ELISA.
Figure 4: ICOS coupling blocks CD28-induced IL-2 and, consequently, reduces IFNy proliferation and secretion.
A - CFSE-labeled CD4 + memory T cells were cultured for 5 days with the different granules alone or in the presence of gradual exogenous rhIL-2 concentration (20 IU / ml and 100 IU / ml) and proliferation was assessed by dilution with CFSE by means of flow cytometry.
B - IL-2 detected by ELISA after five days of culture with different granules without exogenous IL-2.
C - CD4 + memory T lymphocytes in the blood of healthy donors were cultured for 5 days with different granules alone or in the presence of exogenous IL-2 (100 IU / ml). The secretions of IL-10 and IFNy were quantified by ELISA.
Figure 5: Absence of ICOS-L expression in breast tumor cell lines and lacerations of primary breast tumor.
A - ICOS-L expression was evaluated by means of flow cytometry in suspensions of breast tumor epithelial cell lines collected in PBS-EDTA in the absence of trypsin to prevent deterioration of Ag.
B - Expression of ICOS-L was evaluated in tumor cells (CD45- cells) 48 h after culture in the presence of control Ab (dashed line) or anti-ICOS Ab (314.8) (continuous line).
Figure 6: Treatment of primary Neul5 breast tumors with an anti-mouse anti-ICOS substitute Ab (17G9, IgG2b) decreases tumor growth.
Figure 7:
A: Treg cell numbers are increased in primary cervical cancer.
B: ICOS + Treg cells are increased in primary cervical cancer.
Figure 8: Increase in Treg that express ICOS in non-Hodgkin's lymphoma (NHL).
HD Hodgkin's disease
FL Follicular Lymphoma
DLBCL Diffuse Large B Cell Lymphoma
MCL Mantle Cell Lymphoma
MZL Marginal Zone Lymphoma
Figure 9: Presence of Treg ICOS + cells in primary breast tumors has a negative impact on survival.
120 samples of primary tumor embedded in paraffin with 10 years of clinical follow-up were tested for their ICOS expression using a commercial anti-ICOS polyclonal rabbit Ab. The mean of ICOS + cells was evaluated on six different points. To perform the statistical analysis, the median was used as a cutoff point to have balanced groups.
The impact of ICOS expression according to the presence of ICOS in the primary tumor in Global Survival (A) or Progression-Free Survival (B) is shown. EXAMPLES EXAMPLE 1: Characterization of antibodies according to the invention Materials and methods I. Cell Biology 1 - Cell Selection / Purification
* Isolation of mononuclear cells from peripheral blood
PBMCs (peripheral blood mononuclear cells) were isolated from peripheral stem cells transplanted from healthy volunteers (Etablissement Francais du sang, Marseille, France) by Lymphoprep gradient (Abcys). In tubes: 2/3 of blood is deposited dropwise on 1/3 of Lymphoprep and centrifuged for 20 minutes at 2000 rpm at 20 ° C without acceleration, so as not to disturb the gradient. After centrifugation, mononuclear cells are recovered and washed twice in 1% PBS, FCS (Calf Fetal Serum) + heparin for 20 min at 1000 rpm at 20 ° C.
The cells were then used immediately or frozen at -80 ° C at 50 x 10 ° cells / ml in RPMI 1640, 50% FCS, 10% DMSO (dimethylsulfoxide). After 24 hours, the cells are transferred to nitrogen for preservation.
* Negative CD4 selection
CD4 + T lymphocytes were purified from PBMCs. After thawing, the cells were washed and diluted in 40 µl of selection buffer (PBS, 0.5% BSA, 2 mM EDTA) for 10 x 10 6 cells. MACS Human CD4 + T Cell Isolation Kit II kits (Miltenyi Biotec) were used: 10 | 11 of a biotin-conjugated monoclonal antibody solution (primary labeling) was added and the mixture was incubated for 10 min at 4 ° C with shaking .
The cells are then placed in contact with 20 | 11 of magnetic granules coupled with anti-biotin (secondary labeling) for 15 min at 4 ° C with agitation. After washing with selection buffer, the cells were selected in an Automacs (Miltenyi). The negative fraction without labeled CD4 + T cells is then isolated. This provides a pure CD4 + population of around 95%. 2 - Cell Activation and Culture
* Pre-activation with CD3 / CD28 granules and then stimulation with mAbs
CD4 + T cells are placed at a concentration of 106 cells / ml of RPMI, 10% FCS, in the presence of CD3 / CD28 granules (Dynabeads, Invitrogen) (1 cell / 1 granule) and incubated for 48 h at 37 ° C . The cells are then separated from the granules with a magnet and Dynal Biotech rests overnight in RPMI, 10% FCS at a concentration of 106 cells / ml.
On the other hand, anti-CD3 mAbs (OKT3), anti-ICOS (ICOS 88-2) and control IgGl mAb (Sigma) were coated on a 96-well flat plate overnight at 4 ° C. The wells are coated with 50 ng / ml of anti-CD3 supplemented with 20 µg / ml of other mAbs, PBS IX 100 µl / well. The next day, the plate is washed with PBS, saturated for two hours with PBS, 5% FCS (200 p.1 per well). CD4 + T cells with previously incorporated CFSE (see below) are distributed on the coated plate at a rate of 2105 cells / 200 | 11 of medium / well and incubated for 72 h at 37 ° C. In 48 hours, supernatants were collected and, in 72 hours, cells were collected for proliferation analysis using flow cytometry (Figure 4).
* Activation by Artificial APC
Magnetic granules (Dynabeads M-450 Epoxy, Invitrogen) were washed in 0.1 M sodium phosphate buffer, then incubated with anti-CD3 mAbs (OKT-3) at a sub-optimal concentration of 1 mg / lx107 granules , representing 5% of the mAbs coupled with the granules, with the anti-CD28 or ICOS mAb (ICOS 88-2 or CD28.2), (2 pig / 1 x 107 granules, 10%). These artificial APC's were incubated with the mAbs in slow rotation overnight at 4 ° C. The next day, two washes are performed in PBS, 0.1% BSA. Artificial APC 's are distributed over a granule to a cell in a 96 well round plate on which 2 x 105 CD4 + / 200 | 11 T lymphocytes per well were deposited, then incubated for 72 h at 37 ° C. CD4 + T cells previously incorporated CFSE. In 48 hours, the supernatants were collected and, in 72 hours, the cells were collected for analysis of proliferation by means of flow cytometry. 3 - Cell proliferation
Lymphocyte proliferation is accompanied by CFSE (Succinimidyl Ester of Carboxifluorescein Diacetate) (Molecular Probes, Invitrogen). CFSE is cell permeable and non-fluorescent. Upon entering the cell, esterases cleave the acetate groups, which become fluorescent, while the cell becomes impermeable.
The characteristic of the CFSE is to be shared equally in each newly formed cell in each division. It emits green radiation and allows the simultaneous analysis of the number, position and differentiation stage of the cells, the fluorescence intensity per cell being proportional to the concentration of CFSE. To label the cells with CFSE, the cell suspension is diluted in ice-cold IX PBS. CFSE addition: 5 | 1M to 10 x 106 cells. The cells are then placed in a 37 ° C water bath.
After 8 to 10 minutes of shaking, the cells were quickly placed on ice to stop the reaction. The cells are then spun twice with 2 ml of PBS IX. Finally, they are collected in the desired volume of RPMI, 10% FCS for culture. Proliferation is determined thanks to flow cytometry. II - Flow Cytometry
CD4 + cells are diluted with 30% BSA, PBS (50 µl / well) in a 96-well plate for 10 minutes at 4 ° C to saturate non-specific sites. They are then incubated for 30 minutes at 4 ° C in the dark with the desired antibodies coupled to a fluorophore.
After two washes in PBS IX, 1% BSA, 0.02% azide (centrifugation at 2100 rpm, 3 min at 4 ° C), the cells were fixed in 200 | 11 PBS, 2% formaldehyde and placed in a flow cytometer (FACS Canto, BD Biosciences). The results are analyzed thanks to the FlowJo software. Ill- ELISA (enzyme linked immunoadsorption assay)
CD4 + T cell culture supernatants are collected in 48 h and stored at -20 ° C for an IL-10, TNFOC and IFNy assay. RESULTS
1 - Characterization of anti-ICOS mAbs
The inventors developed 5 anti-ICOS abs. Its isotype was evaluated by ELISA. To obtain an indirect analysis of their affinity for their receptors, mAbs were tested using stable transfectants that express ICOS. JICOS.l cells were in the presence of an increasing range of anti-ICOS mAbs labeled with a probe coupled to a fluorophore (PE-GAM: goat anti-mouse PE) and the analysis was done thanks to flow cytometry.
Thus, it was possible to determine the ED50, that is, the concentration of mAbs in which 50% of the sites are saturated. mAbs with the lowest ED50 are those with the highest apparent affinity.
Next, the inventors tested the ability of anti-ICOS mAbs to inhibit the binding of ICOS-L (a recombinant form of the human IgGl Fc domain) brought by JICOS.l cells.
They used an anti-ICOS mAbs concentration gradient and revealed ICOS-L fixation to the Fc thanks to a probe coupled to a fluorophore (GAH-PE: Goat anti-human PE). The analysis was performed using flow cytometry. The inventors thus determined the ID50, that is, the dose that inhibits 50% of the ICOS-L Fc binding in ICOS.
The lower the ID50, the more easily the mAb competes with
Recombinant ICOS Fc.
The inventors thus found that ICOS R 314-8 and ICOS R 53-3 have a high affinity for their binding sites (ED50 <0.5 pg / ml) and a significant blocking potential (ID50 <1 mg / ml ).
The ICOS R 314-8 antibody was therefore chosen to be coupled to the Alexa Fluor 647 fluorophore and used in flow cytometry analysis.
2 - anti-ICOS mAbs differ in their ability to induce IL-10 production by activated CD4 + T cells
The inventors tested the ability of mAbs to act as agonist antibodies, that is, to be able to have the same action as the natural ICOS ligand using functional tests. The studied parameter was the secretion of IL-10, since ICOS induces the production of IL-10 by LT.
The potential agonist of anti-ICOS mAbs was tested on CD4 + T cells, which were pre-activated with CD3 / CD28 granules for 48h and distributed on a plate where anti-CD3 mAb was coated to continue stimulation along with the various anti mAbs -ICOS.
The culture supernatants were then assayed for 48 h for IL-10 and the secretion of IL-10 induced by the different anti-ICOS mAbs was compared based on the secretion of IL-10 induced by a commercially available anti-ICOS mAb (ICOS c).
Anti-ICOS mAbs 53-3, 88-2 and 92-17 significantly increased IL-10 secretion of CD4 + and are therefore agonist antibodies. Regarding anti-ICOS 145-1 and 314-8 mAbs, no significant increase was detected in IL-10 production.
The inventors have finally shown that anti-ICOS mAbs 53-3, 88-2 and 92-17 are better agonists than commercially available anti-ICOS. In fact, if we consider the commercially available anti-ICOS mAb as a reference, anti-ICOS 88-2 mAb causes an increase in IL-10 secretion of + 61%, anti-ICOS 92-17 mAb of + 20% and the + 14% anti-ICOS 53-3 mAb.
The results are summarized in the following table:
EXAMPLE 2: Use of an antagonist antibody of the invention and an agonist antibody of the invention Materials and methods Immunohistochemistry
Frozen primary breast tumor sections were stained with mouse anti-FOXP3 or anti-K167 and developed using the anti-mouse Ig Peroxidase immPRESS kit (Abcys, Paris, France) according to the supplier and DAB instructions. Then, the second primary antibody (anti-mouse ICOS (53.3), anti-CD3, anti-BDCA2) was added and developed with the ImmPRESS and Histogreen kit (Abcys). Staining specificity was assessed using the mouse isotype controls instead of the first or second primary antibody.
Purification of mononuclear cells from breast tumors, tonsils and healthy blood
Mononuclear cells (MNCs) were purified from healthy peripheral blood obtained from EPS or from enzymatic fragments of primary breast tumors or samples from tonsils by means of Ficoll density gradient centrifugation.
Phenotypic analysis of pDC and T cell subsets
For extensive phenotypic analysis, pDC were identified among total MNCs as CD4 + CD123 + cells using FITC or anti-CD123 PE and anti-CD4 PE-Cy5 and PE-coupled antibodies against CD40, CD86 or ICOSL. T cells were identified as CD3 + CD4 + cells. Treg were identified either by the multicolored phenotype CD4 + CD127 ~ CD25altu or by its expression of FoxP3 after activation in CD3 + CD4 + T cells.
The proliferation of Ta-Treg and Ta-Tconv or its counterpart in the blood was evaluated by means of multicolored analysis, allowing the characterization of Treg CD4T (CD127 ~ CD25alt0) and Tconv (CD4 + CD127 + CD25BalX0 ™) associated with the staining of the Ab Ki67.
Flow cytometric analysis was performed on a FACScan (BD Biosciences) or an ADP Cyan (Beckman Coulter) and the data were analyzed using the Cell Quest Pro (BD Biosciences) or FlowJo (Treestar) software. PDC purification
pDC's were purified from MNCs enriched in (Lin) -negative lineage through magnetically activated cell separation using the CD304 / BDCA-4 Microbeads kit or negative depletion using the pDC Isolation kit (Miltenyi Biotec) or FACS separation (cytometer from flow FACSVantage SE ™ DiVa, BD Biosciences) as Lin-CD4 + CD11c- cells. Purity was routinely> 98%.
In vitro generation of DC-derived monocytes (MoDC)
MoDC's were obtained from blood purified monocytes after 7 days of differentiation in GM-CSF (100 ng / ml) + IL-4 (50 IU / ml) (Schering Plow, Kenilworth, USA).
Purification of CD4 + and Treg memory T cells
CD4 + memory T cells (purity> 95%) were obtained from MNCs after magnetic depletion, including anti-CD45RA Ab, as described (Gobert et al., 2009). Treg CD4 + CD25alt0CD12 7 ~ and conventional CD4 + T cells CD4 + CD25 ~ CD127 low / + were selected by FACS® from purified CD4 + memory T cells (purity> 98%).
To monitor their proliferation in vitro, purified CD4 + memory T cells were stained on day 0 with CFSE. Viable cells were selected by exclusion with DAPI or Live and Dead reagent in the case of cell permeabilization (200,000 and 5,000 minimal events were activated on the total cell population and on the purified cells, respectively).
DC-T cell co-cultures
Allogeneic CD4 + memory T cells, Treg or Tconv CD4 + cells were cultured in 3 x 104 to 5 x 104 cells in medium complete with IL-2 (100 IU / ml) and TApDC, healthy pDC or highly purified MoDC, which were pre- activated for 24 hours with IL-3, GM-CSF (10 ng / ml) in the presence of R848. The addition of T lymphocytes in subsets of pre-activated DC's was performed in triplicate in 96-well round-bottom plates in the proportion of 1: 5 (DC / T cells) and co-cultured for 5 days. Proliferation was evaluated either by dilution with CFSE in FoxP3 expression analysis experiments or by DNA synthesis analyzed by the uptake of 3H-TdR.
The impact of the ICOS / ICOSL coupling was assessed by adding Ab Ctrl, Ab anti-ICOS commercial (ISA-3) or particular (314.8) or Ab anti-ICOSL (MIH12) in the cultures. To assess T cell cytokine secretion by ELISA, cells were co-cultured with pDC or TApDC and supernatants collected on day 5 were centrifuged and stored at -20 ° C.
Tconv and Treg stimulation with artificial granules
Artificial APCs were produced as described in Example 1. Treg (3 x 10) or Tconv (1 x 10) chosen from tonsils or Ta-CD4 T cells (1 x 10) purified from tumors were cultured for 5 days with artificial granules in a 1: 1 ratio (artificial APC: T cell) in the presence of IL-2 (100 IU / ml) in 96-well U-bottom plates at 200 | 11. Proliferation was assessed either by dilution with CFSE or DNA synthesis analyzed by the uptake of 3H-TdR.
Detection of cytokines in T cell culture supernatants by ELISA
IL-10, IFN-y and IL-2 in 5-day culture supernatants were quantified by ELISA using commercial kits from Bender Medsystems according to the manufacturer. RESULTS
The data presented below are intended to analyze the impact of two antibodies against ICOS (i.e., blocking mAb 314.8; agonist mAb 88.2) developed by the inventors, in order to validate: i) blocking ICOS by antagonist mAb 314.8 as a new promising drug candidate to cancel the immunosuppressive response seen in breast cancer; and ii) the coupling of ICOS by the mAb agonist 88.2 in CD4 + T cells to favor the amplification of Treg that would be of interest in the field of autoimmunity.
Ta-Treg that expresses highly ICOS are present within lymphoid aggregates in primary breast tumors and proliferate in situ
The inventors previously demonstrated that the presence of tumor-associated regulatory T cells (Ta-Treg) that express CD25alt0 and FoxP3 in primary breast tumors within lymphoid aggregates correlates with a poor prognosis and increased risk of metastasis (Gobert et al., 2009). These Ta-Treg, which represent 15% to 25% of total CD4 + TaT cells, are highly activated, as they express ICOS, CD39, GITR and HLA-DR and suppress TaTconv proliferation and cytokine secretion (IL-2 , IFNy).
These Ta-Tregs proliferate within the primary breast tumor environment in situ (Gobert et al., 2009), as demonstrated either by the presence of Treg ICOS + that co-express Ki67 in frozen sections of tumor (Figure IA) or by a higher proportion of Ki67 + cells within purified Ta-Treg and blood Treg (8% and 4%, respectively) compared to Ta-Tconv and Tconv (3% and 0.3%, respectively) (Figure 1B).
In contrast to these in vivo results, the inventors demonstrated that in vitro stimulation of purified Ta-Treg with expanded granules (granules coated with anti-CD3 and anti-CD28 Ab agonist) is not able to favor the amplification of Ta-Treg, in contrast to that observed with purified TaTconv or purified Treg or Tconv from blood from healthy donors (Figure 1C).
The inventors imagined that the coupling of ICOS is essential for the proliferation and functions of Ta-Treg. A) Use of an antagonist antibody of the invention
Blocking ICOS / ICOS-L interaction through the ICOS antagonist mAb (314.8)
Ta-Treg interact in situ with Ta-pDC within lymphoid aggregates in primary breast carcinoma
Several studies have reported the expression of ICOS-L, the specific ICOS ligand, in pDC (Janke et al., 2006). Using immunohistochemistry on frozen sections of tumor, the inventors observed that Ta-CD3 + T cells present within the lymphoid aggregates surrounding the tumor are interacting with Ta-pDC BDCA2 + (Figures 1D and 1E). A double stain with Ab FoxP3 and BDCA2 revealed that Ta-Treg are in close contact with Ta-pDC in these lymphoid aggregates, suggesting that this interaction would favor the coupling of ICOS by ICOS-L in tumors (Figure 1F).
Ta-pDC are activated, but do not express ICOS-L as a potential consequence of ICOS / ICOS-L interaction in situ
After purification of tumor fragments, Ta-pDC shows an activated phenotype, since they express positively regulated levels of CD86 and CD40, compared with healthy blood and blood pDC's from the corresponding patient. As reported by several groups (Ito et al., 2007; Janke et al., 2006), recently isolated healthy blood pDC's express low levels of ICOS-L which is strongly unregulated after exposure to IL-3 or binding to TLR7 / 8, which is not observed in other subsets of DCs (mDC, MoDC). Interestingly, in contrast to its activated state (CD86 + CD40 +), Ta-pDC are devoid of ICOS-L expression in the membrane. In contrast, freshly isolated corresponding patient's blood pDCs or healthy blood pDCs express ICOS-L. After a 24-hour culture period in IL-3 or by binding to TLR7 / 8, selected Ta-pDCs regain a strong expression of ICOS-L, demonstrating their ability to modulate this expression of ICOS-L (data not shown). Among CD3 + TaT cells, ICOS is strongly expressed in Ta-Treg (69.9% MFI: 361), in contrast to TaTconv (23% MFI: 83) or TaCD8 + (2% MFI: 50). These results indicate that ICOS / ICOS-L interaction in situ leads to ICOS-L overload in the Ta-PDC membrane.
Blocking ICOS / ICOS-L interaction through antagonist anti-ICOS mAb (314.8) cancels ICOS-L overload in pDC
To test this hypothesis, healthy blood T cells were cultured with pDCs activated by TLR7 purified from the tonsils. The inventors observed, after the 24h culture period with increased T: pDC ratio, a dose-dependent overload of ICOS-L over pDC. Interestingly, the addition of the antagonist mAb of the invention against ICOS (314.8) completely nullifies this overloading of ICOS-L onto pDCs, a result that is not reproduced using the commercial anti-ICOS antibody (ISA-3) (data not shown).
The results demonstrate the interactions Ta-pDC and Ta-Treg through ICOS / ICOS-L and indicate that the binding of ICOS may be involved in the activation and proliferation of Ta-Treg.
Co-culture of CD4 + T cells, as well as purified Treg with activated pDCs, but not MoDC, induced proliferation of Treg that is blocked with 314.8
To test the ability of ICOS / ICOS-L interactions to induce Treg amplification, the inventors cultured total CD4 + memory T cells with TLR7 / 8 (R848) activated allogeneic healthy blood purified pDC or mDC. Among the purified CD4 + T cells, 3.5% expressed FoxP3 (data not shown). After 5 days of co-culture with pDC's, the proportion of cells expressing FoxP3alt °, corresponding to Treg, rises to 12.3% and the addition of Ab 314.8 blocks this enrichment in FoxP3alt ° cells by 80%. In contrast, co-culture of CD4 + T cells with activated mDCs was unable to favor a distinct subpopulation of FoxP3alt ° between CD4 + T cells and the addition of 314.8 has no significant effect (6.3% to 8%) •
Similar results were obtained with purified CD4 + T cells from the tumor. Ta-Treg Foxp3 + represent 9% of newly purified TaT CD4 + cells (data not shown). Its co-culture with R848 activated pDCs increases the proportion of Ta-Treg to 14.5%, while the addition of 314.8 leads to a decrease in the proportion of Ta-Treg to 4.5%, below the initial level.
Populations of purified Treg or Tconv selected by FACS stained with CFSE were cultured with pDCs activated by R848 or MoDC's activated by LPS to analyze their proliferation capacity by means of flow cytometry (dilution of CFSE expression). First, the inventors observed that, in the absence of exogenous IL-2, activated MoDC's do not induce the proliferation of purified Treg, whereas Tconv proliferates strongly. In contrast, co-culture with activated pDCs is capable of inducing a strong proliferation of purified Treg and Tconv.
The addition of anti-ICOS mAb 314.8 strongly reduces the proliferation of Treg and Tconv when pDCs are used as APC, while the proliferation of Tconv remains unchanged in co-cultures with MoDC. In this experiment, blocking ICOS or ICOS-L with commercial antibodies (mAb ISA-3 or mAb MIH-12) does not affect either the proliferation of Treg or Tconv in co-cultures of pDC / T.
These data demonstrate that the anti-ICOS mAb 314.8 neutralizes the ICOS coupling on Treg and cancels its expansion induced by pDCs.
Blocking ICOS and ICOS-L cancels IL-10 secretion
interfere strongly on the MoDC / T co-culture
mAb 314.8 also reduces the proliferation of Tconv in response to stimulation with activated pDC's. The inventors determined the impact of 314.8 on the secretion of IFNy and IL-10 by ELISA during allogeneic R848 activated Tconv and pDC 's (Figure 2A) or LPS activated MoDC (Figure 2B). Under these conditions, the secretion of IL-10 is completely canceled out by mAb 314.8 (217 ± 31 pg / ml in the control and 13 ± 6 pg / ml with 314.8). Meanwhile, IFNy secretion is slightly reduced by adding mAb 314.8 in co-cultures with pDC (32% reduction, 507 ± 53 pg / ml in the control condition and 341 ± 73 pg / ml with 314.8) (Figure 2A). In Tconv / MoDC co-cultures, inhibition of ICOS leads to a slight increase in the secretion of IL-10 and IFNy (Figure 2B). B) Use of an agonist antibody according to the invention
Use of agonist anti-ICOS mAb (88.2) to mimic ICOS coupling
To improve their understanding of the ICOS functions in Treg and Tconv, the inventors generated an artificial APC model using granules coated with agonist mAbs, leading to the signaling of CD3 (OKT-3), CD28 (CD28.2) and / or ICOS (88.2, Table 1) in purified T cells.
Coupling ICOS with an agonist mAb (88.2) in Treg induced its proliferation and its ability to secrete high amounts of IL-10
First, the inventors observed that Treg's from healthy donors proliferate in response to anti-CD3 / 88.2 granules in the presence of exogenous IL-2 (Figure 3A). As previously reported (Simpson et al., 2010; Ito et al., 2008), upon activation by coupling TCR and ICOS in the presence of IL-2, both the subpopulations of purified Tconv and Treg secrete high amounts of IL-10 (311 ± 22 pg / ml and 426 ± 48 pg / ml, respectively) and low levels of IFNy (205 ± 8 pg / ml and 381 ± 12 pg / ml). This result contrasts with the data obtained using the anti-CD3 / anti-CD28 granules. In this model, Tconv secretes large amounts of IFNy (1213 ± 72 pg / ml) and low levels of IL-10 (69 ± 58 pg / ml), while Treg secretes IL-10 and low levels of IFNy (422 ± 36 pg) / ml and 305 ± 31 pg / ml, respectively) (Figure 3B).
Similar experiments with T cells purified from tumor demonstrated that CD4 + TaT cells produce similar levels of IL-10 in response to ICOS and CD28, while IFNy levels are weaker in response to ICOS compared to CD28 coupling (Figure 3C).
ICOS coupling blocks CD28-induced IL-2 and consequently reduces IFN-y proliferation and secretion
While CD4 + memory T cells proliferate in response to stimulation of anti CD3 / anti CD28 regardless of exogenous IL-2, no proliferation is seen in response to stimulation with anti CD3 / 88.2 (Figure 4A). The addition of hIL-2 rescues this proliferation in a dose-dependent manner. Interestingly, the co-coupling of ICOS and CD28 in the absence of IL-2 significantly decreases the proliferation of CD4 + memory T cells, compared to the CD28-only coupling, and this is completely rescued in the presence of 100 IU / ml IL- two. Interestingly, the binding of ICOS through mAb 88.2 cancels the secretion of IL-2 detected by stimulation with anti CD3 / anti CD28 (Figure 4B). Taken together, this argues for a reduction in spontaneous secretion of IL-2 when ICOS and CD28 are co-coupled, compared to CD28-only coupling, suggesting an ICOS inhibitory function on CD28-induced IL-2 secretion .
Furthermore, even in the presence of exogenous IL-2, the inventors observed a 50% reduction in the IFN-y produced by Tconv, when ICOS and CD28 are triggered, compared to anti-CD3 anti-CD28 granules (Figure 4C).
In contrast, although the secretion of IL-10 is strictly dependent on IL-2 when cells are activated under stimulation with ICOS, as described previously (Ito 2008, Paulos 2010), the addition of an ICOS signal does not affect IL secretion. -10 induced by anti CD3 / anti CD28 (Figure 4C).
All of these results together demonstrate that the binding of ICOS reduced the ability of anti CD3 / anti CD28 to favor Thl polarization (through reduced IFN-y production), but maintains the production of IL-10, favoring the development of an immunosuppressive environment.
Coupling of ICOS through mAb 88.2 increased the suppressive function of Treg
To assess whether ICOS coupling may be associated with an immunosuppressive T cell response, the inventors developed suppression assays in the absence of exogenous IL-2 to compare the efficiency of anti CD3 / anti CD28 / IgG and anti CD3 / anti CD28 / granules 88.2. The addition of signaling to ICOS (88.2) strongly increases the suppressive function of Treg, compared to anti CD3 / anti CD28 / IgGl (51% inhibition in the condition of a Treg to 4 Tconv anti
CD3 / anti CD28 / 88.2, compared to 21% inhibition with anti CD3 / anti CD28 / IgG). All of these results together demonstrate that ICOS coupling favors an immunosuppressive T cell response that can result either from an increased sensitivity of Tconv to suppression or a stronger suppressive capacity of Treg. EXAMPLE 3: Analysis of the prognostic impact of detection of ICOS + Treg cells in primary breast tumors
120 paraffin-embedded primary tumor samples with 10 years of clinical follow-up were tested for ICOS expression using a commercial rabbit polyclonal anti-ICOS Ab (Spring Biosciences). ICOS + cells were quantified in double blind in 6 different replicates for each tumor and the average of the results was compiled (data not shown). To perform the statistical analysis, the inventors used the median as a cutoff point to have balanced groups.
In univariate analysis, the inventors demonstrated that the presence of ICOS + cells (> 1.66 ICOS + cells / dot) correlated with a high degree of tumor (p = 0.007), expression of the estrogen receptor by tumor cells (p = 0.018) , molecular subtypes A / B luminals (p <0.001) and absence of overexpression of Her2 / neu (p = 0.035).
The impact of detecting ICOS + cells in primary breast tumors on overall survival (OS) or progression-free survival (PFS) was investigated.
While 6/59 deaths were observed in the ICOS ~ group, 14/61 patients died in the ICOS + group, demonstrating the significant prognostic value of detecting ICOS + on OS (p-value of the Log Classification test = 0.0465) (Figure 9A). A similar analysis performed on PFS showed that ICOS + cells were associated with a lower overall survival, with progression on 11/59 in the ICOS ~ group, while 20/61 patients progressed in the ICOS + group (p = 0.0285) (Figure 9B ).
EXAMPLE 4: Confirmation of the existence of in situ interaction of pDC with Treg ICOS + in the tumor environment
Ex vivo co-culture of tumor cell fragments in the presence of anti-ICOS mAb 314.8 or Ab Ctrl for 48 hours in the presence of IL-3 (20 ng / ml). At the end of the culture period, the expression of ICOS-L in pDC is observed only in the presence of anti-ICOS mAb 314.8 and not with control Ab, demonstrating that the overloading of ICOS-L in pDC is mediated by an interaction with cells ICOS + (data not shown).
EXAMPLE 5: Breast tumor epithelial cells from either established cell lines or fresh tumor samples do not express ICOS-L, in contrast to melanoma or glioma tumor cells, even after ex vivo culture with anti-ICOS antibody 314.8
Breast tumor epithelial cell lines were collected in PBS with EDTA to prevent degradation associated with Ag trypsin and the cells were stained with anti-ICOS-L antibody to evaluate expression on the cell surface using flow cytometry. It was found that none of the tested cell lines was positive for ICOS-L (Figure 5A). Similar analyzes were performed on fragments of primary tumor grown 48h in the presence of anti-ICOS Ab (314.8) or control Ab plus IL-3 (20 ng / ml) (Figure 5B).
EXAMPLE 6: Impact of an anti-mouse ICOS anti-mouse substitute Ab (17G9, IgG2b) on breast tumor growth in a model of syngeneic breast tumor
The mouse mammary tumor model was obtained in female FVB mice 28-35 days after orthotopic injection of the Neu 15 cell line. The tumors generated appeared to be significantly infiltrated by activated Ta-pDC, Treg ICOSdlto TATreg and TATconv at rest.
Injection of 17G9 antibody (50 µg / ml) intraperitoneally three times a week from day 11 after tumor implantation results in a reduction in Neul5 tumor size at later time points compared to the control Ab injection (LTF2, IgG2b) (p = 0.053) (Figure 6). EXAMPLE 7: A: Treg cell numbers are increased in primary cervical cancer.
Cervical samples were obtained from patients with either dysplasia (CIN2 / 3, n = 18) or cancer (n = 14). Normal cervical tissue was used as a control (n = 11). The samples were obtained by enzymatic and physical dissociation. After washing, mononuclear cells were incubated with labeled mAbs and Tregs enumerated as CD12 7baiX0CD25alai0CD4 + T cells. The percentage of Treg's within the subset of CD4 + cells is represented. Treg were increased in cervical cancer samples compared to normal and dysplastic tissue. Consequently, this increase is associated with the development of cancer (Figure 7A). B: ICOS + Treg cells are increased in primary cervical cancer.
Cervical samples were obtained from patients with either dysplasia (CIN2 / 3, n = 5) or cancer (n = 12). Normal cervical tissue was used as a control (n = 5). The samples were obtained by enzymatic and physical dissociation. After washing, mononuclear cells were incubated with labeled mAbs using ICOS and the Tregs were enumerated. The percentage of Treg ICOS within the CD4 + subset is represented. Treg's ICOS + are present within tissues with only a tendency to increase in cervical cancer, due to the limited number of samples analyzed (Figure 7B). EXAMPLE 8: Increased ICOS expressing Treg in Non-Hodgkin's Lymphoma (NHL)
The inventors analyzed the Treg numbers and the ICOS expression in Treg in NHL samples. Fresh lymphoma cells extracted from lymph nodes were collected from 45 patients with informed consent. The lymphoma samples correspond to Hodgkin's disease (HD, n = 11), follicular lymphoma (FL, n = 13), diffuse large B cell lymphoma (DLBCL, n = 10), mantle cell lymphoma (MCL, n = 5) and marginal zone lymphoma (MZL, n = 6). Detection of Treg cells was carried out by incubating for 20 min at 4 ° C with anti-ICOS-PE (Becton Dickinson ™), anti-CD3-ECD, anti-CD4-Pacific Blue (Beckman Coulter®), anti- CD127 FITC, anti-CD25 APC-Cy7 and fixable LIVE / DEAD® dead cell staining kit (Invitrogen ™). After staining, each cell preparation was washed twice with PBS, fixed with 2% paraformaldehyde and analyzed on a FACS LSR2 flow cytometer (Becton Dickinson ™). The data were analyzed using the FlowJo Software (TreeStar ™). Treg's were increased in all lymphoma samples, except HD. Most Treg's exhibited increased expression of ICOS compared to control lymph nodes (Figure 8). EXAMPLE 9: Sequencing of Icos 314.8 (CNCM 1-4180)
Total RNA was extracted from hybridoma cells supplied frozen and cDNA was synthesized. Then, RT-PCR was performed to amplify the variable regions (the heavy and light chains) of the mAb. The variable regions of the heavy and light chains of the MAb were separately cloned into a cloning vector, then the sequences obtained were analyzed to deduce the mAb sequences.
Materials
Hybridoma cells ICOS 314.8 (CNCM 1-4180); RNA purification system TRlzol® Plus (Invitrogen, Cat. No. 15596-026); Superscript ™ III first tape synthesis system (Invitrogen, Cat. No. 18080-051). Methods Total RNA extraction
Total RNA was isolated from the hybridoma cells according to the technical manual of the TRIzol® Plus RNA Purification System. The total RNA was verified using gel electrophoresis.RT-PCR
Total RNA was reverse transcribed into cDNA using isotype-specific antisense primer or universal primer and the entire procedure was in accordance with the Superscript ™ First Tape Synthesis System technical manual. The antibody fragment will be amplified according to the standard operating protocol of the GenScript RACE method. Cloning of Antibody Genes
The PCR target products of antibody genes were cloned into the cloning vector separately, according to conventional molecular cloning procedures. Screening and Tracking
Colony screening was employed to select clones with correct sized inserts and no less than ten independent positive colonies were sequenced for each antibody fragment. Results and Analysis Total RNA extraction
The total RNA of the sample was run on a DL3000 DNA marker on a 1.5% agarose electrophoresis gel / GelRed ™. Antibody Gene PCR Product
4 | 11 of PCR products from each sample were run on the DL3000 DNA marker on a 1.5% agarose electrophoresis gel / GelRed ™. Sequencing results and analysis
The sequencing results are as follows. The consensus DNA sequences and corresponding amino acid sequences are listed below:
Heavy chain: DNA sequence (426 bp): leader sequence-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4
ATGGGATGGCGCTGTATCATCCTCTTCTTGGTATCAACAGCTACAGGTGTCCAC TCCCAGGTCCAACTACAGCAGCCTGGGACTGAACTTATGAAGCCTGGGGCTTCAGTGAA GC T GT CCT GCAAGGC TTC TGGCTACACCTTCACCACCTACTGGATGCACTGGGTGAAGC AGAGGC CT GGACAAGGC CTT GAGTGGATCGGAGAGATTGATCCTTCTGATAGTTATGTT AACTACAATCAAAACTTTAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCCAGCAC AGCCTACATACAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTTTGTGCGA GATCCCCTGATTACTACGGTACTAGTCTTGCCTGGTTTGATTACTGGGGCCAAGGGACT CTGGTCACTGTCTCTACA (SEQ ID NO: 13) Heavy chain: the amino acid sequence (142 AA): leader sequence-Frl-CDRL-FR2-CDR2-FR3-CDR3 -FR4 MGWRCIILFLVSTATGVHSQVQLQQPGTELMKPGASVKLSCKASGYTFTTYWMH WVKQRPGQGLEWIGEIDPSDSYVNYNQNFKGKATLTVDKSSSTAYIQLSSLTSEDSAVY FCARSPDYYGTSLAWFDYWGQGTLVTVST (SEQ ID NO: 14) light chain: DNA sequence (396 bp): Frl-1íder sequence CDR1-FR2-CDR2-FR3-CDR3-FR4 ATGAGGTGCCTAGCTGAGTTCCTGGGGCTGCTTGTGCTCTGGATCCCTGGAGTC ATTGGGGATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTC AGTATCCATCTCC TGCAGGTCTAGTAAGAGTCCCCTGCATAGTAACGGCAACATTTACT 5 TATATTGGT TCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCC AACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTACTTTCAC ACTGAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATC TAGAATATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 15)
Light chain: amino acid sequence (132 AA): leader sequence-FRl-CDR1-FR2-CDR2-FR3-CDR3-FR4 MRCLAEFLGLLVLWIPGVIGDIVMTQAAPSVPVTPGESVSISCRSSKSPLHSNG NIYLYWFLQRPGQSPQLLGYGSHGGGGGGGGGGGGLGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGVGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGMKKKKTKKKKKKKKKKKKKTKKKTKKTKKTKTKTKTKTKTKTKTTKTTKTTKTTKTTKTTKTTKTTKTTKTTTKTTTTTTTTTTTTTTTTLK
Thus, the sequences of ICOS 314.8 (CNCM 1-4180) can be summarized as follows:


EXAMPLE 10: Sequencing of Icos 88.2 (CNCM 1-4177)
Total RNA was extracted from hybridoma cells supplied frozen and cDNA was synthesized. Then, RT-PCR was performed to amplify the variable regions (heavy and light chains) of the mAb. The mAb variable regions of the heavy and light chains were cloned into a cloning vector separately, then the sequences obtained were analyzed to deduce the mAb sequences. Materials
ICOS 88.2 hybridoma cells (CNCM 1-4177); TRIzol® RNA system and purification (Invitrogen, Cat. No. 15596-026); Superscript ™ III first tape synthesis system (Invitrogen, Cat. No. 18080-051). Methods
Total RNA extraction Total RNA was isolated from the hybridoma cells according to the technical manual of the TRIzol® Plus RNA Purification System. The total RNA was verified using gel electrophoresis. RT-PCR
The total RNA was reverse transcribed into cDNA using isotype-specific antisense primer or universal primer and the entire procedure was in accordance with the Superscript ™ III first-strand Synthesis System technical manual. The antibody fragment will be amplified according to the standard operating protocol of the GenScript RACE method.
Cloning of Antibody Genes
The PCR target products of antibody genes were cloned into the cloning vector separately, according to conventional molecular cloning procedures.
Sorting and Sequencing
Colony screening was employed to select clones with correct sized inserts and no less than ten independent positive colonies were sequenced for each antibody fragment. Results and Analysis Total RNA extraction
The total RNA of the sample was run together with a DL3000 DNA marker on a 1.5% agarose electrophoresis gel / GelRed ™.
Antibody Gene PCR product 4 | 11 of PCR products from each sample were run together with the DNA marker DL3000 on a 1.5% agarose electrophoresis gel / GelRed ™.
Sequencing results and analysis
The sequencing results are as follows. The consensus DNA sequences and corresponding amino acid sequences are listed below:
Heavy chain: DNA sequence (429 bp): leader sequence-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCAC TCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAA GC T GT CCT GCAAGGC TTC TGGCTACAGTTTCACCAGCTACTGGATAAACTGGGTGAAGC AGAGGC CT GGACAAGGC CTT GAGTGGATCGGAAATATTTATCCTTCTGATAGTTATACT AACTACAATCAAATGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAACAC AGCCTACATGCAGCTCACCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTACAA GATGGAATCTTTCTTATTACTTCGATAATAACTACTACTTGGACTACTGGGGCCAAGGC ACCACTCTCACAGTCTCCTCA (SEQ ID NO: 29) Heavy chain: the amino acid sequence (143 AA): leader sequence-Frl-CDRL-FR2-CDR2-FR3-CDR3 -FR4 MGWSCIILFLVATATGVHSQVQLQQPGAELVRPGASVKLSCKASGYSFTSYWIN WVKQRPGQGLEWIGNIYPSDSYTNYNQMFKDKATLTVDKSSNTAYMQLTSPTSEDSAVY YCTRWNLSYYFDNNYYLDYWGQGTTLTVSS (SEQ ID NO: 30) light chain: DNA sequence (396 bp): Frl-leader sequence, CDRL FR2-CDR2-FR3-CDR3-FR4 ATGAGGTGCCTAGCTGAGTTCCTGGGGCTGCTTGTGCTCTGGATCCCTGGAGCC ATTGGGGATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTC AGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTAATGGCAACACTTACT TGTATTGG TTCCTGCAGAGGCCAGGCCAGTCTCCTCAACTCCTGATATATCGGATGTCC AACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCAC ACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATC TAGAATATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 5 (SEQ ID NO: 31)
Light chain: the amino acid sequence (132 AA): Frl-sequence 11der-CDR1-FR2-CDR2-FR3-CDR3-FR4 MRCLAEFLGLLVLWIPGAIGDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNG NTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC MQHLEYPWTFGGGTKLEIK (SEQ ID NO: 32) Thus, the sequences of ICOS 88.2 (CNCM 1-4177) can be summarized as follows: DNA sequence Amino acid sequence H-CDR1 GGC T AC AGT TTC AC C AGC T AC T GGAT AAAC (SEQ ID NO: 17) GYSFTSYWIN (SEQ ID NO: 23) H-CDR2 AATATTTATCCTTCTGATAGTTATAC TAAC TACA ATACATG (SEQ ID NO: 18) NIYPSDSYTNYNQMFKD (SEQ ID NO: 24) H-CDR3 TGGAATCTTTCTTATTACTTCGATAATAACTACT ACTTGGACTAC (SEQ ID NO: 19) WNLSYYFDNNYYLDY (SEQ ID NO: 25) L-CDRACT AGGTTAGGTA ID NO: 26) L-CDR2 CGGATGTCCAACCTTGCCTCA (SEQ ID NO: 21) RMSNLAS (SEQ ID NO: 27) L-CDR3 ATGCAACATCTAGAATATCCGTGGACG MQHLEYPWT (SEQ ID NO: 22) (SEQ ID NO: 28)

权利要求:
Claims (7)
[0001]
1. Antibody directed against ICOS characterized by the fact that said antibody is selected from the group consisting of Icos 145-1 and Icos 314-8, respectively obtained from the hybridomas deposited at the CNCM on July 2, 2009 under the numbers of access CNCM 1-4179 and CNCM 1-4180.
[0002]
2. Antibody according to claim 1, characterized by the fact that said antibody neutralizes the involvement of ICOS in Tregs and revokes the proliferation of Treg induced by plasmocytoid dendritic cells.
[0003]
3. Antibody directed against ICOS characterized by the fact that the referred antibody has the following 6 complementarity determining regions (CDRs): - H-CDR1 - amino acid sequence GYTFTTYWMH (SEQ ID NO: 7); - H-CDR2 - amino acid sequence EIDPSDSYVNYNQNFKG (SEQ ID NO: 8); - H-CDR3 - FDY amino acid sequence (SEQ ID NO: 9); - L-CDR1 - amino acid sequence RSSKSPLHSNGNIYLY (SEQ ID NO: 10); - L-CDR2 - amino acid sequence RMSNLAS (SEQ ID NO: 11); - L-CDR3 - MQHLEYPYT amino acid sequence (SEQ ID NO: 12).
[0004]
4. Antibody according to claim 3, characterized by the fact that the nucleotide sequences encoding the 6 CDRs are as follows: - H-CDR1 - DNA sequence GGCTACACCTTCACCACCTACTGGATGCAC (SEQ ID NO: 1); - H-CDR2 - DNA sequence GAGATTGATCCTTCTGATAGTTATGTT AACTACAATCAAAACTTTAAGGGC (SEQ ID NO: 2); - H-CDR3 - DNA sequence TTTGATTAC (SEQ ID NO: 3); - L-CDR1 - DNA sequence AGGTCTAGTAAGAGTCCCCTGCATAGTAAC GGCAACATTTACTTATAT (SEQ ID NO: 4); - L-CDR2 - CGGATGTCCAACCTTGCCTCA DNA sequence (SEQ ID NO: 5); - L-CDR3 - DNA sequence ATGCAACATCTAGAATATCCGTACACG (SEQ ID NO: 6).
[0005]
Use of an antibody as defined in any one of claims 1 to 4, characterized in that it is for the manufacture of a medicament for the treatment of a disease or a condition associated with the suppression of the Treg-mediated immune response.
[0006]
6. Use, according to claim 5, characterized by the fact that said disease or condition is selected from cancers and chronic infections.
[0007]
7. Use, according to claim 6, characterized by the fact that said cancer is selected from human malignant lymphoma, ovarian cancer, cervical cancer and breast cancer.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2019-07-02| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-10-15| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-05-19| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-10-27| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 29/03/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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EP11305380|2011-03-31|

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EP11305380.5.|2011-03-31|

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PCT/EP2012/055735|WO2012131004A2|2011-03-31|2012-03-29|Antibodies directed against icos and uses thereof|

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