EP2566511A2

EP2566511A2 - Use of an anti-cd71 antibody for preparing a medicament

Info

Publication number: EP2566511A2
Application number: EP11723558A
Authority: EP
Inventors: Laurence Boumsell
Original assignee: LFB Biotechnologies SAS
Current assignee: LFB SA
Priority date: 2010-05-03
Filing date: 2011-05-03
Publication date: 2013-03-13
Also published as: WO2011138557A3; FR2959416A1; US20130216476A1; FR2959416B1; WO2011138557A2

Abstract

The present invention relates to the use of an anti-CD71 monoclonal antibody or of a fragment of an abovementioned antibody, capable of binding to the CD71 antigen, for preparing a medicament for preventing or treating myelomas.

Description

Use of anti-CD71 antibodies for the preparation of a medicament

The present invention relates to the use of anti-CD71 antibodies or anti-CD71 antibody fragments for the preparation of a medicament for the prevention or treatment of cancers.

CD71 is a Type II glycoprotein that exists as a 180 kDa homodimer. CD71 is a receptor for transferrin, a protein that transports iron from the intestine to liver stores and reticulocytes.

Ferrotransferrin, Fe3 + associated transferrin, is bound to CD71 at neutral pH, and is internalized to the endosomal compartment at approximately pH5. The Fe3 + ions are then transported to the cytoplasm by an unknown mechanism. After the release of Fe3 + ions, the apotransferrin, transferrin without Fe3 +, remains fixed at CD71 at pH5 and returns to the cell membrane at approximately pH 7.4. At neutral pH conditions, apotransferrin has no affinity for CD71. The dissociation of the complex formed by the apotransferrin and the CD71 makes it possible to begin another cycle of transport.

CD71 plays an important role in cell proliferation because it controls the consumption of iron, an essential process in many metabolic pathways. This control is implemented by the binding and endocytosis of transferrin by CD71. The expression of CD71 is post-transcriptionally regulated by the stability of CD71 RNA, and the intracellular level of iron. In the presence of an iron deficiency signal, IRP-1 and IRP-2 (iron response protein) are fixed on specific sequences, designated iron response elements (IREs), located in the 5'UTR region of RNA. of CD71. This binding stabilizes the CD71 RNA. In contrast, when the iron level is sufficiently high, the affinity of IRP-1 and IRP-2 for IREs is decreased and the CD71 RNA becomes more sensitive to degradation by RNases.

Myeloma, also known as multiple myeloma, or Kahler's disease, is a cancer of the plasma cells. These are produced in the spleen and migrate into the bone marrow. Plasma cells are specialized B cells that normally produce antibodies. Normal plasma cells are characterized by an inability to proliferate. Nevertheless, when these cells become malignant, they acquire a great capacity for proliferation and secrete all the same type of immunoglobulin. In most cases, this immunoglobulin is IgG or IgA, but it can also be IgD, IgE or IgM. Therefore, the blood or urine of patients with myeloma often has a very large amount of a single type of antibody called paraprotein. When a sample of blood or urine from such a patient is analyzed by electrophoresis, these paraproteins often form a monoclonal peak. Sometimes, instead of the secretion of a complete immunoglobulin, the tumor plasmocytes secrete an incomplete part of the so-called "Kappa or Lambda light chain" immunoglobulin. In the latter case, there is little or no monoclonal peak in the blood, however there is a severe hypogammaglobulemia and proteinuria in the urine, made of Kappa or Lambda chain, called "BENCE JONES".

In France, the incidence of this disease is about 4000 new cases per year. The average survival is 5 years, but it depends on the stage of the disease.

Myeloma can be divided into 3 stages according to the Durie and Salmon classification established in 1986. This classification is based on the amount of paraprotein produced, the severity of the bone lesion, the blood calcium level and the insufficiency of bone marrow production ( hemoglobin). Stage I corresponds to a low tumor mass myeloma, whereas Stage III is a high tumor mass myeloma. A possible impairment of renal function adds a subclassification, in which stage A has no renal involvement and stage B is characterized by renal failure.

In recent years, the new classification systems based on the detection of genetic markers make it possible to further define the prognosis for the survival of a patient. For example, deletion of chromosome 13q (Fonseca et al., Blood 2003; 101: 4569-4575) or locus 17pl3 (locus for a tumor suppressor gene) (Avet-Loiseau et al., Blood 2007; 109: 3489-3495). ) in the plasma cells may announce a very poor prognosis.

The classical treatment of myeloma is mainly based on a chemotherapy treatment, which aims to control the proliferation of tumor cells. Currently, chemotherapeutic agents used in the treatment of myelomas are alkylating agents, such as melphalan or cyclophosphamide, often combined with cortisone drugs, such as prednisone, or proteasome inhibitors, such as bortezomib (Velcade®). ), or anti-angiogenic agents, such as thalidomide. Although these drugs have provided some prolongation of patient survival, the side effects caused by these are also considerable. In addition, these medications can rarely bring lasting healing.

Another approach to treating myeloma involves autologous stem cell techniques. These techniques involve the use of healthy stem cells from the peripheral blood or bone marrow of the subject himself. The stem cells thus recovered can be frozen until the moment of use and then thawed and reinjected or retransplanted into the patient's body intravenously. About two weeks after the injection, the stem cells can begin to produce new blood cells and regenerate the bone marrow. In the past 10 years, autologous stem cell transplantation has become a preferred treatment. Because, if the patients are able to support this treatment, this method makes it possible to give a better survival of these patients. However, since autologous stem cell transplantation is often combined with chemotherapy before transplantation at high doses, this method can lead to very serious side effects, such as a sharp decrease in white blood cells.

Therefore, it is necessary to develop new drugs or new approaches to increase the survival of patients with myeloma.

A first aspect of the invention aims to provide a new drug for the prevention or treatment of myeloma.

Another aspect of the invention is to provide an in vitro diagnostic method for myeloma.

The present invention relates to the use of an anti-CD71 monoclonal antibody or a fragment of a said antibody capable of binding to the CD71 antigen, said antibody or fragment of said antibody comprising:

at least the variable region of a heavy chain comprising or consisting of the amino acid sequence SEQ ID NO: 1, and

at least the variable region of a light chain comprising or consisting of an amino acid sequence chosen from SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 13,

SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17,

for the preparation of a medicament for the prevention or treatment of myeloma.

The present invention is based on an unexpected finding made by the inventors in a study on the level of expression of CD71 on tumor cell lines of myeloma. Indeed, CD71 is overexpressed on plasma cells that become malignant. The invention shows both in vitro and in vivo that blocking CD71 by an anti-CD71 antibody can inhibit the development of myelomas and prolong the survival of mice bearing myeloma.

Several patterns of mechanisms may be involved in inhibiting the development of myelomas by an anti-CD71 antibody. The first mechanism is the inhibition of proliferation of myeloma cells by blocking the delivery of iron to tumor cells via the anti-CD71 antibody.

An anti-CD71 antibody can also trigger or activate an immune response, such as the production of cytokines, or the induction of apoptosis in tumor cells, thereby destroying cancer cells.

Myeloma cells can also be specifically removed by effector cells by the antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) process.

In the invention, the term "antibody" refers to an immunoglobulin, a multimeric protein consisting of 4 chains participating in the acquired immune response.

Immunoglobulins are well known to those skilled in the art and consist of an assembly of two dimers each consisting of a heavy chain and a light chain. The multimeric complex is assembled by the binding of a light chain and a heavy chain by a disulfide bridge between two cysteines, the two heavy chains being itself also interconnected by two disulfide bridges.

Each of the heavy chains and light chains consists of a constant region and a variable region. The assembly of the chains which compose an antibody makes it possible to define a characteristic three-dimensional scree in Y, where

the base of Y corresponds to the constant region Fc which is recognized by the complement and the Fc receptors, and

the end of the Y arms corresponds to the respective assembly of the variable regions of the light and variable chain of the heavy chain.

More precisely, each light chain consists of a variable region (VL) and a constant region (CL). Each heavy chain consists of a variable region (VH) and a constant region consisting of three constant domains CH1, CH2 and CH3. The association of the two domains CH2 and CH3 composes the domain Fc.

The variable region of the light chain consists of three domains determining antigen recognition (CDR) surrounded by four framework domains (FR). The three-dimensional folding of the variable region is such that the 3 CDRs are exposed on the same side of the protein and allow the formation of a specific structure recognizing a specific antigen. The term "the ability to bind to the CD71 antigen" means the ability of an antibody to recognize and specifically bind to an antigen. This specificity is conferred by the variable region of the light chain or heavy chain.

The ability of an antibody to recognize and specifically bind to an antigen can be assayed by techniques such as ELISA, indirect immunofluorescence, or western blot.

"A fragment of a said antibody capable of binding to the CD71 antigen" may be a Fab fragment, F (ab) '2, scFV, ScFv dimer or a diabody, a triabody or a tetrabody

A Fab fragment consists of a light chain comprising a constant region of a light chain of a human immunoglobulin and a variable region of a murine immunoglobulin, and a heavy chain comprising a constant region of a chain heavy with a human immunoglobulin and a variable region of a murine immunoglobulin.

An F (ab) '2 fragment consists of the disulfide bonding of two Fabs described above.

An scFv fragment consists of the combination of a heavy chain variable fragment and a light chain variable fragment of an immunoglobulin.

A scFv dimer consists of two covalently bound ScFv fragments, each ScFv being themselves a variable fragment of a heavy chain and a variable fragment of a light chain.

A diabody consists of the non-covalent association of two polypeptides containing themselves two variable chains of the same nature (VL-VL and VH-VH) which may be of the same or different specificity.

In the same way, a triabody or a tetrabody can be constituted.

In one embodiment, the invention relates to the use of an anti-CD71 monoclonal antibody or a fragment of a said antibody capable of binding to the CD71 antigen, said antibody or fragment of said antibody comprising :

at least the variable region of a heavy chain comprising or consisting of an amino acid sequence encoded by a nucleic sequence SEQ ID NO: 3, and

at least the variable region of a light chain comprising or consisting of an amino acid sequence encoded by a nucleic sequence chosen from SEQ ID NO: 4 and SEQ ID NO: 12,

for the preparation of a medicament for the prevention or treatment of myeloma. In the present invention, the nucleic acid sequence SEQ ID NO: 3 encodes the amino acid sequence SEQ ID NO: 1. The nucleic sequences SEQ ID NO: 4 and SEQ ID NO: 12 respectively coding the amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 11. Advantageously, the invention relates to the use of an anti-CD71 antibody as defined above, wherein the constant regions of said light and heavy chains are constant regions of a human antibody.

Said human antibody may be an IgG, IgA, IgD, IgE or IgM antibody.

This embodiment of the invention makes it possible to reduce the immunogenicity of the antibody in humans and thereby to improve its effectiveness during its therapeutic or prophylactic administration in humans.

The constant region of each of the heavy chains of the antibody according to the invention is in particular the constant region of human IgG1, IgG2, IgG3, IgG4, IgM, IgD, IgE, IgA1 or IgA2.

Advantageously, the constant region of each of the heavy chains of the antibody according to the invention is the constant region of a human immunoglobulin of Gl type, because such an antibody shows an ability to generate ADCC activity in the greatest number of of individuals (humans).

Advantageously, the constant region of each of the light chains of the antibody according to the invention is the constant region of the κ (kappa) or λ (lambda) chain.

In a particular embodiment, the invention relates to the use of an anti-CD71 antibody defined above, in which:

at least the constant region of a light chain comprises or consists of the amino acid sequence chosen from SEQ ID NO: 6 and SEQ ID NO: 14

at least the constant region of a heavy chain comprises or consists of the amino acid sequence chosen from SEQ ID NO: 5 or SEQ ID NO: 9.

In another embodiment, the invention relates to the use of an anti-CD71 antibody as defined above, wherein:

at least the constant region of a light chain comprises or consists of the amino acid sequence encoded by the nucleic acid sequence SEQ ID NO: 8,

at least the constant region of a heavy chain comprises or consists of the amino acid sequence encoded by a nucleic sequence chosen from SEQ ID NO: 7 or SEQ ID NO: 10. In the present invention, the nucleic acid sequence SEQ ID NO: 8 encodes the amino acid sequence SEQ ID NO: 6. The nucleic sequences SEQ ID NO: 7 and SEQ ID NO: 10 respectively encode the amino acid sequences SEQ ID NO: 5 and SEQ ID NO: 9. In a particular embodiment, the anti-CD71 antibody for the implementation of the invention may be an antibody where:

the variable region of each of its light chains comprises or consists of the amino acid sequence SEQ ID NO: 2, and

the variable region of each of its heavy chains comprises or consists of the amino acid sequence SEQ ID NO: 1, and

the constant region of each of its light chains comprises or consists of the amino acid sequence SEQ ID NO: 14, and

the constant region of each of its heavy chains comprises or consists of the amino acid sequence chosen from SEQ ID NO: 5 or SEQ ID NO: 9.

Advantageously, the anti-CD71 antibody for the implementation of the invention is produced by the hybridoma BA120g, deposited on June 14, 2005 at the CNCM under number CNCM 1-3449. An anti-CD71 monoclonal antibody for the practice of the present invention may be produced by conventional methods known to those skilled in the art, for example, the construction of a DNA vector which expresses:

- the heavy chain, or

a fragment of the heavy chain, or

- the light chain, or

- a fragment of the light chain, or

- the heavy chain and the light chain, or

a fragment of the heavy chain and a fragment of the light chain.

These vectors are, for example, plasmids, cosmids, artificial chromosomes of yeasts, artificial chromosomes of bacteria and artificial chromosomes derived from bacteriophage PI, vectors derived from viruses. In one embodiment of the invention, a fragment of an anti-CD71 antibody implemented in the use described above may be Fab, F (ab) '2, scFV, ScFv dimer or a diabody. , a triabody or a tetrabody.

These fragments can be recombinant fragments, synthetic, or produced by enzymatic cleavage according to methods known to those skilled in the art. These fragments can also be produced by introducing one or more stop codons into the gene encoding an anti-CD71 antibody. For example, when a stop codon is introduced after the CH1 domain of the gene encoding the heavy chain, the chain thus produced is a heavy chain capable of forming an Fab fragment with a light chain. On the other hand, if a stop codon is added after the hinge region downstream of the CH1 domain of a heavy chain, this truncated chain may constitute an F (ab) '2 fragment with a light chain.

In another particular embodiment of the invention, the anti-CD71 antibody used in the use described above is coupled to a bioactive molecule.

This bioactive molecule may be a radioisotope, for example a range radiation emitter, a beta radiation emitter or an alpha emitter.

Advantageously, a radioisotope coupled to the anti-CD71 antibody or a fragment of the anti-CD71 antibody is a radioisotope that can be used for a therapeutic or diagnostic purpose, such as Actinium 225, Actinium 227 or Arsenic 72. , Asiate 211 (PET (Positron Emission Tomography)), Bismuth 212 or 213, Bromine 75 (PET), Bromine 77, Cobalt 55 (PET), Copper 61 (PET), Copper 64 (PET and treatment), Copper 67 (PET), Iodine 123 (PET), Iodine 131, Lutetium 177 (PET and treatment), Osmium 194, Radon 223, Rhenium 186, Ruthenium 105, Terbium 149, Thallium 228 and 229, Yttrium 90 and 91.

These radioisotopes can be directly linked to the antibody or via a chelator, such as DTA (9,10-dithioanthracene), or DTPA (diethylene triamine penta acid).

A bioactive molecule can also be a non-radioactive metal.

The said bioactive molecule can also be:

a toxin, for example ricin, abrin, diphtheria toxin, Phytolacca antiviral protein, or a functional fragment of a toxin;

a nucleic acid such as an antisense RNA, a siRNA, or a miRNA;

a cyto-toxic agent, for example mitomycin C, methotrexate, adriamycin, doxorubicin, daunorubicin, vinblastine, bleomycin, taxol, taxotere, navelbine; an enzyme such as Raise;

a galenic vector, for example liposomes, nanoparticles, polymers, cationic emulsions, anionic emulsions, neutral emulsions, or dendritomes;

another antibody or a fragment of another antibody which recognizes an antigen other than CD71;

biotin, avidin or streptavidin.

A bioactive molecule mentioned above can be directly linked to the antibody or via a linker.

A particular aspect of the invention relates to the use

an anti-CD71 monoclonal antibody or a fragment of a said antibody capable of binding to the CD71 antigen, said antibody or fragment of the above-mentioned antibody comprising:

at least the variable region of a light chain comprising or consisting of an amino acid sequence chosen from SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, and

effector cells,

for the preparation of a medicament for the prevention or treatment of myeloma.

Effector cells are used simultaneously with the anti-CD71 antibody for the preparation of a medicament for the prevention or treatment of myelomas.

These effector cells are cells capable of expressing Fc domain receptors, such as human T lymphocytes, human NK lymphocytes, human monocytes, human granulocytes, or human macrophages. The interaction of the Fc receptor on the effector cells with the Fc domain of the anti-CD71 antibody makes it possible to induce an ADCC activity targeting the target cells, that is to say the cells recognized by the specific antibody. , and eliminate them afterwards.

Effector cells may be autologous or allogeneic cells.

Advantageously, effector cells are cells from a patient in need of myeloma treatment. Effector cells are separated from peripheral blood taken from a donor or patient according to conventional methods well known to those skilled in the art (Boyum et al, Scand J Clin Lab Invest, Suppl 1976. 5 : 9-15).

In an advantageous embodiment, the invention relates to the use of an anti-CD71 antibody described above and effector cells for the preparation of a medicament for the prevention or treatment of myelomas, wherein the constants of the light and heavy chains of said antibody are constant regions of a human antibody.

In a more advantageous embodiment, the invention relates to the use of an anti-CD71 antibody described above and effector cells for the preparation of a medicament for the prevention or treatment of myeloma, wherein:

at least the constant region of a light chain comprises or consists of the amino acid sequence SEQ ID NO: 14,

More advantageously, the invention relates to the use of effector cells and of an anti-CD71 antibody produced from hybridoma BA120g, deposited on June 14, 2005 at CNCM under number CNCM 1-3449, for the preparation of a medicament for the prevention or treatment of myeloma. In another advantageous embodiment of the invention, the invention relates to the use of an anti-CD71 antibody described above and effector cells for the preparation of a medicament for the prevention or treatment of myelomas. wherein the antibody is coupled to a bioactive molecule selected from:

- radioisotopes,

- non-radioactive metals,

toxins selected from ricin, abrin, diphtheria toxin,

the nucleic acids chosen from antisense ARs,

the cytotoxic agents chosen from mitomycin C, methotrexate,

adriamycin,

enzymes such as RNases,

biotin, avidin or streptavidin.

In a particular embodiment, the anti-CD71 antibody described above is intended for the prevention or treatment of a myeloma selected from stage I myeloma (low mass tumor), stage II myeloma (tumor mass). intermediate), or stage III myeloma (high tumor mass).

The classification of myeloma is based on the criteria established by Dr. Durie and Dr. Salmon in 1986. It is a classification according to the quantity of paraprotein produced, the severity of bone lesion, the blood calcium level and the insufficient marrow production (hemoglobin).

Stage I corresponds to a low tumor mass myeloma, in which all the following criteria are present:

- Hemoglobin> 100 g / 1

- Calcium <120 mg / l (3 mmol / l)

- Absence of bone lesion or isolated bone plasmocytoma

- Low monoclonal Ig level:

* IgG <50 g / 1

* IgA <30 g / 1

Proteinuria of Bence Jones <4 g / 24 h.

Stage II does not meet the definition of Stage I or Stage III.

Stage III is a myeloma of high tumor mass, which has at least one of the following criteria:

- Hemoglobin <8.5 g / dl - Calcium> 120 mg / l (> 3 mmol / l)

- Multiple bone lesions (> 3)

- High level of monoclonal Ig:

* IgG> 70 g / 1

* IgA> 50 g / 1

* Proteinuria of Bence Jones> 12 g / 24 H

Another aspect of the present invention is to provide an anti-CD71 antibody for the treatment of myelomas.

In one embodiment, the invention relates to an anti-CD71 monoclonal antibody or a fragment of a said antibody capable of binding to the CD71 antigen, said antibody or fragment of said antibody comprising

at least the variable region of a light chain comprising or consisting of an amino acid sequence chosen from SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17,

for the treatment of myeloma.

In another embodiment, the invention relates to a monoclonal antibody

CD71 or a fragment of a said antibody capable of binding to the CD71 antigen, said antibody or fragment of said antibody comprising:

for the treatment of myeloma.

In an advantageous embodiment, the invention relates to an anti-CD71 antibody for the treatment of myelomas, wherein the constant regions of said light and heavy chains are constant regions of a human antibody.

In a more advantageous embodiment, the invention relates to an anti-CD71 antibody, wherein: at least the constant region of a light chain comprises or consists of the amino acid sequence SEQ ID NO: 14,

at least the constant region of a heavy chain comprises or consists of the amino acid sequence chosen from SEQ ID NO: 5 or SEQ ID NO: 9,

for the treatment of myeloma.

In another more advantageous embodiment, the invention relates to an anti-CD71 antibody, wherein:

at least the constant region of a heavy chain comprises or consists of the amino acid sequence encoded by a nucleic sequence chosen from SEQ ID NO: 7 or SEQ ID NO: 10,

for the treatment of myeloma.

In a particularly advantageous embodiment, the invention relates to an anti-CD71 antibody, wherein:

the constant region of each of its heavy chains comprises or consists of the amino acid sequence chosen from SEQ ID NO: 5 or SEQ ID NO: 9,

for the treatment of myeloma.

In an advantageous embodiment, the antibody used for the treatment of myeloma is produced from a hybridoma BA120g, deposited on June 14, 2005 at CNCM under number CNCM 1-3449.

The anti-CD71 antibody described above for the treatment of myelomas may be associated with pharmaceutically acceptable containers or vehicles.

The anti-CD71 antibody described above for the treatment of myeloma can be administered intravenously, for example by injection or infusion. In a particular embodiment, an anti-CD71 antibody described above for the treatment of myelomas, is coupled to a bioactive molecule chosen from:

- radioisotopes,

- non-radioactive metals,

toxins selected from ricin, abrin, diphtheria toxin,

the nucleic acids chosen from antisense ARs,

the cytotoxic agents chosen from mitomycin C, methotrexate, adriamycin,

enzymes such as RNases,

biotin, avidin or streptavidin

Another aspect of the invention relates to a product containing:

at least one anti-CD71 monoclonal antibody or a fragment of the above-mentioned antibody capable of binding to the CD71 antigen, said antibody or fragment of said antibody comprising:

at least the variable region of a light chain comprising or consisting of an amino acid sequence chosen from SEQ ID NO: 2 and SEQ ID NO: 11,

- an anticancer agent selected from Velcade ^®, ^® Melphalan, Prednisone ^® as combination product for simultaneous, separate or spread out over time in tumor therapy.

In a particular embodiment, the invention relates to a product containing:

the constant regions of the light and heavy chains derived from a human antibody, an anticancer agent chosen from Velcade, Melphalan, Prednisone, as a combination product, for simultaneous, separate or spread over time use in tumor therapy.

In a particular embodiment, the invention relates to a product containing: at least one anti-CD71 monoclonal antibody or a fragment of the above-mentioned antibody capable of binding to the CD71 antigen, said antibody or fragment of said antibody comprising:

at least one constant region of a light chain comprises or consists of the amino acid sequence SEQ ID NO: 6 and SEQ ID NO: 14,

- an anticancer agent selected from Velcade ^®, ^® Melphalan, Prednisone ^®,

as a combination product, for simultaneous, separate or spread over time use in tumor therapy.

In a particular embodiment, the invention relates to a product containing:

- an anticancer agent selected from Velcade ^®, ^® Melphalan, Prednisone ^®, as a combination product, for simultaneous, separate or spread over time use in tumor therapy.

In a particularly advantageous embodiment, the invention relates to a product containing:

the anti-CD71 antibody produced from a hybridoma BA120g, deposited on June 14, 2005 at CNCM under number CNCM 1-3449,

- an anticancer agent selected from Velcade ^®, ^® Melphalan, Prednisone ^®,

the anti-CD71 antibody produced from a hybridoma BA120g, deposited on June 14, 2005 at CNCM under the number CNCM 1-3449, and

- Velcade ^® .

In another embodiment, the invention relates to a product containing:

at least the variable region of a light chain comprising or consisting of an amino acid sequence chosen from SEQ ID NO: 2, SEQ ID NO: 11 and SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17

effector cells, and

- an anticancer agent selected from Velcade ^®, ^® Melphalan, Prednisone ^®,

In a particular embodiment, the invention relates to a product containing:

at least one anti-CD71 monoclonal antibody or a fragment of the above-mentioned antibody capable of binding to the CD71 antigen, said antibody or fragment of said antibody comprising: at least the variable region of a heavy chain comprising or consisting of the amino acid sequence SEQ ID NO: 1, and

the constant regions of the light and heavy chains derived from a human antibody,

- an anticancer agent selected from Velcade ^®, ^® Melphalan, Prednisone ^®, and

effector cells,

In a particular embodiment, the invention relates to a product containing:

at least the variable region of a light chain comprising or consisting of an amino acid sequence chosen from SEQ ID NO: 2 and SEQ ID NO: 11, and

at least one constant region of a light chain comprises or consists of the amino acid sequence SEQ ID NO: 6 or SEQ ID NO: 14,

- an anticancer agent selected from Velcade ^®, ^® Melphalan, Prednisone ^®,

effector cells,

as a combination product, for simultaneous, separate or spread over time use in tumor therapy. In a particular embodiment, the invention relates to a product containing:

at least one anti-CD71 monoclonal antibody or a fragment of the above-mentioned antibody capable of binding to the CD71 antigen, said antibody or fragment of said antibody comprising: the variable region of each of its light chains comprises or consists of the amino acid sequence SEQ ID NO: 2, and

effector cells,

- an anticancer agent selected from Velcade ^®, ^® Melphalan, Prednisone ^®,

effector cells,

Another aspect of the invention aims to provide a method for in vitro diagnosis of myeloma.

This method is based on the unexpected finding made by the inventors that CD71 is often overexpressed on myeloma cells. Therefore, overexpression of CD71 on plasma cells may mean that these cells become malignant.

This method includes:

(i) incubation of a cell sample taken from a patient suspected of having myeloma, with an anti-CD71 antibody or a fragment of a said antibody capable of binding to the CD71 antigen, said antibody or said fragment of said antibody comprising:

at least the variable region of a heavy chain comprising or consisting of the amino acid sequence SEQ ID NO: 1, and at least the variable region of a light chain comprising or consisting of an amino acid sequence chosen from SEQ ID NO: 2, SEQ ID NO: 11 or SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17

(ii) measuring the amount of CD71 antigens present on the surface of cells present in said sample,

(iii) comparing the value obtained in the previous step with a reference value established in a healthy cell sample.

Advantageously, the anti-CD71 antibody used in this method is an antibody in which:

More preferably, the anti-CD71 antibody used in this method is produced from a hybridoma BA120g, deposited on June 14, 2005 at CNCM under number CNCM 1-3449.

The anti-CD71 antibody used in this method can be used directly or coupled with a marker.

When this antibody is used directly, the presence of this antibody can be detected by another detection antibody, the latter being linked to a marker, such as a radioactive agent, or an enzyme capable of catalyzing a substrate emitting fluorescence radiation. or capable of emitting radiation at a wavelength different from that of absorption, or any other marker making it possible to detect the binding of the detection antibody to the anti-CD71 antibody.

The anti-CD71 antibody used in this method may be coupled beforehand with a marker for detecting the binding of this antibody to the plasma cells. This marker may be a radioactive agent, or an enzyme capable of catalyzing a substrate emitting fluorescence radiation or capable of emitting radiation at a wavelength different from that of absorption, or any other marker for detecting fixation. anti-CD71 antibody on the plasma cells. The invention is illustrated by the following examples and figures. The following examples are intended to clarify the object of the invention and to illustrate advantageous embodiments, but in no case is intended to restrict the scope of the invention.

Figure 1: Figure 1 shows a calibration curve for measuring the ability of an antibody to bind to cells. The x-axis represents the logarithm of MFI (mean fluorescence intensity) of coupled secondary antibody. The y-axis represents the logarithm of the ability of an antibody to bind to cells. The crosses represent the data actually obtained by the measurement.

Figure 2: Figure 2 shows the measurement of the survival of mice tested in 5 groups. Group 1 (black square) receives nothing. Group 2 (black spot) receives an irrelevant antibody at 0.048 mg / kg / injection. Group 3 (triangle) receives an irrelevant antibody at 0.24 mg / kg / injection. Group 4 (reverse triangle) receives an anti-CD71 antibody at 0.048 mg / kg / injection. Group 5 (empty square) receives an anti-CD71 antibody at 0.24 mg / kg / injection. The x-axis represents the number of days after injection of tumor cells. The y-axis represents the percentage of living mice.

Figure 3: Figure 3 shows the measurement of the survival of mice tested in 5 groups. Group 1 (square in black) receives twice a week an injection of 1.2 mg of an irrelevant antibody. Group 2 (black spot) receives twice a week an injection of 1.2 mg of the anti-CD71 antibody. Group 3 (triangle) receives twice a week an injection of 1.2 mg of the anti-CD71 antibody and an injection of 0.5 mg / kg of VELCADE®. Group 4 (reverse triangle) receives twice a week an injection of 0.5 mg / kg of VELCADE®. Group 5 (empty square) receives no treatment. The x-axis represents the number of days after injection of tumor cells. The y-axis represents the percentage of living mice.

RESULT

1. Detection of CD71 antigen by anti-CD71 antibody

The detection of the CD71 antigen by the anti-CD71 antibody was measured on the ARH-77 cell line, a myeloma line.

This cell line is cultured in suspension at 37 ° C. in a humidified atmosphere (5% of C0 ₂ , 95% humidity). The culture medium is RPMI (Ref # BE12-702F, Lonza, Verviers, Belgium) containing 2mM L-glutamine and supplemented with 10% fetal bovine serum (Ref # DE14-801E, Lonza).

Possible contamination of the cell culture by mycoplasmas is detected using MycoAlert® Mycoplasma Detection Kit (Ref # LT07-318, Lonza) according to the manufacturer's instructions. This kit allows a selective biochemical test that highlights the enzymatic activity of mycoplasma. The cell culture supernatant is recovered and tested by MycoAlert® kit. The test is repeated twice and the result is compared with a negative control and a positive control (Mycoalert® Assay Control Set, Ref # LO07-518, Lonza). Viable mycoplasmas are lysed and released enzymes react with the MycoAlert® kit substrate, catalyzing the conversion of ADP to ATP. Measuring the level of ATP before and after the addition of MycoAlert® substrate provides a ratio indicating the presence or absence of mycoplasma.

The detection of anti-CD71 antibodies is carried out by flow cytometry (GUAVA

Personal Cell Analysis-96 (PCA-96) Systems). The data obtained is analyzed by FCS Express software (De Novo Software).

The negative control antibody is IgG1 (MAT002). The positive control antibody is an anti-CD71 antibody from MAT (MAT201). The antibody to be tested is the anti-CD71 antibody produced by the hybridoma BA120g, diluted to 1 μg / ml, 1 μg / ml, 0 μg / ml, 0 μg / ml, 0, 0 ^ g / ml, 0.005 μg / ml The secondary antibody coupled is an anti-mouse IgG PE antibody The dilution and washing buffer (PBS (phosphate buffered saline) 2% FCS (fetal calf serum)) is prepared from 490ml of PBS (wo Ca2 + / Mg2 +) and 10ml of defrosted SVF, PBS (Cambrex Biowhittaker BE 17-516F) is stored at room temperature until use, and SVF (Cambrex Ref5SB0008) is stored at -20 ° C until use Trypan Blue (Sigma T8154) is stored at room temperature.

The concentration of control antibody is 10 μg / ml.

About 100,000 cells in a volume of ΙΟΟμΙ are first incubated respectively with anti-CD71 antibody in different dilution or control antibody. The anti-CD71 antibodies are then revealed by a secondary antibody coupled to a fluorescent agent (anti-mouse IgG-PE 1/100). The results are shown in the following Table 1.

Table 1

Primary antibody CD71-% CD71 +% MFI

- 94.8 5.2 6.2

IgG1 controls 10 μg / ml 96.3 3.7 4.9

Anti-CD71 ΙΟμ ^ ηύ 5.3 94.7 32.4

Anti-CD71 ^ g / ml 10.6 89.4 28.8

Anti-CD71 0.25 g / ml 25.2 74.8 17.6 Anti-CD71 0.05μ§ / ηι1 34.3 65.7 12.7

Anti-CD71 0.0 ^ g / ml 59.9 40.1 6.6

Anti-CD71 0.005 μg / ml 70.0 30.0 5.6

The anti-CD71 antibody can bind specifically to the cell membrane of ARH-77 cells relative to the IgG1 control antibody. When the concentration of anti-CD71 antibody is greater than 1 g / ml, this antibody can recognize about 90% of ARH-77 cells in the sample.

2. Determination of the number of CD71 antigens on the surface of ARH-77 cells The number of CD71 antigens on the surface of ARH-77 cells is determined by indirect immunofluorescence using QIFIKIT kit (Dakocytomation, Trappes, France) according to the manufacturer's instructions. The result of immunofluorescence is detected by flow cytometry.

The ARH-77 cells are first incubated respectively with the purified anti-CD71 antibody from the BA120g hybridoma and an irrelevant antibody (anti-mouse IgG1) as a negative control. The white particles provided in the kit are used to establish the background noise. Four reference particle populations, having a different number of control antibodies, are used to establish the calibration curve. The antibodies binding to the cells are then revealed by a second antibody coupled with a fluorescent agent (anti-mouse IgG-FITC).

The mean fluorescence intensity (MFI) measured for the four reference particle populations makes it possible to establish a calibration curve (FIG. 1). This curve varies according to the device used. In the present experiment, the formula corresponding to this curve is as follows:

Y = A * X ² + B * X + C,

in which :

- A is equal to 0.013248;

- B egaie 449,282318;

C is -31.588965;

X is MFI (mean fluorescence intensity) derived from the flow cytometry reading;

Y is the total capacity of an antibody to bind to a cell population. The ability of anti-CD71 antibodies to bind specifically to ARH-77 cells (SABC) is obtained according to the following formula: SABC = ABC-BAE, wherein BAE is background and ABC is the ability to anti-CD71 antibody to bind to ARH-77 cells. Background noise is the ability of an irrelevant antibody to bind non-specifically to cells.

Table 2 below shows the result of determining the number of CD71s on ARH-77.

Table 2

3. In vivo test of anti-tumor efficacy of anti-CD71 antibody in mice with severe combined immunodeficiency

3.1 Effect of anti-CD71 antibody on the weight of mice bearing ARH-77 tumor cells

The female mice with severe combined immunodeficiency (SCID), aged 6 to 7 weeks, weighing 16 to 20 g are obtained from Charles River (France). The mice used in the experiments are first observed for 7 days in an animal care unit free of specific pathogenic organisms (EOPS). This animal care unit is authorized by the French Ministry of Agriculture and the French Ministry of Research (Agreement No.A21231011). Animal experiments are implemented according to the European Animal Testing Directive and the UK Animal Welfare Guidelines for Experimental Neoplasia.

The tumor-expressing ARH-77 cells on the cell surface are suspended in RPMI 1640 medium (Ref # BE12-702F, Lonza, Verviers, Belgium) containing 2mM L-glutamine and supplemented with 10% fetal bovine serum (Ref. # DE14-801E, Lonza).

Severe combined immunodeficiency (SCID) female mice were first injected intravenously with a volume of 0.2 ml of RPMI 1640 medium containing 5 million suspended ARH-77 tumor cells. The injection was carried out between 24 to 72 hours after exposure of the mice tested in this experiment to radiation gamma (1.8 Gy, Co, BioMEP sari, France).

The day of injection of the tumor cells is considered as day 0. The treatment starts on day 5.

The 40 mice are then separated into 5 groups:

Table 3 below summarizes the treatment schedule for each group.

Table 3

Treatment takes place on days 5, 8, 11 and 14. The results are shown in Figure 2.

In group 1 (mice not receiving any treatment), a mouse is found dead at day 28, the 7 others are sacrificed between day 25 and day 32 after the injection of the ARH-77 cells, because of the presence of pathological signs.

In group 2 (mice receiving irrelevant antibody at 0.048 mg / kg / injection), three mice died on day 35 and were sacrificed between day 29 and day 96 after injection of ARH-77 cells and were sacrificed and finally 2 mice were sacrificed at the end of study at day 106.

In group 3 (mice receiving irrelevant antibody at 0.24 mg / kg / injection), one mouse died at day 28, 6 are sacrificed between day 29 and 32 after injection of the cells ARH-77 and were sacrificed and finally 1 mouse was sacrificed at the end of study at day 106.

In group 4 (mice that received the anti-CD71 antibody at 0.048 mg / kg / injection), one mouse was found dead at day 102, two were sacrificed at day 57 and 67 because they had pathological signs and the other five survived the study and are sacrificed on day 106.

In group 5 (mice given the anti-CD71 antibody at 0.24 mg / kg / injection) a mouse is sacrificed at day 43 because of the presence of pathological signs and the 7 others survive the study and are sacrificed to day 106. During the experiments, the animals will be killed by cervical dislocation under isoflurane anesthesia if any of the following signs present:

- the signs of suffering (cachexia, weakness, difficulty of movement and eating);

- compound toxicity (convulsion)

- 15% loss of body weight for 3 consecutive days or 20% of body weight in a day.

An autopsy by microscopy is carried out on all the animals sacrificed or dead during these experiments. The autopsy focuses on the heart, lungs, liver, spleen, kidney, and intestine.

Isoflurane forene (Minerve, Bondoufle, France) is used to anaesthetize mice before intravenous tumor cell injections and during sacrifice of animals by cervical dislocation. Viability, clinical signs and behaviors are noted daily during the experiments. The body weight of each mouse is measured and recorded twice a week.

3.2 Effect of anti-CD71 antibody on the survival of mice bearing ARH-77 tumor cells

Seventy-one (1) ml of RPMI 1640 medium containing intravenous injection of five mice with severe combined immunodeficiency (SCID) was injected intravenously with five million ARH-77 suspended tumor cells. The injection was carried out between 24 to 72 hours after exposure of the whole body of the mice to gamma radiation (1.8 Gy, ⁶⁰ Co, INRA, Dijon, France).

The day of injection of tumor cells is considered as day 0 (D0). The treatments with the anti-CD71 antibody and with the irrelevant antibody begin on day J1. The Velcade® treatment begins on day 4 and is therefore performed on Days D4, D7, D11, D14, D18, D25, D28 and D32.

The 40 mice are separated into 5 groups. Table 4 below summarizes the treatment schedule for each group.

:

Table 4

The injection volume of irrelevant antibodies and anti-CD71 antibodies is

200μί / 8ουή8 / ίη | ^' 6ΰίίοη. Velcade® injection volume (10ml / kg) is adjusted to most individuals.

The surviving mice during the experiment are sacrificed after 144 days after injection of tumor cells. An autopsy is performed on all mice found dead or sacrificed during the experiment because of the presence of one of the pathological signs specified in the above section.

In group 1 (mice that received irrelevant antibody), a mouse died at 2g ^ith _our j _^ ^ _sister _{[s exhibited} signs of disease between 24 ^th day and 56 ^th day after injection of ARH-77 cells and were sacrificed and finally 2 mice were sacrificed at study at J144.

In group 2 (Mice that received anti-CD71 antibody), a mouse is found dead in the 42 ^th day, one is sacrificed 98 ^th day as it showed signs of disease and the other 6 have survived to the study and are sacrificed to J144.

In group 3 (mice given anti-CD71 antibody and Velcade®, all mice survive the study and are sacrificed on J144.

In Group 4 (mice only Velcade®), one mouse died at the 27 ^th day, the remaining 7 are sacrificed because of the presence of signs of disease between day 24 and day 31. The autopsy revealed different signs (tumors on the bladder, ovary, and kidney, small spleen or inflammation on right axillary lymph nodes, and on left inguinal ganglia).

In group 5 (untreated mice), the mice are sacrificed between day 24 and day 45 due to the presence of pathological signs.

The results are shown in Figure 3.

Table 5 below compares the average survival of the mice in the 5 dead or sacrificed groups before the end of the experiment because of the presence of the pathological signs specified above.

Table 5

Treatment with anti-CD71 antibody alone inhibits the development of myeloma in mice with severe combined immunodeficiency and prolongs survival. mice 29.6 days, for untreated mice, more than 70 days for mice treated with the injection of anti-CD71 antibodies.

The combination of anti-CD71 antibodies with Velcade® has a synergistic effect on the inhibition of myeloma development and the prolonged survival of affected mice, as compared to the use of anti-CD71 alone, or Velcade antibodies. ® alone (Figure 3). In all the mice that received this treatment, this combination succeeded in inhibiting the development of myeloma.

Claims

1. Use of an anti-CD71 monoclonal antibody or a fragment of a said antibody capable of binding to the CD71 antigen, said antibody or fragment of said antibody comprising:

for the preparation of a medicament for the prevention or treatment of myeloma.

The use of an antibody according to claim 2, wherein the constant regions of said light and heavy chains are constant regions of a human antibody.

3. Use of an antibody according to claim 1 and 2, wherein

at least the constant region of a light chain comprises or consists of the amino acid sequence chosen from SEQ ID NO: 6 or SEQ ID NO: 14,

4. Use of an antibody according to claims 1 to 3, wherein said fragment is Fab, F (ab) '2, Fd, scFV, ScFv dimer, diabody, triabody or tetrabody.

The use of an antibody according to claims 1 to 4, wherein said antibody or said antibody fragment is coupled to a bioactive molecule selected from:

- radioisotopes,

- non-radioactive metals,

toxins selected from ricin, abrin, diphtheria toxin,

the nucleic acids chosen from antisense RNAs,

the cytotoxic agents chosen from mitomycin C, methotrexate, adriamycin,

enzymes such as RNases,

biotin, avidin or streptavidin.

6. Use of an antibody according to claims 1 to 5, wherein the myeloma is selected from stage I myeloma (low tumor mass), stage II myeloma (intermediate tumor mass), stage III myeloma (strong). tumor mass).

7. Product containing:

at least one anti-CD71 monoclonal antibody or a fragment of the above-mentioned antibody capable of binding to the CD71 antigen, said antibody or fragment of said antibody comprising

an anticancer agent chosen from Velcade®, Melphalan®, Prednisone®.

The product of claim 7, wherein the constant regions of said light and heavy chains are constant regions of a human antibody.

The product of claim 8, wherein:

at least the constant region of a light chain comprises or consists of the amino acid sequence SEQ ID NO: 6 or SEQ ID NO: 14,

10. In vitro diagnostic method of myeloma, comprising:

at least the variable region of a heavy chain comprising or consisting of the amino acid sequence SEQ ID NO: 1, and at least the variable region of a light chain comprising or consisting of an amino acid sequence chosen from SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17,