FR2980110A1 - Composition, useful for treating tumor including malignant tumor, leukemia and non-Hodgkin lymphoma, comprises bendamustine and an anti-cluster of differentiation 20 antibody, which is a monoclonal antibody - Google Patents

Abstract

Composition comprises bendamustine and an anti-cluster of differentiation 20 (anti-CD20) antibody, which is a monoclonal antibody, whose light chain includes an amino acid sequence of SEQ ID NO. 7 (a 213 amino acid sequence fully defined in the specification) and heavy chain includes an amino acid sequence of SEQ ID NO. 8 (a 448 amino acid sequence fully defined in the specification). An independent claim is included for the anti-CD20 antibody for treating a tumor, in combination with bendamustine. ACTIVITY : Cytostatic. MECHANISM OF ACTION : None given.

Description

The invention relates to the combination of anti-CD20 antibodies and bendamustine, and the therapeutic applications of such a combination. The CD20 antigen is a transmembrane protein present on pre-B lymphocytes as well as on mature B cells. The CD20 antigen is present on the surface of more than 90% of peripheral B lymphocytes and lymphoid organs. It is found on normal and tumor B lymphocytes. More particularly, the CD20 antigen is expressed on more than 90% of non-Hodgkin's lymphomas and is absent from hematopoietic stem cells, pro-B lymphocytes, plasma cells or other healthy tissues.

The CD20 antigen plays a role in the proliferation and / or differentiation of B lymphocytes, but this is not well known. However it represents an interesting target of targeted therapy to control and kill B cells involved in cancers or autoimmune diseases. IDEC Pharmaceuticals Corporation, U.S has developed a chimeric anti-CD20 monoclonal antibody, Rituximab, marketed as Rituxan® or MabThera®. This antibody is now used in the treatment of chronic lymphocytic leukemia and has many applications in the treatment of immunological diseases mediated by B-lymphocytes, such as for example malignant tumors, such as non-Hodgkin's lymphoma, certain autoimmune diseases such as autoimmune haemolytic anemia, idiopathic thrombocytopenic purpura and inflammatory diseases such as rheumatoid arthritis or multiple sclerosis. Several other anti-CD20 monoclonal antibodies have been developed, among which the EMAB603 antibody as described in the patent application WO2006 / 064121. This antibody is produced by the clone R603 deposited on November 29, 2005 under the registration number CNCM 1-3529 to the National Collection of Cultures of Microorganisms (CNCM, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15). Bendamustine, which is 4- {5- [bis (2-chloroethyl) amino] -1-methyl-2-benzimidazolyl} butyric acid, was synthesized initially in the 1960s in the German Democratic Republic (DDR), then was available from 1971 to 1992 as Cytostasan® and was subsequently marketed in Germany as Ribomustine® for the treatment of chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, myeloma, and Bendamustine is marketed in the United States as Treanda® by Cephalon and combinations of anti-CD20 and bendamustine antibodies have been proposed (WO2010 / 083365 and WO2011 / 018224). An object of the invention is an anti-CD20 antibody, for use in the treatment of a tumor, in combination with bendamustine, said anti-CD20 antibody being a monoclonal antibody whose light chain comprises the amino acids SEQ ID 10 NO: 7 and the heavy chain comprises the amino acid sequence SEQ ID NO: 8. Preferably the antibody is produced by the clone R603 deposited on November 29, 2005 under the registration number CNCM 1-3529 to the National Collection of Cultures of Microorganisms (CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15 ). In another embodiment, the antibody comprising the SEQ ID NO: 7 light chain sequence and the SEQ ID NO: 8 heavy chain sequence is a recombinant antibody produced by animal transgenesis, preferably in the milk of a transgenic goat. The antibody and bendamustine are intended for separate, simultaneous or sequential administration. The antibody and bendamustine can be combined within the same pharmaceutical composition. The invention then provides a pharmaceutical composition comprising bendamustine and an anti-CD20 antibody, said anti-CD20 antibody being a monoclonal antibody comprising the light chain sequence SEQ ID NO: 7 and the heavy chain sequence SEQ ID NO: 8 , preferably produced by the clone R603 deposited November 29, 2005 under the registration number CNCM 1-3529. Preferably the pharmaceutical composition is for intravenous or intraperitoneal administration.

The combination of the invention is useful in the treatment of a tumor, preferably a malignant tumor expressing the CD20 antigen, such as lymphoma or leukemia. According to a particular embodiment, the tumor is a non-Hodgkin's lymphoma. Key to the Figures Figure 1 is a graph that represents the tumor volume in mice bearing xenografts of a tumor line derived from human follicular lymphoma RL, treated with an anti-CD20 antibody (rituximab or R603) and bendamustine, alone or in combination. The tumor cells are injected on day 1. The arrows indicate the days of the administration of the treatment. The stars indicate a significant difference, in terms of tumor volume, of p <0.05, between the rituximab + bendamustine and R603 + bendamustine treatments, at the given day. DETAILED DESCRIPTION OF THE INVENTION Surprisingly, the inventors have shown that a combination of the antiCD20 antibody comprising the light chain sequence SEQ ID NO: 7 and the heavy chain sequence SEQ ID NO: 8, with bendamustine, had a greater therapeutic effect than a combination of rituximab (another anti-CD20 antibody) and bendamustine, at the same doses. On this basis, the inventors propose associating this anti-CD20 antibody with bendamustine, in particular for treating a tumor. Thus, a method for treating a tumor is described herein comprising administering to a patient a combination of the anti-CD20 antibody comprising the light chain sequence SEQ ID NO: 7 and the heavy chain sequence SEQ ID NO : 8, preferably from clone R603, with bendamustine. Preferably, the patient is a human, but may also be a non-human mammal. The anti-CD20 antibody used in the invention is an immunoglobulin which comprises the light chain sequence SEQ ID NO: 7 and the heavy chain sequence SEQ ID NO: 8.

Preferably, the antibody has two light chains whose variable domain is encoded by the murine nucleic acid sequence SEQ ID NO: 1 and the human constant region is encoded by the nucleic acid sequence SEQ ID NO: 4, and two heavy chains whose variable domain is encoded by the murine nucleic acid sequence SEQ ID NO: 2 and the constant region is encoded by the human nucleic acid sequence SEQ ID NO: 3. Each of the light chains of the antibody is encoded by murine-human chimeric nucleic acid sequence SEQ ID NO: 5, and each of the heavy chains is encoded by the murine-human chimeric nucleic acid sequence SEQ ID NO: 6. The chimeric nucleic acid sequence murine-human SEQ ID NO: 7 coding for each of the light chains of the antibody is obtained by fusion of the murine nucleic acid sequence SEQ ID NO: 1 coding for the variable domain of each of the light chains of the antico rps and the human nucleic acid sequence SEQ ID NO: 4 encoding the constant region of each of the light chains of the antibody. The murine-human chimeric nucleic acid sequence SEQ ID NO: 6 coding for each of the heavy chains of the antibody is obtained by fusion of the murine nucleic acid sequence SEQ ID NO: 2 coding for the variable domain of each of the heavy chains of the antibody and the human nucleic acid sequence SEQ ID NO: 3 coding for the constant region of each of the heavy chains of the antibody. The peptide sequence of each of the light chains, deduced from the nucleic acid sequence SEQ ID NO: 5, is the sequence SEQ ID NO: 7 and the peptide sequence of each of the heavy chains, deduced from the SEQ nucleic acid sequence. ID NO: 6, is the sequence SEQ ID NO: 8. The amino acid located at position 106 is a lysine (K) in the sequence SEQ ID NO: 7. The antibody used in the invention can be produced by any technique known to those skilled in the art. In a first embodiment, the antibody is a monoclonal antibody produced by the clone R603, deposited on November 29, 2005 under the registration number CNCM 1-3529. It then has a fucosylation of a content of about 50%. In another embodiment, the antibody is a recombinant antibody obtained by genetic engineering.

It can thus be produced by any technique known to those skilled in the art, for example by recombination in a host cell, transformed with one or more vectors which allow the expression and / or the secretion of the nucleotide sequences encoding the heavy chain and / or the light chain of the antibody. The vector generally comprises a promoter, translation initiation and termination signals, as well as appropriate transcriptional regulatory regions. It is stably maintained in the host cell and may optionally have particular signals that specify the secretion of the translated protein. These different elements are chosen and optimized by those skilled in the art depending on the cellular host used. Such vectors are prepared by methods commonly used by those skilled in the art, and the resulting clones can be introduced into a suitable host by standard methods, such as lipofection, electroporation, the use of polycationic agents, thermal shock, or chemical methods. The cellular host may be chosen from prokaryotic or eukaryotic systems, for example bacterial cells, but also yeast cells or animal cells, in particular mammalian cells. Mammalian cells preferred for the production of the monoclonal antibody are the YB2 / 0 rat line, the CHO hamster line, in particular the CHO dhfr- and CHO Lecl3 lines, PER.C6TM (Crucell), 293, K562, NSO, SP2 / 0, BHK or COS. Insect cells can also be used. Another mode of production is the expression of the recombinant antibody in transgenic organisms, for example in plants (Ayala M, Gavilondo J, Rodriguez M, Fuentes A, Enriquez G, Perez L, Cremata J, Pujol M. Production of Plantibodies in Nicotiana plants, Methods Mol Biol 2009; 483: 103-34.) or especially in the milk of transgenic animals such as the rabbit, goat or pig (Pollock, DP, JP Kutzko, E. Birck- Wilson, JL Williams, Y. Echelard and HM Meade, (1999), Transgenic milk as a method for the production of recombinant antibodies, Journal of Immunological Methods, 231: 147-157). According to a preferred embodiment, the antibody is produced in the milk of non-human transgenic mammals genetically modified to produce this glycoprotein. Preferably, it is the milk of a rabbit or a transgenic goat, preferably in the milk of a transgenic goat.

The secretion of the antibody by the mammary glands, allowing its presence in the milk of the transgenic mammal, involves controlling the expression of the antibody in a tissue-dependent manner. Such control methods are well known to those skilled in the art. The control of the expression is carried out thanks to sequences allowing the expression of the glycoprotein in a particular tissue of the animal. These include promoter sequences of "WAP", "I3-casein", "13-lactoglobulin" and optionally signal peptide type sequences. Preferably, the antibody is produced in the mammary glands of a transgenic goat, using an expression vector comprising the sequence of both chains, under the control of a 5'-3-casein promoter. A method of extracting proteins of interest from transgenic animal milk is described in EP 0 264 166. Advantageously, the antibody produced by animal transgenesis, in particular in the mammary glands of a mammalian genetically modified animal. transgenic goat, exhibits glycosylation with a high galactosylation rate, for example greater than 70%. The term "tumor" preferably includes malignant tumors, namely cancers. Cancers include lymphomas, Hodgkin's disease, leukemias, lung cancer, non-small cell lung cancer (NSCL), bronchoalveolar lung cancer, bone cancer, pancreatic cancer, cancer of the lungs, skin, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal margin, cancer of the stomach, duodenum cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tube, endometrial cancer, cervical cancer, carcinoma of the uterus, carcinoma of the uterus, carcinoma of the vagina, vulvar carcinoma, esophageal cancer, small bowel cancer, endocrine cancer, thyroid cancer, parathyroid gland cancer, adrenal gland cancer , soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer or ureter, renal cell carcinoma, pelvic carcinoma, mesothelioma, hepatocellular carcinoma, bile duct carcinoma, central nervous system (CNS) tumors, axillary tumors spinal, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymones, medulloblastomas, meningiomas, pituitary adenoma, including refractory versions of all the above cancers, or a combination of one or more of the highest cancers. Tumors expressing the CD20 antigen are preferred, i.e. tumors in which a significant level of expression of the CD20 antigen is observed in a cell, preferably on the surface of T cells or cells. B, preferably a B cell, a tumor or a cancer, preferably a non-solid tumor. Patients with a tumor expressing CD20 can be determined by standard assays known in the art. For example, the expression of CD20 antigens can be measured using immunohistochemistry (IHC), flow cytometry (FACS) or PCR based on detection of the corresponding mRNA. "Cancers or cancers expressing the CD20 antigen" refers to all tumors or cancers in which the cancer cells show expression of the CD20 antigen. This includes lymphomas (preferably B-cell non-Hodgkin's lymphoma (NHL)) and leukemias (preferably chronic lymphoid leukemia and B-cell lymphoblastoma). Such lymphomas and leukemias include, for example: a) follicular lymphomas, b) Burkitt's lymphoma (including Burkitt's endemic lymphoma, sporadic Burkitt's lymphoma, and non-Burkitt's lymphoma). C) Marginal zone lymphomas (including including B-cell lymphoma of the extraganglionic marginal area, mucosal-associated lymphatic tissue lymphoma (MALT), ganglionic marginal zone B-cell lymphoma, splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphomas (including diffuse large B-cell lymphoma (DLCL), diffuse mixed-cell lymphoma, immunoblastic lymphoma, primitive B-cell mediastinal lymphoma, f) hairy cell leukemia, g) B-cell lymphoblastic lymphoma, h) Waldenstrom's disease, i) acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) / lymphocytic lymphoma (SLL), prolymphocytic leukemia re B, j), multiple myeloma, plasmacytoma j) Hodgkin's disease. In another embodiment, it is a non-Hodgkin's B-cell lymphoma (NHL). In another embodiment, it is a mantle cell lymphoma (LCM), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large cell B-cell (DLCL), Burkitt's lymphoma, Tricho leukocyte leukemia, Follicular lymphoma, Multiple myeloma, Marginal zone lymphoma, Post-transplant lymphoproliferative disorders (SLP), HIV-associated lymphoma, Waldenstrom's disease, or primary CNS lymphoma .

According to the invention, the term "treatment" means curative or prophylactic treatment of a disease, and includes the improvement of the symptoms of the disease, and the decrease in the risk of relapse, and the risk of metastasis.

The anti-CD20 antibody and the bendamustine may be combined within the same pharmaceutical composition, or used as separate pharmaceutical compositions, which may be administered simultaneously or sequentially. In particular they can be administered separately, ie either concomitantly or independently, for example, with a time lag. According to the invention, the two therapeutic agents are used in combination in order to potentiate the antiproliferative effects of one and the other. In the same way, the two therapeutic agents are used in combination to potentiate the cytotoxic effects of both.

The compositions according to the invention may be administered by any appropriate route of administration, for example parenterally, orally, sublingually, vaginally, rectally, transdermally, preferably by intravenous, subcutaneous or intradermal injection. Intramuscular, intraperitoneal, intrasynovial, intrathecal or intratumoral injection is also possible. Injections can be performed as a bolus, or by continuous infusion. When the anti-CD20 antibody composition and the bendamustine composition are administered separately, these compositions may be in the same or different administration form. The compositions of the invention may comprise or be combined with other chemotherapeutic or cytotoxic agents, but this is not preferred.

The pharmaceutical compositions according to the invention advantageously comprise one or more excipients or vehicles, which are pharmaceutically acceptable. For example, saline, physiological, isotonic, buffered, etc., solutions compatible with a pharmaceutical use and known to those skilled in the art may be mentioned. The compositions may contain one or more agents or vehicles selected from dispersants, solubilizers, stabilizers, preservatives, etc. Agents or vehicles that can be used in formulations (liquid and / or injectable and / or solid) include methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, and the like. acacia, etc. The compositions may be formulated as injectable suspensions, gels, oils, tablets, suppositories, powders, capsules, capsules, etc., optionally using dosage forms or devices providing sustained and / or delayed release. For this type of formulation, an agent such as cellulose, carbonates or starches is advantageously used. A pharmaceutical composition useful in the invention comprises, for example, an anti-CD20 antibody, a polysorbate, a buffer and mannitol and / or glycine and has a pH of between 5.0 and 7.0. The terms "combination" or "co-administration" refer to the administration of anti-CD20 antibody and bendamustine as a single formulation or as two separate formulations. Administration can be simultaneous or sequential, in any order. The two therapeutic agents can be administered simultaneously (at the same time) or successively, for example via intravenous injection (iv) by continuous infusion (one for the anti-CD20 antibody and possibly one for the bendamustine). Advantageously, the anti-CD20 antibody is administered before bendamustine. When the two therapeutic agents are administered sequentially, the dose is administered on the same day, by at least two separate administrations, or one of the agents is administered on the first day and the second on the following day, or the day of treatment. after, for example. In a particular protocol, bendamustine can be administered twice a week and the antibody once a week.

In another particular protocol, bendamustine may be administered for two days, every twenty-eight days. Depending on the type and severity of the disease, a dosage of about 50 to 5000 mg / day, or about 50 to 2000 mg / day, preferably about 100 to 500 mg / day (for example 300 mg / day) may be provided. / day) anti-CD20 antibody and 10 to 500 mg / m2 / day, or about 50 to 200 mg / m2 / day, (eg 300 mg / m2 / day) bendamustine, for concomitant administration both drugs for the patient. In a preferred embodiment, the dosage of the antibody is in the range of about 0.05mg / kg to about 80mg / kg body weight, about 0.5mg to about 8g per day.

In a preferred embodiment, the dosage of bendamustine is in the range of from 0.01 mg / kg to about 500 mg / kg, for example 0.1 mg / kg to 300 mg / kg, or about 0.1 mg / kg. at 20 g per day. Alternatively, a higher initial loading dose followed by one or more lower doses may also be administered. Alternatively, a lower initial loading dose followed by one or more higher doses may also be administered. In a particular embodiment, the anti-CD20 antibody and the bendamustine can be used in an antibody / bendamustine ratio of 3/1 to 1/1.

The following figures and examples illustrate the invention without limiting its scope. EXAMPLES Example 1 Antitumor Activity on a Model of Human Follicular Lymphoma (Non-Hodgkin's Lymphoma): Protocol: Subcutaneous dose of 2.106 cells of the RL line issued was injected into mice (SCID CB17 mice 6 weeks old) of an exponentially growing human follicular lymphoma, in a volume of 200 g of RPMI 1640 medium containing 10% of fetal calf serum. When the tumors became palpable (median volume 100 mm 2), the mice were randomly divided into groups of ten mice.

Table 1: Study protocol Compound group Assay Number of mice Route Number of cumulative dose (mg / kg) of administration treatments (mg / kg) 1 Vehicle (PBS) - 10 ip, q7d 4 - / - 2 R603 ( LFB) 60 10 iv, q7d 4 240 3 Rituximab (Roche) 60 10 iv, q7d 4 240 4 Bendamustine 30 10 ip, q7d 4 120 (Pro Bio Sint) 4 240 R603 60 10 iv, q7d Bendamustine 30 ip, q7d 4 120 4 240 6 Rituximab 60 10 iv, q7d Bendamustine 30 ip, q7d 4 120 iv: intravenous ip Intraperitoneal q7d: 1 injection per week The treatments started 21 days after transplantation of lymphoma cells. The anti-CD20 antibody (R603 or rituximab) and the vehicle are administered i.v. once a week. Bendamustine is administered intraperitoneally once a week on the same day as the antibody. The study aimed to compare the evolution of tumor volumes according to different treatments.

Results: Inhibition of tumor growth was calculated by comparing the treated groups to the control group. Table 2 below shows that the combination of the R603 antibody with bendamustine offers 100% inhibition on the 31st day. Table 2: Inhibition of tumor growth at day 31 Treatment Dose (mg / kg) Inhibition of tumor growth R603 60 87% Rituximab 60 82% bendamustine 30 77% R603 + 60 + 30 100% bendamustine Rituximab + 60 + 30 100% In addition, Figure 1 shows that the R603 antibody exhibits greater activity against cancer tissue xenografts, compared to rituximab, in combination with bendamustine. The figure shows that a 7-day delay in tumor growth is observed between the two antibodies in combination with bendamustine.

Claims (12)

REVENDICATIONS1. Pharmaceutical composition comprising CLAIMS1. A pharmaceutical composition comprising bendamustine and an antiCD20 antibody, said anti-CD20 antibody being a monoclonal antibody whose light chain comprises the amino acid sequence SEQ ID NO: 7 and the heavy chain comprises the amino acid sequence SEQ ID NO : 8. 2. The pharmaceutical composition according to claim 1, wherein the antibody is produced by the clone R603 deposited on November 29, 2005 under the registration number CNCM 1-3529. The pharmaceutical composition of claim 1, wherein the antibody is a recombinant antibody produced by animal transgenesis. 4. Pharmaceutical composition according to one of claims 1 to 3, for an intravenous or intraperitoneal administration. Anti-CD20 antibody for use in treating a tumor in combination with bendamustine, said anti-CD20 antibody being a monoclonal antibody comprising the light chain sequence SEQ ID NO: 7 and the heavy chain SEQ ID NO: 8. 6. An anti-CD20 antibody for use in treating a tumor, in combination with bendamustine, according to claim 5, said anti-CD20 antibody being a monoclonal antibody produced by the R603 clone deposited on November 29, 2005 under the registration number CNCM 1-3529. An anti-CD20 antibody for use in treating a tumor, in combination with bendamustine, according to claim 5, wherein the antibody is a recombinant antibody produced by animal transgenesis. 8. An anti-CD20 antibody for use in the treatment of a tumor, in combination with bendamustine, according to one of claims 5 to 7, the antibody and the bendamustine being intended for separate administration, simultaneous or sequentially. 9. Anti-CD20 antibody for use in the treatment of a tumor, in combination with bendamustine, according to one of claims 5 to 8, the antibody and the bendamustine being combined within the same pharmaceutical composition . An anti-CD20 antibody for use in treating a tumor, in combination with bendamustine, according to one of claims 5 to 8, the tumor being a malignant tumor expressing the CD20 antigen. An anti-CD20 antibody, for use in treating a tumor, in combination with bendamustine, according to claim 10, wherein the tumor is lymphoma or leukemia. An anti-CD20 antibody, for use in treating a tumor, in combination with bendamustine, according to claim 11, the tumor being a non-Hodgkin's lymphoma.