HK1164345B - Truncated cd20 protein, deltacd20 - Google Patents

Description

Truncated CD20 protein, DeltaCD20

Technical Field

The invention relates in particular to an alternatively spliced protein derived from the gene encoding CD20, a nucleic acid encoding a protein according to the invention, a mutated form of the CD20 gene, and medicaments, diagnostic tools, diagnostic methods and therapeutic methods using the protein and nucleic acid sequences according to the invention.

Background

The gene encoding the CD20 protein expressed on B lymphocytes belongs to a family located in the q12 region of chromosome 11. This region identified a gene cluster designated MS4A (transmembrane domain 4 Subfamily (Membrane Spanning4 domain Subfamily a)) with 12 subclasses designated MS4a1 to MS4a 12. This gene cluster has 600 kb. The entire CD20 gene has 41.17kb and is scattered into 8 exons separated by 7 introns.

The precursor mRNA of CD20 has a size of 14.95KB with 416bp of untranslated 5 'sequence (5' UTR). The 3' UTR region has 2291bp following the polyA extension, and appears to correspond to regulatory sequences. This sequence potentially encodes a protein with 297 Amino Acids (AA), with a predicted molecular weight of 33.0 KDa. In the 5' UTR region, more specifically in exon 1, various splice sites have been identified, resulting in three forms of transcripts with size changes of 2.8, 2.6 and 3.4 kb.

The various transcript forms encode CD20 protein having a molecular weight of 33 KD; nevertheless, the presence of three isoforms of 33, 34.5 and 36kD, corresponding to splice variants but to post-transcriptional modifications, phosphorylation, has been demonstrated by Western blotting and immunoprecipitation. This protein consists of a very hydrophobic region, 4 transmembrane segments (AA68 to 84), defining an extracellular region (AA encoded by exon VI) and an intracellular region (AA encoded by exons III, V, VII and VIII).

Although widely used as a marker for peripheral blood B lymphocytes, for a lymphocyte population identification technique or for diagnosis of a disease or a B blood disease (B haemophathy), the function of the CD20 protein has not been elucidated so far. Nevertheless, the protein structure of the human or mouse CD20 molecule is similar to other proteins, such as rhodopsin, gap junction protein or certain adrenergic receptors, all involved in signaling, suggesting a similar role for this protein. The intracellular portion of this protein contains many phosphorylated sequences and is associated with Src family tyrosine kinases (Fyn, Lyn, Lck).

Functional studies in vitro and knock-out (KO) mouse models on CD20 have shown that this protein is involved in inter-membrane Ca + + transport. Binding of anti-CD 20 antibody to this molecule also induces an increase in c-Myc and B-Myb oncogenes, an increase in intracellular protein phosphorylation, and an increase in CD18, CD58 and MHC class II molecules, with tyrosine kinase activation causing B cell adhesion. These functions are attributed to the CD20 protein and remain controversial, as B cell development and function have not been reported as normal and exhibit no specific abnormalities in performance in the KO mouse model for CD 20.

Further function is attributed to the CD20 protein, cell cycle regulation in B Lymphocyte (BL) differentiation and its activation/maturation into plasma cells. In B lymphocyte ontogeny, CD20 is abundantly expressed on the surface of pre-B cells (absent on progenitor B cells) following rearrangement of the gene encoding the Ig heavy chain, with continued membrane expression to the mature late B stage. CD20 is not expressed on hematopoietic stem cells, on progenitor B cells and plasma cells, except in certain pathological cases, on a small cluster of cells that may be associated with plasmablasts. Finally, note that the CD20 ligand is unknown, making determination of its function difficult.

Expression on the surface of B lymphocytes makes it possible to identify this population by flow cytometry or by immunomagnetic purification techniques. Expression of the CD20 molecule on most B cells is implicated in malignant disease, making it the target of choice for therapy for several reasons:

-the label is present on the BL and absent on the stem and plasma cells

It is expressed in large quantities on the cell surface

It is not secreted or released into the circulation after proteolysis

-after immobilization of anti-CD 20, the CD20/Ab complex is not internalized

Rituximab (Rx, trade name: Mabthera)^TM) Is a humanized mouse chimeric antibody against the CD20 antigen. It is active on malignant cells presenting the CD20 antigen, i.e. follicular lymphoma at stages III-IV and invasive diffuse large B-cell non-hodgkin's lymphoma, positive for CD 20. It is also used, optionally in combination with chemotherapy, and more experimentally in other contexts, such as certain autoimmune diseases, such as lupus or rheumatoid arthritis.

The Fc portion of human IgH has been selected for its ability to fix complement and induce ADCC (Antibody-dependent cell-Mediated) cytotoxicity). The factors that influence its efficacy are diverse: CD20 surface expression density, antibody diffusion, therapeutic anti-CD 20 antibody capture, antibody/target binding, FcgR3 receptor polymorphism.

Disclosure of Invention

The present invention is based on the discovery that alternative splicing of the gene encoding CD20 results in the expression of a truncated form of CD 20. This polypeptide (i.e., deltaCD20 or deltaCD20) lacks all or part of the transmembrane portion of native CD20 (or wtCD20), such that deltaCD20 is not immobilized on the B lymphocyte membrane.

Thus, in a first embodiment, the invention relates to a polypeptide, characterized in that it comprises a sequence identical to the sequence of SEQ ID NO: 2 substantially the same amino acid sequence.

In the context of the present invention, the term "substantially identical" refers to two sequences having more than 90%, preferably 95%, more preferably 99% and most preferably 100% homology.

In the context of the present invention, the term "polypeptide" refers to an amino acid optionally comprising post-translational modifications. Preferably, the polypeptide according to the invention is obtainable by synthetic or genetic engineering methods. In the latter case, the term recombinant protein is used.

Preferably, the polypeptide according to the invention has an amino acid sequence identical to SEQ ID NO: 2 substantially identical amino acid sequence; and more preferably it is a polypeptide having the sequence SEQ ID NO: 2.

For the sake of clarity, the polypeptide according to the invention differs in particular from wtCD20 (the amino acid sequence of wtCD20 is indicated by SEQ ID NO: 4) in that it is not associated with the cell membrane. Thus, it is also possible to identify polypeptides according to the invention which do not comprise at least twenty consecutive amino acid sequences from the amino acid sequence set forth in SEQ ID NO: twenty contiguous amino acid sequences found between amino acid 43 and amino acid 209 of position 4 (i.e., the transmembrane portion of wtCD 20).

The invention also relates to a nucleic acid sequence, characterized in that it encodes a polypeptide according to the invention. As a preferred embodiment, the nucleic acid sequence according to the invention comprises a nucleotide sequence identical to SEQ ID NO:1, or a nucleic acid sequence identical thereto. As described in a more preferred embodiment, the nucleic acid sequence according to the invention is identical to SEQ ID NO:1 are identical.

In the context of the present invention, the term "nucleic acid sequence" refers in particular to a natural or synthetic DNA or RNA sequence. Preferably, the nucleic acid sequence according to the invention is obtained or synthesized by genetic engineering methods. As described in a further preferred embodiment, it consists of a completely or partially purified nucleic acid sequence.

The invention also relates to a recombinant vector comprising a nucleic acid sequence according to the invention, placed under the control of one or more elements required for its expression in a host cell. Recombinant vectors are well known to those skilled in the art and they make possible, inter alia, the production of recombinant proteins and/or the amplification of nucleic acid sequences. A large number of recombinant vectors are available in the prior art, in particular plasmids and viral vectors. Thus, as described in a preferred embodiment, the recombinant vector according to the invention is selected from the group comprising plasmids and viral vectors.

As described in the first embodiment, the recombinant vector according to the present invention is a viral vector. Viral vectors are well known to those skilled in the art and have been used in human clinical practice (e.g., MVA, adenovirus, retrovirus). They generally consist of a vector containing all or part of the viral genome modified to incorporate foreign sequences. As described in a preferred embodiment, the viral vector according to the invention is selected from the group comprising adenoviral vectors, retroviral vectors, poxvirus vectors, herpesvirus derived vectors, vectors derived from viruses associated with adenoviruses and alphavirus derived vectors. The invention also relates to viral particles comprising a recombinant vector according to the invention.

Preferably, the recombinant vector according to the invention is associated with one or more compounds facilitating its introduction into a host cell. Compounds that facilitate introduction of the vector into the host cell are well known to those skilled in the art and some are commercially available, the term transfection agent also being used. In a particularly preferred embodiment, the compound which facilitates introduction of the recombinant vector according to the invention into a host cell is selected from the group comprising cationic lipids, calcium salts, cationic polymers and polypeptides.

In the context of the present application, the term "elements required for expression in a host cell" refers to nucleic acid sequences for translation and transduction (transduction) of nucleic acid sequences and to nucleic acid sequences for increasing said transduction and translation. As described in a preferred embodiment, the elements required for expression in the host cell are selected from the group comprising introns, polyadenylation sites and promoters.

In the context of the present invention, the term host cell refers in particular to prokaryotic and eukaryotic cells. Among these cells, bacteria, yeasts, insect cells (e.g., sf9) and animal cells (e.g., CHO, 293, PERC6) are listed. The invention also relates to a host cell comprising a recombinant vector according to the invention.

Applicants have also revealed that the expression of deltaCD20 is correlated with the presence of certain diseases. Still further, it has been disclosed that the expression level of deltaCD20 is correlated with the development of these diseases. This applies more particularly to diseases associated with B lymphocyte dysfunction (also known as B hematological disorders), which represents 85 to 90% of lymphohematological disorders. These consist in particular of Follicular Lymphomas (FL), Chronic Lymphocytic Leukemia (CLL), B-cell Acute Lymphocytic Leukemia (ALL), mantle cell lymphomas (ML), B-cell lymphomas, myelomas, Waldenstrom's disease (F: (M))Disease, WD).

Thus, the invention also relates to an in vitro diagnostic method using a biological sample from a patient, characterized in that it comprises the measurement of the expression level of a polypeptide according to the invention and/or the measurement of the expression level of an mRNA encoding a polypeptide according to the invention.

The term "biological sample" refers to any fluid or tissue containing B lymphocytes from a patient. Preferably, the biological sample is blood or bone marrow of a patient.

The method according to the invention can be carried out with untreated biological samples (i.e. samples taken without any modification), but can also be carried out after the biological samples have been treated by any method deemed necessary by the person skilled in the art. These treatments include red blood cell lysis, monocyte isolation (e.g., on Ficoll), and/or the addition of molecules for protecting biological samples (e.g., anti-protease). Techniques such as red blood cell lysis and monocyte isolation in particular enable an increase in the ratio of B lymphocytes compared to other cells present in the biological sample. Thus, as described in a preferred embodiment, the diagnostic method according to the present invention further comprises a step for increasing the ratio of B lymphocytes compared to other cells present in the biological sample.

The polypeptide may be composed of protein, intact or cleaved before or after the post-translational modification. The method involves the detection of all or part of the polypeptide and therefore it is possible to detect only part of the mRNA or polypeptide, since the expression level of this part represents the expression level of the whole molecule.

In this application, the term "expression level" refers to the amount of deltaCD20 expressed by the cell. Expression levels can be measured quantitatively or semi-quantitatively. In practice, it is not necessary to know the exact amount of deltaCD20 expressed by the cells, but only to determine whether this amount is significantly greater than a particular value. Using a panel of healthy subjects and measuring the expression level of deltaCD20 in the cells of these subjects can be readily determined by one skilled in the art. If the method according to the invention reveals that the level of expression of deltaCD20 is greater than normal in cells of a patient who will be predisposed to B lymphocyte dysfunction.

As a preferred embodiment, the measurement of the expression level of a polypeptide according to the invention is a measurement of the expression level of mRNA encoding said polypeptide. Techniques for measuring the amount of mRNA of a particular coding molecule are well known to those skilled in the art. These techniques include quantitative RT-PCR, semi-quantitative RT-PCR, Northern Blot (Northern Blot) and microarray techniques. The design and generation of probes and oligonucleotides required to carry out these techniques is within the purview of one skilled in the art. These oligonucleotides include in particular SEQ ID NOs: 14 to SEQ ID NO:17, which is also the subject of the present invention. Thus, as described in a more preferred embodiment, the expression level of mRNA encoding a polypeptide according to the invention is measured by quantitative RT-PCR, semi-quantitative RT-PCR, real-time RT-PCR, northern blot or by microarray techniques.

According to a further preferred embodiment, said measurement of the expression level of a polypeptide according to the invention and/or of an mRNA encoding a polypeptide according to the invention is a measurement of the expression level of a polypeptide according to the invention and/or of an mRNA encoding a polypeptide according to the invention in B-lymphocytes.

As a preferred embodiment, said measuring of the expression level of a polypeptide according to the invention is carried out immunologically. In the context of the present application, the term "immunological method" refers to a protein detection technique using specific antibodies directed against the protein. In the context of the present application, the term "antibody" refers to a polypeptide comprising at least one paratope. Antibodies include T cell receptors, immunoglobulins, chimeric antibodies, human antibodies, monoclonal antibodies, humanized antibodies (humanized antibodies), recombinant antibodies, and antibody fragments. Antibody fragments include Fab, Fab ', F (ab)2, F (ab') 2, Fv and scFv.

Thus, as described in a more preferred embodiment, the diagnostic method according to the invention further comprises a step in which a polypeptide according to the invention is placed in contact with a specific antibody directed against said polypeptide.

Techniques for measuring the expression level of a protein in a cell are well known to those skilled in the art. They include, in particular, ELISA, flow cytometry, western blotting. Thus, as described in a more preferred embodiment, the immunological method is selected from the group comprising ELISA, flow cytometry, Western blotting.

Immunological methods can be used in particular on cell lysates or on accessible cells permeated by the membrane to render antibodies inside the cells.

According to a preferred embodiment, the diagnostic method according to the invention is a method for diagnosing a disease associated with a dysfunction of B-lymphocytes in one or more patients. As described in a preferred embodiment, the disease associated with a dysfunction of one or more B-lymphocytes is selected in a group comprising Follicular Lymphoma (FL), Chronic Lymphocytic Leukemia (CLL), B-cell Acute Lymphocytic Leukemia (ALL), mantle cell lymphoma (ML), B-cell lymphoma, myeloma, Waldenstrom's Disease (WD).

In solid organ transplantation, including kidney transplantation, allogenic graft (allogenic graft) loss remains a major problem. The role of the donor's allo-antibody mechanism in hyperacute rejection, the role of early and delayed rejection associated with T cells, is well known and in part controlled. Nevertheless, treatments affecting the T lymphocyte compartment have a reduced impact on long-term graft survival, suggesting other target effector mechanisms.

Splicing of the identified CD20 gene results in a deleted transcript, the sequence of which remains in-frame. Transcription or translation of spliced forms of mRNA into protein can interfere with the expression of the normal CD20 protein and impair its surface expression, which can modulate the efficacy of rituximab therapy. This has recently been reported in the literature, where the acquisition of tolerance to anti-CD 20 antibody (rituximab) in lymphoma cell lines is linked to pre-and post-transcriptional regulation phenomena or epigenetic (epigenetic) phenomena. As such, the deltaCD20 protein therefore represents a new therapeutic target for improving the efficacy of rituximab therapy and prevents escape and recurrence.

In renal transplantation, the study of high-throughput transcriptomes on patients with acute rejection makes it possible to determine the characteristic gene cluster of the B-lymphocyte population, characteristic of certain types of acute rejection, which is not usually distinguishable in light microscopy. Supplementary studies with immunohistochemical techniques have demonstrated a strong presence of CD20+ B lymphocytes infiltrating the graft. Finally, the persistence of CD20+ cells infiltrating the transplanted kidney was demonstrated after treatment with rituximab, while the pool of circulating B lymphocytes was removed.

Following this development, a team has described a study reporting anti-CD 20 antibody (rituximab) treatment for kidney or heart transplantation with adrenocortical hormone, ATG and plasmapheresis, with the result being encouraging, being 85% survival of the graft at 2 years.

As a general rule, there is an increasing value in the B lymphocyte compartment involved in allogeneic antibody production in solid organ transplantation. Rituximab naturally demonstrates its value in inhibiting B-responses (B-responses) in agents targeting this B-lymphocyte population, both for prophylactic (pre-transplant) and for delayed therapy (targeting memory B-cells). The use of diagnostic methods as described herein (e.g., in biopsy or non-invasive urine samples) and our immunotherapeutic strategies targeting Δ CD20 protein are useful in predicting acute rejection and improving rituximab therapy.

Thus, as described in a preferred embodiment, the disease associated with the dysfunction of one or more B lymphocytes is acute transplant rejection.

Thus, as described in a further preferred embodiment, the diagnostic method according to the invention is a method for assessing the efficacy of a treatment comprising the use of an anti-CD 20 antibody. As described in a preferred embodiment, the anti-CD 20 antibody is rituximab.

The present application also relates to kits for carrying out the diagnostic methods according to the invention. Thus, the invention also relates to a diagnostic kit comprising at least one specific antibody directed against a polypeptide according to the invention. In deltaCD20, the presence of at least one neoepitope associated with wild-type form CD20 makes possible the production of specific antibodies against deltaCD 20. It is well within the ability of those skilled in the art to produce such antibodies by a variety of methods available in the art. These include in particular the screening of phage libraries expressing scFv. Thus, the invention also relates to a specific antibody directed against a polypeptide according to the invention. As described in a preferred embodiment, the antibodies according to the invention are directed against the wild-type form of CD20, and more specifically against a polypeptide having the sequence as shown in SEQ ID NO: 4, is not specific.

According to a further embodiment, the diagnostic kit according to the invention comprises at least one specific oligonucleotide probe for an mRNA encoding a polypeptide according to the invention. As described in a further embodiment, the oligonucleotide probe comprises a nucleotide sequence identical to SEQ ID NO:14 or SEQ id no:17, and (b) 17. As described in a particularly preferred embodiment, the oligonucleotide probe has a sequence identical to SEQ ID NO:14 or SEQ ID NO:17, and (b) 17.

The invention also relates to the use of a diagnostic kit according to the invention for detecting a disease associated with a dysfunction of one or more B lymphocytes. As described in a preferred embodiment, the disease associated with dysfunction of one or more B lymphocytes is selected from the group comprising Follicular Lymphoma (FL), Chronic Lymphocytic Leukemia (CLL), B cell Acute Lymphocytic Leukemia (ALL), mantle cell lymphoma (ML), B cell lymphoma, myeloma, Waldenstrom's Disease (WD). As a further preferred embodiment said disease associated with a dysfunction of one or more B lymphocytes is acute transplant rejection.

The invention also relates to the use of a diagnostic kit according to the invention for assessing the efficacy of a treatment comprising the use of an anti-CD 20 antibody. As described in a preferred embodiment, the anti-CD 20 antibody is rituximab.

The polypeptides according to the invention are ideal targets for immunotherapeutic strategies (vaccination). More specifically, for diseases associated with treatment with anti-CD 20 antibodies (e.g., rituximab), this immunotherapeutic strategy is useful for preventing tolerance and improving treatment. Indeed, the inventors demonstrated with western blotting that in the case of induction of rituximab tolerance in vitro (line B with rituximab selection pressure), the increase in signal generated by the truncated protein (truncated protein) correlated with the increase in signal obtained with quantitative RT-PCR using mRNA from the same population. This means that most of the cells expressing deltaCD20 escaped and were resistant to standard rituximab therapy. For cytotoxic T cell responses, the truncated protein, deltaCD20, is a potential protein target. Similarly, spliced mRNA is also described as a valuable target using antisense oligonucleotide technology or using the RNA silencing pathway (SiRNA). The purpose of both approaches is to allow this selective splicing to disappear in vivo. Thus, the invention also relates to a method for improving therapy comprising the use of an anti-CD 20 antibody, comprising the use of an antisense oligonucleotide and/or siRNA, capable of inhibiting, completely or partially, the expression of a polypeptide according to the invention by cells of a patient.

Peptide vaccination (peptide vaccination) or immunotherapy is currently the subject therapeutic approach with major value in cancer prevention or treatment. The principle is based on immunization with polypeptides, which in turn generate T epitopes of tumor antigens recognized by Cytotoxic T Lymphocytes (CTLs), playing an important role in the elimination of tumor cells expressing these antibodies on their surface.

CTLs do not recognize whole protein antigens, but recognize peptide fragments thereof, presented by Major Histocompatibility Complex (MHC) molecules expressed on the surface of various cells. These peptide fragments form the T epitope.

The presentation of these peptides is the result of a complex process, called "antigen preparation", involving 3 main steps: 1/degradation of cytoplasmic antigens by a multienzyme complex called proteasome; 2/transporting the peptide obtained from said degradation in the endoplasmic reticulum by the TAP transporter; 3/these peptides associate with MHC to form a stable peptide/MHC complex for export to the cell surface.

The epitopes presented by major histocompatibility complex i (mhc i) generally have 8 to 11 amino acids and are recognized by CD8+ T cells and represent the major component of the cytotoxic response.

With respect to these epitopes, in particular (considering the essential role of the CD8+ response in cytotoxic action) those presented by MHC I, the identification thereof thus represents an essential step with respect to the development of antitumor immunotherapy.

A variety of tumor antigens are currently known to be capable of inducing CTL responses. Certain of the T epitopes of these antigens have been identified and the efficacy of peptide-based (peptide-based) vaccines that reproduce these T epitopes has been demonstrated in a number of cases.

Following the discovery of a novel alternative splicing of the human CD20 gene (deltaCD20) and the discovery of the protein encoded by the truncated mRNA, applicants discovered the impact of such targets on anti-tumor immunotherapy. Indeed, the results obtained demonstrate the role of this target protein (not expressed on B lymphocytes of healthy donors) in tumorigenesis and its role in resistance to anti-CD 20 antibody (rituximab) therapy.

In addition to the standard treatment of diseases associated with B lymphocyte dysfunction or acute transplant rejection (anti-CD 20 antibody (rituximab)), the discovery of the Delta20 protein and induction of T cytotoxic responses against polypeptides overlapping with the splice binding zone (splice junction zone) made it possible to carry out vaccination immunotherapy. Such vaccination may be performed directly with the peptide or with a recombinant vector encoding the peptide.

Thus, the invention also relates to the use of all or part of a polypeptide according to the invention, or of a recombinant vector according to the invention, for the preparation of a medicament. As described in a preferred embodiment, the polypeptide comprises at least one polypeptide selected from the group consisting of SEQ ID NOs: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO:12 and SEQ ID NO:13, in the sequence of the group of 13. As described in a more preferred embodiment, the polypeptide consists of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO:12 and SEQ ID NO:13, or a polypeptide of a sequence of group 13.

The invention also relates to the use of a nucleic acid sequence encoding a polypeptide comprising at least one polypeptide selected from the group comprising SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO:12 and SEQ ID NO:13, in the sequence of the group of 13. As described in a preferred embodiment, the use of a nucleic acid sequence encoding a polypeptide having a sequence encoded by a sequence selected from the group consisting of SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO:12 and SEQ ID NO:13, or a sequence consisting of the sequences of the group of 13.

As a more preferred embodiment of the use for the preparation of a medicament, said polypeptide comprises at least one polypeptide such as SEQ ID No:9, and (c) 9. As a particularly preferred embodiment, the use according to the invention is characterized in that said polypeptide consists of a polypeptide having a sequence such as SEQ ID No: 9.

As described in a preferred embodiment, the pharmaceutical product is for use in improving the efficacy of a treatment comprising the use of an anti-CD 20 antibody. As a more preferred embodiment, the anti-CD 20 antibody is rituximab.

As described in a preferred embodiment, the medicament is for the treatment or prevention of a B-hematological disorder, Follicular Lymphoma (FL), Chronic Lymphocytic Leukemia (CLL), B-cell Acute Lymphocytic Leukemia (ALL), mantle cell lymphoma (ML), B-cell lymphoma, myeloma, Waldenstrom's Disease (WD).

The invention also relates to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO:12 and SEQ ID NO:13, in the sequence of the group of 13.

A further approach may be gene transfer, encoding an antibody according to the invention, thus re-targeting T lymphocytes against this protein. The development of monoclonal antibodies against this protein will make it possible to identify in parallel the sequences encoding the hypervariable regions (CDR3) of the light and heavy Ig chains. The identified sequences can thus be transfected with T cells by gene transfer. A synergistic suicide gene may make it possible to control an anti- Δ CD20T response, although it will be restricted to activated tumor T cells expressing this form of protein. Thus, the invention also relates to the use of an antibody according to the invention for the preparation of a medicament, or the use of a nucleic acid encoding said antibody for the preparation of a medicament, or the use of a vector comprising said nucleic acid sequence for the preparation of a medicament, and preferably for the preparation of a medicament for the treatment of a disease from the list, associated with B-lymphocyte dysfunction as described in the present application.

CD20 is also a membrane marker because it is expressed on the surface of B cells, but is also a "susceptible" gene, encoding a target molecule for anti-CD 20 antibody therapy (rituximab) in certain blood disorders. Both attributes can be used for gene therapy in order to modify T cells (not normally expressing CD20) in vitro (e.g. by retroviruses) or in vivo. This is particularly useful for regulating and controlling complications following bone marrow allogeneic transplantation caused by T Lymphocytes (TLs). Thus, after genetic modification, cells can be selected (e.g., by means of an immunomagnetic system) on the basis of CD20 expression (not normally expressed by TLs) and can be targeted in vivo by anti-CD 20 antibodies (rituximab) if complications (graft versus host disease, GvHD) are present.

Although the use of the CD20 gene is valuable as a selectable marker and as a susceptible gene in gene modification rules, various studies using the retroviral LTR promoter as a susceptible gene have demonstrated instability in expression of this gene. The generation of alternative splicing of the CD20 gene may be the source of expression regulation. Furthermore, we demonstrated the presence of selective transcription in retroviral packaging lines transfected with vectors carrying "full length" CD20 cDNA. This line will therefore produce Δ CD20 retroviral particles that can infect target cells, but without surface CD20 expression, which would limit the efficacy of retroviral transduction. Further, the transduced target cells express spliced forms of the CD20 transcript, which can impair their sensitivity to rituximab.

Thus, the discovery of alternative splicing, and the discovery of donor and acceptor sites for such splicing, makes it possible to generate a nucleic acid sequence that encodes native CD20 and that includes modifications on the donor and/or acceptor sites to prevent the alternative splicing. Thus, the present invention relates to a nucleic acid sequence encoding CD20, characterized in that it does not comprise an alternative splice site. As described in a preferred embodiment, the sequence comprises a sequence such as SEQ ID NO:5, and particularly preferably the sequence consists of a sequence such as SEQ ID NO: 5. The invention also relates to a recombinant vector as defined above, comprising said nucleic acid sequence. The invention also relates to viral particles comprising said recombinant vector, said recombinant vector being associated with one or more compounds facilitating its introduction into a host cell, as defined above, and the host cell (as defined above) comprising said recombinant vector. The invention also relates to the use of said nucleic acid sequence or said recombinant vector for the preparation of a medicament and preferably for the preparation of a medicament for improving the efficacy of a treatment comprising the use of an anti-CD 20 antibody, and more preferably said anti-CD 20 antibody is rituximab.

The invention also relates to pharmaceutical compositions comprising a polypeptide, a nucleic acid sequence, an antibody and/or an oligonucleotide according to the invention and a pharmaceutically acceptable buffer.

Detailed Description

Experiment of

1. Demonstration of alternative splicing of the gene encoding CD20

To shut down the cDNA encoding CD20 in the plasmid backbone of the retrovirus, we amplified a segment corresponding to the entire coding part (CDs) of the CD20 protein by RT-PCR using RNA extracted from the DAUDI B line, using 2 primers covering the "start" and "stop" codons, respectively.

Electrophoresis of the PCR products revealed 2 distinct bands, one with the expected size of 894bp and the other with a size less than 393bp and the same length.

Sequencing and alignment of the NCBI Genbank revealed a sequence corresponding to the fragment encoding CD20 gene, with 501bp deletion in its middle portion, retaining the "start" and "stop" codons. Deletion analysis on the protein demonstrated that the transmembrane portion was virtually completely deleted, which is disadvantageous for the fixation of this protein on the cell membrane surface.

The truncated protein has a sequence of 131 amino acids, a predicted molecular weight of 15kD, and includes an intracytoplasmic C-terminal domain, a small portion of the first transmembrane domain, and an intracytoplasmic N-terminal domain terminus. By transfection of cell lines with vectors expressing wtCD20/GFP or Δ CD20/GFP fusion proteins, as demonstrated by confocal microscopy images, the potentially truncated protein was not immobilized on the membrane due to the lack of expression of the 4 transmembrane segments. After transfection with pcDNA3.1CT topological expression vector (Invitrogen), the wtCD 20. delta./GFP or CD20/GFP protein was expressed in 293T eukaryotic cells. The GFP protein was cloned in the CD20 sequence (. DELTA.or WT) reading range. GFP protein was detected by direct excitation (green) and wtCD20 protein was detected by anti-CD 20 antibody conjugated to TIRTC (red color). Thus, in cells infected with the truncated form of CD20, the cytoplasm was traced, and substantial co-localization of the green and red markers corresponding to GFP and wtCD20 to the membrane was observed. This confirms that Δ CD20 is restricted to the cytoplasm.

Using the splice site, splice donor site and acceptor site prediction software ((NNSPLICE v0.9 software-http://www.fruitfly.org/seq_tools/splice.htm) Analysis of the wild-type sequence encoding the CD20 gene (WRCD20) demonstrated the presence of a Donor Site (DS) and a cryptic Acceptor Site (AS), corresponding precisely to the sequence encoding the deleted CD20 gene (Δ CD 20). More in depth, the branching site was detected by a series of other software (Netgene 2-http:// www.cbs.dtu.dk/services/Netgene2) and was located at one twenty base pairs of the acceptor site.

1.Expression of Δ CD20 protein

a. Western blot detection

For the presence of the truncated protein (without transmembrane part and therefore cytoplasmic) encoded by the spliced transcript, we tested with a B-cell line by means of Western blotting using an available antibody (Thermo Fischer, # RB9013) against CD20 which recognizes the C-terminal part of the protein. Thus, we demonstrated the presence of a band of the desired size (. about.17 kD) corresponding to Δ CD20 in addition to the desired band corresponding to wild-type CD20 protein. This protein is not expressed in the T line.

2.Δ CD 20: for B-hematopathy or complications after kidney allogeneic transplantation (acute rejection, lymph) Tumor) diagnostic and/or prognostic molecular biomarkers

a. Selective Δ CD20 transcript and hematologic B disorders

Using qualitative PCR assays, amplifying the entirety of the coding sequence (flCD20 PCR), we screened other lines derived from various types of B hematological diseases (Burkitt, pre-B acute lymphocytic leukemia, transformed B-EBV) and demonstrated that CD20 transcript in short form was present in all B lines and absent in the T line. We then developed a sensitive and specific PCR tool (Δ CD20 PCR) by designing primers that cover the splice binding sites (splicing sites). This new tool can only detect short forms of mRNA and thus avoids PCR competition that is incompatible with high sensitivity.

Using this more efficient PCR tool, we tested the short form of CD20 on PBMCs, or for greater sensitivity, CD20 on CD19+ or CD20+ B cells from 5 healthy donors, and did not detect the same. Using our sensitive PCR tools, specific screens for truncated transcripts (truncated transcript) showed positive signals on cell lines and negative signals on PBMCs, indicating that in EBV transformed cells, in malignant cells, this splicing mechanism is present and absent in normal B cells. This truncated form of CD20RNA is therefore a signature of the B cell activation state associated with its malignant phenotype (phenotype).

b. Diagnostic quantification of Δ CD20 in various types of B-hematologic disorders

To elucidate the possibility of using Δ CD20 transcript quantification in certain hematological diagnoses, we applied our QRTPCR assay to various types of B hematological diseases. Quantification of both forms (full length and truncated) was performed in a series of dilution ranges of known amounts of standard plasmid containing CD20 in either the wt or delta form. The calculation of the ratio R ═ Δ CD20/(wtCD20+ Δ CD20) ] x100 makes it possible to express a relative quantification of CD20 transcripts with respect to the wild type form. We therefore demonstrate differential expression of these alternative transcripts in various B blood diseases. In the in vitro transformed B-EBV line (2.9 ± 4.51%, n ═ 6), in purified CD19+ tonsillectomy (tonsillectomy) cells (9 ± 2.2%, n ═ 7) and in vitro generated B naive cells (blasts) (14 ± 7.8%, n ═ 5), we quantified the spliced form of Δ CD20 [ expressed as% of Δ CD 20: r ═ x100 (Δ CD20/wtCD20+ Δ CD 20). In an advantageous manner, we quantified Δ CD20, 3.6 ± 5.1% in B acute lymphocytic leukemia (n ═ 27) and 3.9 ± 5.3% in follicular lymphoma (n ═ 5); 2.9 ± 4.5% in mantle cell lymphoma (n ═ 6); 3.2 ± 2.2% in high grade lymphoma (n ═ 5); and 0.1 ± 0.2% in B-CLL (n ═ 8).

c. Δ CD 20: residual Disease Marker (RDM)

A further possibility would also be to use our real-time PCR quantification assay to monitor the efficacy of the treatment (e.g. chemotherapy optionally associated with rituximab-Rx-). This test is very suitable for hematological disorders that do not have characteristic molecules or phenotypic markers. The quantitative kinetics of Δ CD20 were compared to the expression of standard molecular markers such as BCR/ABL (p190) transcript and cyclin D1 for acute lymphoblastic leukemia-type B (ALL-B) and mantle cell lymphoma, respectively. We demonstrated a correlation between the usual markers (cyclin D1 and BCR/Abl p190) and Δ CD 20.

3.Δ CD20 protein, a potential therapeutic target

a. Tolerance to anti-CD 20 antibody (rituximab)

In vitro we established Rx tolerance with lines B by exposing them to different doses and times. The acquisition of tolerance was confirmed in an in vitro sensitivity test.

In a very advantageous manner, by means of western blotting, we demonstrated that the signal of the truncated form of CD20 protein is enhanced as a function of the Rx tolerance gain of the population. More specifically, we quantified mRNA encoding Δ CD20 in cells of patients with follicular lymphoma (n-3) or mantle cell lymphoma (n-3). We observed that rituximab tolerance was obtained in each case in correlation with an increase in the amount of mRNA encoding Δ CD20 (mean x 3.38).

b. Construction of a Delta CD 20-specific oligopeptide

Thus, using translation of the Δ CD20mRNA sequence, we used the SYFPEITHI library (http:// www.uni-tuebingen. de/uni/kxi /) to design peptides (AA30 to AA39) that overlap with the alternative splicing binding band, which were thus specific for the Δ CD20 form. Using the predicted proteasome cleavage tool (http:// paproc. de), two potentially immunogenic peptides of value (No.4-RMS and No.7-SLE) were detected as they were potentially produced by proteasome enzymes (proteoasome enzymes) and restricted by HLA-A0201.

The peptides corresponding to these sequences were synthesized by MILLEGEN (LABEGE, france) and dissolved in 20% DMSO and stored at-80 ℃.

In vitro, the still unresolved affinity of the binding of peptides identified as being directed against MHC molecules, and the stability of the peptide/MHC I molecule complex were confirmed (data not shown).

c. Induction of T-cytotoxic response following peptide vaccination

For H-2 class I and II, the immunogenicity of the Δ CD20a peptide was assessed by generating CTLs on HLA-A2/DR1 and Knockout (KO) transgenic mice.

Mice (4 per group) were immunized with 2 or 3 peptides (2 injections at tail roots, 8 days apart): 50. mu.g of each peptide, 100. mu.g of helper peptide (20 mers peptide from CMV-specific pp 65), all emulsified together with Freund's incomplete adjuvant (incomplete Freud adjuvant).

Seven days after the second immunization, the spleens of the mice were removed and the splenocytes were restimulated in vitro with 4 μ g/ml of each peptide, respectively. On the fifth day of culture, the responding population was tested to determine the specific cytotoxin. Cells with eventual cytotoxicity were re-stimulated in vitro at one week intervals to obtain specific CTLs.

RMAS-HHD cells were used as targets to study cytotoxins. These target cells were filled with 10. mu.g/ml of the peptide under test, or an irrelevant control peptide, at 37 ℃ for 90 minutes, and labeled with 100. mu. Ci of 51Cr for 90 minutes, and washed three times. Splenocytes were plated in 96-well V-plates (3 × 103 cells/well in 100 μ l RPMI 1640+ 10% fetal calf serum). Then, 100. mu.l of effector cells (effector/target ratio: 30: 1; 10: 1; 3: 1 and 1: 1) were added to the wells and the plates were incubated at 37 ℃ for 4 hours. After incubation, 50 μ l of supernatant was collected, transferred to a special plate (Lumaplate) and the radioactivity was determined in a gamma counter. The specific cracking percentage was calculated using the formula: [ (experimental 51Cr release-spontaneous 51Cr release)/(maximum 51Cr release-spontaneous 51Cr release) ] x 100.

Three of the 4 mice from immunization group a generated T cytotoxic responses against peptide No.4 and no response to the third party peptide BMLF1, which was specific for EBV, thus indicating the specificity of the response.

These results demonstrate that immunization with the RMS peptide results in CTLs killing the RMAS-HHD target bearing the peptide, rather than cells bearing an unrelated peptide.

These studies clearly demonstrate that we are able to obtain an anti- Δ CD20 CTL response that can be used to improve anti-CD 20 antibody therapy and prevent or eliminate the persistence of CD20+ tumor B lymphocytes (expressing Δ CD20 protein).

4. Generation of site-directed mutagenesis of sequences encoding CD20, unsuitable for alternative splicing

We therefore sought to mutate the wild-type sequence of the human CD20 gene to limit the formation of these alternative transcripts. Mutation of the receptor site to modify the nucleotide sequence and maintain the reading frame, and thus the amino acid sequence — AA Gln encoded with CAG or CAA codons. Mutation at nucleotide 612 (coordinates based on ATG — first base of start codon): nt612G > A. After directed mutagenesis, we confirmed the mutation of a single base pair at the receptor site by sequencing. Finally, we demonstrated that the mutated CD20 gene sequence (mutCD20) did not further produce alternative transcripts. Flow cytometric analysis of CD20 expression demonstrated that expression was maintained and that the protein was actually expressed on the membrane.

Claims (20)

1. Diagnostic kit, characterized in that it comprises at least one oligonucleotide having a sequence identical to SEQ ID NO. 14 or SEQ ID NO. 17.

2. The in vitro use of a diagnostic kit according to claim 1 for the preparation of a device for the detection of the dysfunction of one or more B lymphocytes.

3. In vitro use of a diagnostic kit according to claim 2, wherein the dysfunction of one or more B-lymphocytes is selected in a group comprising Follicular Lymphoma (FL), Chronic Lymphocytic Leukemia (CLL), B-cell Acute Lymphocytic Leukemia (ALL), mantle cell lymphoma (ML), B-cell lymphoma, myeloma and Waldenstrom's Disease (WD).

4. In vitro use of a diagnostic kit according to claim 1 for the preparation of a device for assessing the efficacy of the use comprising an anti-CD 20 antibody.

5. The in vitro use of the diagnostic kit of claim 4, wherein said anti-CD 20 antibody is rituximab.

6. Use of a polypeptide for the preparation of a medicament, characterized in that the polypeptide consists of a polypeptide having a sequence selected from the group consisting of SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12 and SEQ ID NO 13.

7. The use according to claim 6, wherein said polypeptide consists of SEQ ID No. 9.

8. The use of claim 6, comprising the use of an anti-CD 20 antibody.

9. The use of claim 8, wherein said anti-CD 20 antibody is rituximab.

10. Polypeptide, characterized in that the sequence of the polypeptide is selected from the group of SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12 and SEQ ID NO 13.

11. Nucleic acid, characterized in that it consists of the sequence of SEQ ID NO. 5.

12. A recombinant vector comprising the nucleic acid sequence of claim 11 under the control of one or more elements required for expression thereof in a host cell.

13. The recombinant vector of claim 12, selected from the group consisting of plasmids and viral vectors.

14. The recombinant vector according to claim 13, which is a viral vector selected from the group consisting of adenoviral vectors, retroviral vectors, poxvirus vectors, herpesvirus derived vectors, adeno-associated virus derived vectors and alphavirus derived vectors.

15. The recombinant vector of claim 13, wherein the recombinant vector forms a complex with one or more compounds that facilitate its introduction into a host cell.

16. The recombinant vector of claim 15, wherein said compound that facilitates its introduction into a host cell is selected from the group consisting of cationic lipids, calcium salts, cationic polymers, and polypeptides.

17. The recombinant vector according to any one of claims 12 to 16, wherein the elements required for expression in the host cell are selected from the group comprising introns, polyadenylation sites and promoters.

18. Use of a nucleic acid sequence according to claim 11 or a recombinant vector according to any one of claims 12 to 17 for the preparation of a medicament.

19. The use of claim 18, for improving the efficacy of a treatment comprising the use of an anti-CD 20 antibody.

20. The use of claim 19, wherein said anti-CD 20 antibody is rituximab.