# Patent

## Title
Antibody method for treatment of a disease in which the target cells are cells which express CD20

## Abstract

The present invention relates to a monoclonal antibody method directed against CD20 antigen including administration of an anti CD20 antibody wherein each of the light chains thereof has the murine-human chimeric amino acid sequence SEQ ID NO: 28, and each of the heavy chains thereof has the murine-human chimeric amino acid sequence SEQ ID NO: 20.

## Background
This application is a continuation of application Ser. No. 11/793,138, filed on Aug. 14, 2008, now U.S. Pat. No. 9,234,045 issued on Jan. 12, 2016, which was filed as PCT International Application No. PCT/FR2005/003123 on Dec. 14, 2005, which claims the benefit under 35 U.S.C. § 119(a) to Patent Application No. 0413320, filed in FRANCE on Dec. 15, 2004, all of which are hereby expressly incorporated by reference into the present application.


REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “20151014_3493_0456PUS2_ST25.txt” created on Oct. 14, 2015 and is 56,116 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
The present invention relates to a monoclonal antibody directed against the CD20 antigen, in which the variable regions of each of the light chains are encoded by sequences which share at least 70% sequence identity with murine nucleic acid sequence SEQ ID No. 5, the variable region of each of the heavy chains are encoded by sequences which share at least 70% identity with murine nucleic acid sequence SEQ ID No. 7, and the constant regions of the light and heavy chains are constant regions from a non-murine species, as well as to the use of such an antibody for activating FcγRIII receptors in immune effector cells and for the manufacture of a drug, in particular for the treatment of leukaemia or lymphoma.
INTRODUCTION AND PRIOR ART
The CD20 antigen is a hydrophobic transmembrane protein with a molecular weight of 35-37 kDa which is present on the surface of mature B lymphocytes (Valentine et al. (1987) Proc. Natl. Acad. Sci. USA 84(22): 8085-8089; Valentine et al. (1989) J. Biol. Chem., 264(19): 11282-11287). It is expressed during the development of B lymphocyte cells (B cells) as from the early pre-B stage until differentiation into plasmocytes, a stage at which this expression disappears. The CD20 antigen is present on both normal B lymphocytes and malign B cells. More specifically, the CD20 antigen is expressed on most phenotype-B lymphomas (80% lymphomas): for example, it is expressed on over 90% non-Hodgkin's B-cell lymphomas (NHL) and over 95% B-type Chronic Lymphocytic Leukaemia (B-CLL). The CD20 antigen is not expressed on haematopoietic stem cells and on plasmocytes.
The function of CD20 has not yet been fully clarified, but it may act as a calcium channel and be involved in the regulation of the first stages of B lymphocytes differentiation (Golay et al. (1985) J. Immunol. 135(6): 3795-3801) and proliferation (Tedder et al. (August 1986) Eur. J. Immunol. 16(8): 881-887).
Therefore, although some uncertainty remains as regards its role in the activation and proliferation of B cells, the CD20 antigen is, because of its location, an important target for the treatment of conditions which involve tumoural B cells, such as NHL or B-CLL for instance, using antibodies which specifically recognise CD20. Furthermore, this antigen is an ideal target since it is a membrane protein for which no expression modulation or polymorphism is known.
Only one non-radioactively labelled anti-CD20 monoclonal antibody, Rituxan® (rituximab, Genentech), is currently commercially available for the treatment of B-cell lymphoma. It shows encouraging clinical results in patients with NHL when associated with chemotherapy. Its effectiveness, however, remains variable and frequently modest when it is used alone (Teeling et al. (2004) Blood 104(6): 1793-1800).
In addition, Rituxan® has only a modest effect on B cells in B-CLL. This low degree of effectiveness has been correlated with various phenomena: on the one hand, B-CLL B cells only express CD20 in relatively low quantities, and on the other hand, Rituxan® only induces very low ADCC (Antibody-Dependent Cellular Cytotoxicity) activity levels against these cells in vitro. These two observations might explain the correlation that has been observed between the level of expression of CD20 on tumours (in quantitative flow cytometry) and response to treatments.
Since B-CLL is the commonest form of leukaemia in western countries, and high-dose chemotherapy treatment sometimes prove to be insufficient and involve side effects which lead to haematopoietic aplasia and immunodeficiency, monoclonal antibodies appear to be an innovative approach. It is therefore of primary importance to develop antibodies which are capable of specifically targeting the CD20 antigen and which allow tumour cells such as B-CLL, which only express this antigen to a limited degree, to be destroyed.
Antibodies 2F2 and 7D8, proposed by Genmab (Teeling et al. (2004) Blood 104(6): 1793-1800) for the treatment of B-CLL, have a capacity to activate the complement which is greater than that induced by Rituxan®. These antibodies, however, have a low ADCC activity, similar to that of Rituxan®. Yet, some clinicians have shown that the complement activity is the cause of deleterious side effects, as the antibodies trigger an activation system which leads to the production of molecules (in particular, anaphylatoxins) which have a wide spectrum of non-specific activities (inflammatory, allergic or vascular reactions etc.). In addition, these antibodies are still at the research stage and their clinical effectiveness has yet to be evaluated.
In application FR03/02713 (WO 2004/029092), the present Applicant describes an anti-CD20 antibody which can be produced in the YB2/0 line and which has been selected for its ability to induce a high ADCC activity and a high level of IL-2 production by the Jurkat-CD16 cell compared to Rituxan®. There is a significant need for new anti-CD20 antibodies which will allow the range of B-cell diseases treated using the currently available immunotherapies to be extended; this is particularly the case with B-cell diseases in which the CD20 antigen is expressed to a small degree on the populations of B cells involved, and for which no satisfactory immunotherapies exist.
It is with this purpose in mind that the present Applicant has developed new CD20 antibodies which exhibit a particularly high ADCC activity compared to Rituxan®.
DESCRIPTION
A first object of the invention therefore relates to a monoclonal antibody directed against the CD20 antigen, in which the variable region of each of the light chains is encoded by a sequence which shares at least 70% identity with murine nucleic acid sequence SEQ ID No. 5, the variable region of each of the heavy chains is encoded by a sequence which shares at least 70% identity with murine nucleic acid sequence SEQ ID No. 7, and the constant regions of the light and heavy chains are constant regions from a non-murine species.
The antibodies are made up of heavy and light chains linked together by disulphide bonds. Each chain is made up, in the N-terminal position, of a variable region (or domain) (encoded by rearranged V-J genes for the light chains and V-D-J genes for the heavy chains) specific to the antigen against which the antibody is directed, and, in the C-terminal position, of a constant region made up from a single CL domain for the light chains, or several domains for the heavy chains.
For the purposes of the invention, the expressions “monoclonal antibodies” or “monoclonal antibody composition” refer to a preparation of antibody molecules having identical and unique specificities.
The antibody according to the invention, in which the variable regions in the light and heavy chains are from a species which is different from that of the constant regions of the light and heavy chains, is referred to as a “chimeric” antibody.
Murine nucleic acid sequences SEQ ID No. 5 and SEQ ID No. 7 code for the variable domain of each of the light chains and the variable domain of each of the heavy chains respectively, of the antibody produced by murine hybridoma CAT-13.6E12, available at the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ) under number ACC 474. This hybridoma produces a murine IgG2a, κ-type monoclonal antibody directed against CD20.
These murine sequences were chosen to derive the sequences of the variable regions of the antibodies according to the invention because of the specificity of the CAT-13.6E12 murine antibody for the CD20 antigen, the antigen also recognised by Rituxan®. The variable regions of the antibodies according to the invention share at least 70% identity with sequences SEQ ID No. 5 and SEQ ID No. 7, with this sequence identity providing the antibodies according to the invention with a specificity which is identical to that of the CAT-13.6E2 murine antibody. Preferably, this sequence identity also provides the antibody according to the invention with an affinity for the target which is identical to that of the CAT-13.6E12 murine antibody.
In addition, the present Applicant has surprisingly shown that the CAT-13.6E12 murine antibody has the ability to induce the secretion of IL-2 in the presence of Jurkat-CD16 cells which express ectodomains of the FcγRIIIA receptor (as shown in FIG. 11), indicating a strong binding between the murine antibody and human CD16 (FcγRIIIA), which again motivated the choice made by the present Applicant.
In addition, in the antibodies according to the invention, the constant regions of the light and heavy chains are from a non-murine species. In this regard all non-murine mammal species and families may be used, in particular humans, monkeys, murine (apart from mice), porcine, bovine, equine, feline, canine, as well as birds.
The antibodies according to the invention may be constructed using standard recombinant DNA techniques, well known to those skilled in the art, and more particularly using the “chimeric” antibody construction techniques described, for example, in Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855, where the recombinant DNA technology is used to replace the constant region of a heavy chain and/or the constant region of a light chain in an antibody from a non-human mammal, with the corresponding regions in an human immunoglobulin. One particular embodiment will be illustrated below.
Advantageously, the variable region of each of the light chains of the antibody according to the invention is encoded by a sequence which shares at least 80% identity with murine nucleic acid sequence SEQ ID No. 5, and the variable region of each of the heavy chains of the antibody according to the invention is encoded by a sequence which shares at least 80% identity with murine nucleic acid sequence SEQ ID No. 7.
Advantageously, the variable region of each of the light chains of the antibody according to the invention is encoded by a sequence which shares at least 90% identity with murine nucleic acid sequence SEQ ID No. 5, and the variable region of each of the heavy chains of the antibody according to the invention is encoded by a sequence which shares at least 90% identity with murine nucleic acid sequence SEQ ID No. 7.
Advantageously, the variable region of each of the light chains of the antibody according to the invention is encoded by a sequence which shares at least 95% identity with murine nucleic acid sequence SEQ ID No. 5, and the variable region of each of the heavy chains of the antibody according to the invention is encoded by a sequence which shares at least 95% identity with murine nucleic acid sequence SEQ ID No. 7.
Advantageously, the variable region of each of the light chains of the antibody according to the invention is encoded by a sequence which shares at least 99% identity with murine nucleic acid sequence SEQ ID No. 5, and the variable region of each of the heavy chains of the antibody according to the invention is encoded by a sequence which shares at least 99% identity with murine nucleic acid sequence SEQ ID No. 7.
Advantageously, the invention also relates to any antibody in which the variable regions of the heavy and light chains include one or more substitution(s), insertion(s) or deletion(s) of one or more amino acids, with these sequence modifications complying with the sequence identity percentage levels defined above, without affecting the antibodys' specificity or affinity for the target.
The antibodies of the invention are also any antibody which includes the CDRs (Complementary Determining Regions) of the CAT-13.6E12 antibody, combined with FR (framework) regions (highly conserved regions of the variable regions, also known as “backbone” regions). Such antibodies have affinities and specificities which are closely comparable with, and preferably identical to, those of the CAT-13.6E12 murine antibody.
Preferably, the variable region of each of the light chains of the antibody according to the invention is encoded by murine nucleic acid sequence SEQ ID No. 5 or by murine nucleic acid sequence SEQ ID No. 25, and the variable region of each of the heavy chains of the antibody according to the invention is encoded by murine nucleic acid sequence SEQ ID No. 7.
In one embodiment of the invention, an antibody according to the invention is therefore a monoclonal antibody directed against the CD20 antigen, in which the variable region of each of the light chains is encoded by murine nucleic acid sequence SEQ ID No. 5, the variable region of each of the heavy chains is encoded by murine nucleic acid sequence SEQ ID No. 7, and the constant regions of the light and heavy chains are constant regions from a non-murine species.
In a second embodiment, the antibody according to the invention is therefore a monoclonal antibody raised against the CD20 antigen, in which the variable regions of each of the light chains are encoded by murine nucleic acid sequence SEQ ID No. 25, the variable regions of each of the heavy chains are encoded by murine nucleic acid sequence SEQ ID No. 7, and the constant regions of the light and heavy chains are constant regions from a non-murine species.
In both embodiments, the antibodies differ in their nucleotide sequences by a single nucleotide: the nucleotide located at position 318 in SEQ ID No. 5 and SEQ ID No. 25, which correspond to a cytosine and an adenine respectively.
The antibodies of the invention according to these embodiments have specificities and affinities for the target antigen, CD20, which are comparable with, and preferably identical to, those of the CAT-13.6E12 murine antibody.
Preferably, the constant regions of each of the light chains and each of the heavy chains of the antibody according to the invention are human constant regions. In this preferred embodiment of the invention, the immunogenicity of the antibody is reduced in humans, and consequently, the antibodys' effectiveness is improved upon therapeutic administration to humans.
According to a preferred embodiment of the invention, the constant region of each of the light chains of the antibody according to the invention is of κ type. Any allotype is suitable for the implementation of the invention, e.g. Km(1), Km(1, 2), Km(1, 2, 3) or Km(3), but the preferred allotype is Km(3).
According to another additional embodiment, the constant region of each of the light chains of the antibody according to the invention is of λ type.
According to one specific aspect of the invention, and in particular when the constant regions of each of the light chains and of each of the heavy chains of the antibody according to the invention are human regions, the constant region of each of the heavy chains of the antibody is of γ type. According to this alternative, the constant region of each of the heavy chains of the antibody may be of γ1, γ2 or γ3 type, with these three constant region types exhibiting the specific feature of binding the human complement, or even of γ4 type. Antibodies which have γ-type constant regions for each of the heavy chains belong to the IgG class. Immunoglobulins G (IgG) are heterodimers made up of 2 heavy chains and 2 light chains, linked together by disulphide bonds. Each chain is made up, in the N-terminal position, of a variable region or domain (encoded by rearranged V-J genes for the light chains and V-D-J genes for the heavy chains) specific to the antigen against which the antibody is directed, and, in the C-terminal position, of a constant region made up of a single CL domain for the light chain, or of 3 domains (CH1, CH2 and CH3) for the heavy chain. Combining the variable domains and the CH1 and CL domains of the heavy and light chains make up the Fab fragments which are linked to the Fc regions through a highly flexible hinge region allowing each Fab fragment to bind its antigen target whilst the Fc region, the mediator for the effector properties of the antibody, remains accessible to effector molecules such as FcγR and C1q receptors. The Fc region, made up of both CH2 and CH3 globular domains, is glycosylated at the CH2 domain, with a lactosamine-type biantennary N-glycan linked to Asn 297 being present on each of the 2 chains.
Preferably, the constant region of each of the heavy chains of the antibody is of γ1 type, as such antibody exhibits the ability to induce ADCC activity in the greatest number of (human) individuals. In this respect, any allotype is suitable for the implementation of the invention, e.g. G1m(3), G1m(1, 2, 17), G1m(1, 17) or G1m(1, 3). Preferably, the allotype is G1m(1, 17).
According to one particular aspect of the invention, the constant region of each of the heavy chains of the antibody is of γ1 type, and is encoded by human nucleic acid sequence SEQ ID No. 23, with the constant region of each of the light chains being encoded by human nucleic acid sequence SEQ ID No. 21. Such an antibody therefore includes a murine variable region and a human constant region, with γ1-type heavy chains. This antibody therefore belongs to the IgG1 sub-class. According to the embodiment of the antibody according to the invention, the antibody has two light chains, the variable domain of which is encoded by murine nucleic acid sequence SEQ ID No. 5 or murine nucleic acid sequence SEQ ID No. 25, and the human constant region of which is encoded by nucleic acid sequence SEQ ID No. 21, and two heavy chains, the variable domain of which is encoded by murine nucleic acid sequence SEQ ID No. 7 and the constant region of which is encoded by human nucleic acid sequence SEQ ID No. 23.
Preferentially, each of the light chains of the antibody according to the invention is encoded by murine-human chimeric nucleic acid sequence SEQ ID No. 13 or by murine-human chimeric nucleic acid sequence SEQ ID No. 27, and each of the heavy chains is encoded by murine-human chimeric nucleic acid sequence SEQ ID No. 19. Murine-human chimeric nucleic acid sequence SEQ ID No. 13, which codes for each of the light chains of the antibody, is obtained by fusing murine nucleic acid sequence SEQ ID No. 5, which codes for the variable domain of each of the light chains of the antibody, to human nucleic acid sequence SEQ ID No. 21, which codes for the constant region of each of the light chains of the antibody.
Murine-human chimeric nucleic acid sequence SEQ ID No. 27, which codes for each of the light chains of the antibody, is obtained by fusing murine nucleic acid sequence SEQ ID No. 25, which codes for the variable domain of each of the light chains of the antibody, to human nucleic acid sequence SEQ ID No. 21, which codes for the constant region of the light chains of the antibody.
Murine-human chimeric nucleic acid sequence SEQ ID No. 19, which codes for each of the heavy chains of the antibody, is obtained by fusing murine nucleic acid sequence SEQ ID No. 7, which codes for the variable domain of each of the heavy chains of the antibody, to human nucleic acid sequence SEQ ID No. 23, which codes for the constant region of each of the heavy chains of the antibody.
According to a particular aspect of the invention, when each of the light chains of the antibody is encoded by murine-human chimeric nucleic acid sequence SEQ ID No. 13, and each of the heavy chains is encoded by murine-human chimeric nucleic acid sequence SEQ ID No. 19, the peptide sequence of each of the light chains, deduced from nucleic acid sequence SEQ ID No. 13, is sequence SEQ ID No. 14 and the peptide sequence of each of the heavy chains, deduced from nucleic acid sequence SEQ ID No. 19, is sequence SEQ ID No. 20.
According to a further particular aspect of the invention, when each of the light chains of the antibody is encoded by murine-human chimeric nucleic acid sequence SEQ ID No. 27, and each of the heavy chains is encoded by murine-human chimeric nucleic acid sequence SEQ ID No. 19, the peptide sequence of each of the light chains, deduced from nucleic acid sequence SEQ ID No. 27, is sequence SEQ ID No. 28, and the peptide sequence of each of the heavy chains, deduced from nucleic acid sequence SEQ ID No. 19, is sequence SEQ ID No. 20.
The peptide sequences SEQ ID No. 14 and SEQ ID No. 28 differ only by the amino acid present at position 106 on each of these two sequences. The amino acid located at position 106 is lysine (K) in sequence SEQ ID No. 28; it is asparagine (N) in sequence SEQ ID No. 14.
The invention also relates to antibodies in which each of the light chains encoded by murine-human chimeric nucleic acid sequence shares at least 70% homology or identity with murine-human chimeric nucleic acid sequence SEQ ID No. 13, and each of the heavy chains encoded by a murine-human chimeric nucleic acid sequence shares at least 70% homology or identity with the murine-human chimeric nucleic acid sequence SEQ ID No. 19, with these modifications adversely impairing neither the specificity of the antibody nor its effector activities, such as ADCC (Antibody-Dependent Cell-mediated Cytotoxicity) activity.
In a particularly advantageous manner, the antibody of the invention is produced by a rat hybridoma cell line. The line which produces the antibody according to the invention is an important characteristic since it provides the antibody with certain of its particular properties. In fact, the method of expression of the antibody induces the post-translational modifications, in particular the glycosylation modifications, which may vary from one cell line to another, and therefore provides antibodies which have identical primary structures with different functional properties.
In a preferred embodiment, the antibody is produced in the rat hybridoma YB2/0 cell line (cell YB2/3HL.P2.G11.16Ag.20, registered at the American Type Culture Collection under ATCC number CRL-1662). This line was chosen because of its ability to produce antibodies with improved ADCC activity compared to antibodies with the same primary structures produced, for example, in CHO cells.
According to a specific embodiment, a preferred antibody according to the invention is antibody EMAB6 produced by clone R509, registered on 8 Nov. 2004 under registration number CNCM I-3314 at the Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France). Each of the light chains of the EMAB6 antibody is encoded by murine-human chimeric nucleic acid sequence SEQ ID No. 13, and each of the heavy chains is encoded by murine-human chimeric nucleic acid SEQ ID No. 19. This chimeric antibody competes with the CAT-13.6E12 murine antibody in binding CD20 and has a cytotoxic activity which is much greater than that of Rituxan®, which may be attributable in part to the specific glycosylation of the N-glycan of the heavy chain of these antibodies (see Example 4). In fact, a specific feature of the R509 clone is that it produces an EMAB6 antibody composition with a fucose/galactose ratio of less than 0.6, which has been shown, in patent application FR 03 12229, to be optimal to provide the antibody with strong ADCC activity. This antibody is therefore particularly interesting as a therapeutic tool for the treatment of conditions in which the cells to be targeted express CD20.
In a further specific embodiment, another preferred antibody according to the invention is antibody EMAB603 produced by clone R603, registered on 29 Nov. 2005 under registration number CNCM I-3529 at the Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France). Each of the light chains of the EMAB603 antibody is encoded by murine-human chimeric nucleic acid sequence SEQ ID No. 27, and each of the heavy chains is encoded by murine-human chimeric amino acid sequence SEQ ID No. 19. This chimeric antibody competes with the CAT-13.6E12 murine antibody in binding CD20 and has a cytotoxic activity which is much greater than that of Rituxan®, which may be attributable in part to the specific glycosylation of the N-glycan of the heavy chain of these antibodies (see Example 3). In fact, a specific feature of the R603 clone is that it produces an EMAB603 antibody composition with a fucose/galactose ratio of less than 0.6, (see Example 4) which has been shown, in patent application FR 03 12229, to be optimal to provide the antibody with strong ADCC activity. This antibody is therefore of particular interest as a therapeutic tool for the treatment of conditions in which the cells to be targeted express CD20.
Another object of the invention relates to vector pEF-EMAB6-K for the expression of the light chain of an antibody according to the invention, having sequence SEQ ID No. 12. This vector is the vector which allows an antibody according to the invention, the light chain of which is encoded by nucleic acid sequence SEQ ID No. 13, the deduced peptide sequence of which is sequence SEQ ID No. 14, to be expressed. This vector is a nucleic acid molecule into which murine nucleic acid sequence SEQ ID No. 5, which codes for the variable domain of each of the light chains of the antibody, and nucleic acid sequence SEQ ID No. 21, which codes for the constant regions of each of the light chains of the antibody, have been inserted to be introduced and maintained in a host cell. It allows foreign nucleic acid fragments to be expressed in the host cell since it has sequences (promoter, polyadenylation sequence, selection gene) which are essential to this expression. Such vectors are well known to those skilled in the art and may be, without implied limitation, an adenovirus, a retrovirus, a plasmid or a bacteriophage. In addition, any mammalian cell may be used as a host cell, that is as a cell which expresses the antibody according to the invention, e.g. YB2/0, CHO, CHO dhfr- (e.g. CHO DX B11, CHO DG44), CHO Lec13, SP2/0, NSO, 293, BHK or COS.
Another object of the invention relates to vector pEF-EMAB6-H for the expression of the heavy chain of an antibody according to the invention, having sequence SEQ ID No. 18. This vector is the vector which allows an antibody according to the invention, the heavy chain of which is encoded by nucleic acid sequence SEQ ID NO 19, the deduced peptide sequence of which is sequence SEQ ID No. 20, to be expressed. This vector is a nucleic acid molecule into which murine nucleic acid sequence SEQ ID No. 7, which codes for the variable domain of each of the heavy chains of the antibody, and human nucleic acid sequence SEQ ID No. 23, which codes for the constant region of each of the heavy chains of the antibody, have been inserted to be introduced and maintained in a host cell. It allows these foreign nucleic acid fragments to be expressed in the host cell since it has sequences (promoter, polyadenylation sequence, selection gene) which are essential to this expression. Just as indicated earlier, the vector may be, for example, a plasmid, an adenovirus, a retrovirus or a bacteriophage, and the host cell may be any mammalian cell, e.g. YB2/0, CHO, CHO dhfr- (CHO DX B11, CHO DG44), CHO Lec13, SP2/0, NS0, 293, BHK or COS.
An antibody produced by co-expressing the pEF-EMAB6-K and pEF-EMAB6-H vectors in the YB2/0 cell is illustrated by the anti-CD20 EMAB6 antibody, produced by clone R509 (registered under registration number CNCM I-3314 at the CNCM). This antibody induces a cytotoxicity which is greater than that induced by Rituxan®, both in cells from patients with B-CLL (on which the CD20 antigen is expressed at lower levels) and in Raji cells used as a model and which express CD20 at higher densities compared to the cells from patients with B-CLL. Furthermore, the EMAB6 antibody induces a secretion of IL-2 (interleukin 2) in Jurkat-CD16 cells which is at much higher levels than with Rituxan®. Since the EMAB6 antibody can be produced by growing the R509 clone in a culture medium and under conditions which allow the vectors described above to be expressed, it is therefore a very interesting tool for advancing the therapy and diagnosis of B-cell diseases in which the CD20 antigen is involved, more specifically B-CLL, as well as for research in this area.
A particular object of the invention is a stable cell line which expresses an antibody according to the invention.
Advantageously, the stable cell line which expresses an antibody according to the invention is selected from the group consisting of: SP2/0, YB2/0, IR983F, a human myeloma such as Namalwa or any other cell of human origin such as PERC6, the CHO lines, in particular CHO-K-1, CHO-Lec10, CHO-Lec1, CHO-Lec13, CHO Pro-5, CHO dhfr- (CHO DX B11, CHO DG44), or other lines chosen from Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK, K6H6, NSO, SP2/0-Ag 14 and P3X63Ag8.653.
The line used is preferably the rat hybridoma YB2/0 cell line. This line was chosen because of its ability to produce antibodies with improved ADCC activity with respect to antibodies with the same primary structure produced, for example, in CHO cells.
According to one particular aspect of the invention, the stable cell line which expresses an antibody according to the invention and which is more specifically chosen from the group described above, has incorporated the two pEF-EMAB6-K and pEF-EMAB6-H expression vectors as described earlier.
One specific aspect of the invention relates to clone R509, registered under registration number CNCM I-3314 at the Collection Nationale de Cultures de Microorganismes (CNCM).
One specific aspect of the invention relates to clone R603, registered under registration number CNCM I-3529 at the Collection Nationale de Cultures de Microorganismes (CNCM).
Another object of the invention relates to a DNA fragment having sequence SEQ ID No. 19 which codes for the heavy chain of an antibody according to the invention. Murine-human chimeric nucleic acid sequence SEQ ID No. 19 codes for each of the heavy chains of the antibody. It is obtained by fusing murine nucleic acid sequence SEQ ID No. 7, which codes for the variable domain of each of the heavy chains of the antibody, to human nucleic acid sequence SEQ ID No. 23, which codes for the constant region of each of the heavy chains of the antibody.
Another specific object of the invention relates to a DNA fragment having sequence SEQ ID No. 13, which codes for the light chain of an antibody according to the invention. Murine-human chimeric nucleic acid sequence SEQ ID No. 13 codes for each of the light chains of the antibody. It is obtained by fusing murine nucleic acid sequence SEQ ID No. 5, which codes for the variable domain of each of the light chains of the antibody, to human nucleic acid sequence SEQ ID No. 21, which codes for the constant region of each of the light chains of the antibody.
Another specific object of the invention relates to a DNA fragment having sequence SEQ ID No. 27, which codes for the light chain of an antibody according to the invention. Murine-human chimeric nucleic acid sequence SEQ ID No. 27, codes for each of the light chains of the antibody. It is obtained by fusing murine nucleic acid sequence SEQ ID No. 25, which codes for the variable domain of each of the light chains of the antibody, to human nucleic acid sequence SEQ ID No. 21, which codes for the constant region of each of the light chains of the antibody.
One specific object of the invention relates to the use of the antibody according to the invention to activate, in vivo or in vitro, the FcγRIIIA receptors of effector immune cells. In fact, the antibodies of the invention have the specific feature and advantage of being cytotoxic. As such, they exhibit the ability to activate FcγRIIIA receptor with their Fc regions. This is of considerable interest as this receptor is expressed on the surface of cells known as “effector cells”: binding of the Fc region of the antibody to its receptor carried by the effector cell causes the activation of FcγRIIIA receptors and the destruction of the target cells. Effector cells are, for instance, NK (Natural Killer) cells, macrophages, neutrophils, CD8 lymphocytes, Tγδ lymphocytes, NKT cells, eosinophils, basophils or mastocytes.
In one specific aspect, the antibody of the invention is used as a drug. Advantageously, such a drug is intended for the treatment of conditions in which the target cells are cells which express CD20, such as malignant B-cell lymphoma.
According to one advantageous aspect, the antibody according to the invention is used to manufacture a drug for the treatment of leukaemia or lymphoma.
One specific object of the invention is the use of the antibody according to the invention for the manufacture of a drug for the treatment of a pathology selected from the group consisting of acute B lymphoblastic leukaemia, B-cell lymphoma, mature B-cell lymphoma, including B-type Chronic Lymphocytic Leukaemia (B-CLL), small B-cell lymphoma, B-cell prolymphocytic leukaemia, lymphoplasmocytic lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone MALT-type lymphoma, lymph node marginal zone lymphoma with or without monocytoid B cells, splenic marginal zone lymphoma (with or without villous lymphocytes), tricholeucocytic leukaemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, as well as any immune dysfunction diseases involving B lymphoid cells, including auto-immune diseases.
Another object of the invention is the use of the antibody according to the invention for the manufacture a drug for the treatment of lymphoid leukaemia.
Another object of the invention is the use of the antibody according to the invention for the manufacture of a drug for the treatment of B-type Chronic Lymphoid Leukaemia (B-CLL). Furthermore, the antibody according to the invention is particularly well suited for the treatment of conditions in which CD20 is less strongly expressed on B cells, and preferably, B-CLL (B-type Chronic Lymphocytic Leukaemia). In this regard, the antibody according to the invention may be used in combination with one or more further antibody(ies), e.g. monoclonal antibodies directed against one or more further antigens expressed on lymphoid cells, such as, for example, antigens HLA-DR, CD19, CD23, CD22, CD80, CD32 and CD52, for the manufacture of a drug for the treatment of leukaemia or lymphoma. Thus, the humanised antibody Campath-1H® (alentuzumab, MabCampathR®) which targets a molecule which is abundantly expressed on lymphoid cells, the CD52 antigen, and which induces cell lysis by mobilising the host effector mechanisms (complement binding, antibody-dependent cytotoxicity) is used in the treatment of CLL (Moreton P., Hillmen P. (2003) Semin. Oncol. 30(4): 493-501; Rawstron A. C. et al, (2004) Blood 103(6): 2027-2031; Robak T. (2004) Leuk. Lymphoma 45(2): 205-219; Stanglmaier M. et al, (2004) Ann. Hematol. 83(10): 634-645). Clinical tests are also underway to evaluate antibodies or immunotoxins which target the antigens HLA-DR, CD22, CD23, CD80 in patients with CLL (Mavromatis B. H., Cheson B. D. (2004) Blood Rev. 18(2): 137-148; Mavromatis B., Cheson B. D. (2003) J. Clin. Oncol. 21(9): 1874-1881, Coleman M. et al, (2003) Clin. Cancer Res. 9: 3991S-3994S; Salvatore G. et al, (2002) Clin. Cancer Res. 8: 995-1002).
In a further embodiment, the antibody according to the invention may be used in combination with cells which express FcγRs, such as NK cells, NKT (Natural Killers T) cells, Tγδ lymphocytes, macrophages, monocytes or dendritic cells, i.e. in combination with a cellular therapy (Peller S., Kaufman S. (1991) Blood 78: 1569, Platsoucas C. D. et al, (1982) J. Immunol. 129: 2305; Kimby E. et al, (1989) Leukaemia 3(7): 501-504; Soorskaar D. et al, Int. Arch. Allery Appl. Immunol. 87(2): 159-164; Ziegler H. W. et al, (1981) Int. J. Cancer 27(3): 321-327; Chaperot L. et al, (2000) Leukaemia 14(9): 1667-1677; Vuillier F., Dighiero G. (2003) Bull. Cancer. 90(8-9): 744-750).
In addition, the antibody according to the invention advantageously allows the doses administered to patients to be reduced: advantageously, the antibody dose administered to the patient is 2 times, 5 times, or even 10 times, 25 times, 50 times or particularly advantageously 100 times less than a dose of Rituxan®. Advantageously, the antibody dose administered to the patient is between 2 and 5 times, between 5 and 10 times, between 5 and 25 times, between 5 and 50 times, or even between 5 and 100 times less than a dose of Rituxan®. Thus, the antibody according to the invention, for instance the EMAB6 antibody, may advantageously be administered at a dose of less than 187.5 mg/m2, 75 mg/m2, 37.5 mg/m2, 15 mg/m2, 7.5 mg/m2, or particularly advantageously less than 3.75 mg/m2. The dose administered is advantageously between 187.5 mg/m2 and 75 mg/m2, or between 75 mg/m2 and 37.5 mg/m2, between 75 mg/m2 and 15 mg/m2, between 75 mg/m2 and 7.5 mg/m2, or even between 75 mg/m2 and 3.75 mg/m2.
Thus, the invention also refers to a method for treating diseases in which the target cells are cells which express CD20, such as malignant B-cell lymphoma, consisting in administering to a patient an effective dose of a composition containing an antibody according to the invention. More specifically, the treatment method is particularly suited to the treatment of leukaemia or lymphoma. Even more specifically, it is a method for treating a pathology chosen from acute B lymphoblastic leukaemia, B-cell lymphoma, mature B-cell lymphoma, including B-type Chronic Lymphocytic Leukaemia (B-CLL), small B-cell lymphoma, B-cell prolymphocytic leukaemia, lymphoplasmocytic lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone MALT-type lymphoma, lymph node marginal zone lymphoma with or without monocytoid B cells, splenic marginal zone lymphoma (with or without villous lymphocytes), tricholeucocytic leukaemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, as well as any immune dysfunction diseases involving cells of the B lymphoid lines, including auto-immune diseases, consisting in administering an effective dosage of an antibody or antibody composition according to the invention.
A particular object of the invention is the use of an antibody according to the invention for the manufacture of a drug for the treatment of chronic graft-versus-host disease in order to treat symptoms which involve the receiver's B cells.
Finally, a last object of the invention is the use of an antibody according to the invention for the manufacture of a drug for a treatment of organ, in particular kidney, transplant rejection.
Recent studies (Ratanatharathorn et al, (August 2003) Biol. Blood Marrow Transplant 9(8): 505-511; Becker et al, (June 2004) Am. J. Transplant. 4(6): 996-1001) have in fact shown the benefits of anti-CD20 antibodies in the treatment of such conditions.
Further aspects and advantages of the invention will be described in the following examples which should be regarded as illustrative examples and do not limit the scope of the invention.



DESCRIPTION OF THE FIGURES
FIG. 1: Schematic representation of the CKHu vector used for the chimerisation of the light chain kappa of antibodies EMAB6 and EMAB603.
FIG. 2: Schematic representation of the light chain pEF-EMAB6-K expression vector used for the production of antibody EMAB6.
FIG. 3: Schematic representation of the G1Hu vector used for the chimerisation of the heavy chain of antibodies EMAB6 and EMAB603.
FIG. 4: Schematic representation of the heavy chain pEF-EMAB6-H expression vector used for the production of antibody EMAB6.
FIG. 5: Competition by the chimeric EMAB6 antibody for the binding of the murine antibody produced by CAT-13.6E12 (CAT13) to CD20 expressed on Raji cells.
FIG. 6A: Complement-dependent cytotoxic activity of the anti-CD20 antibodies on Raji cells. Rituxan®: open triangle, EMAB6: closed lozenge. Cell lysis is estimated by measuring the intracellular LDH released into the supernatant. Results are expressed as percentage lysis, with 100% being the value obtained with Rituxan® (at 5,000 ng/mL anti-CD20 antibody). Mean of 5 tests. FIG. 6B: Complement-dependent cytotoxic activity of the anti-CD20 antibodies on Raji cells. Comparison of the complement-dependent cytotoxic activities of EMAB6 (closed lozenge) and EMAB603 (open lozenge).
FIG. 7A: ADCC activity induced by anti-CD20 antibodies in the presence of Raji cells. Rituxan®: open triangle, EMAB6: closed lozenge. Cell lysis is estimated by measuring the intracellular LDH released into the supernatant. Results are expressed as percentage lysis, with 100% being the value obtained with Rituxan® (at 250 ng/mL anti-CD20 antibody). Mean of 3 tests. FIG. 7B: ADCC activity induced by anti-CD20 antibodies in the presence of Raji Cells. Comparison of ADCC induced by EMAB6 (closed lozenge) and EMAB603 (open lozenge).
FIG. 8: ADCC activity induced by anti-CD20 antibodies in the presence of B lymphocytes from patients with B-CLL. Rituxan®: open triangle, EMAB6: closed lozenge. E/T ratio=15. Cell lysis is estimated by measuring the calcein released into the supernatant. Results are expressed as percentages, with 100% being the value obtained with Rituxan® (at 500 ng/mL anti-CD20 antibody). Mean of 4 experiments corresponding to 4 different patients.
FIG. 9A: Activation of CD16 (FcγRIIIA) induced by anti-CD20 antibodies in the presence of Raji cells. Rituxan®: open triangle, EMAB6: closed lozenge. Results are expressed as percentage of IL-2, as measured in supernatants using ELISA; with 100% being the value obtained with Rituxan® (at 2,500 ng/mL anti-CD20 antibody). Mean of 4 tests. FIG. 9B: Activation of CD16 (Fcg RIIIA) induced by anti-CD20 antibodies in the presence of Raji cells. Comparison between the activation of CD16 (FcγRIIIA) as induced by EMAB6 (closed lozenge) and EMAB603 (open lozenge).
FIG. 10: Activation of CD16 (FcγRIIIA) induced by anti-CD20 antibodies in the presence of B lymphocytes from patients with B-CLL. Rituxan®: open triangle, EMAB6: closed lozenge. Results are expressed as percentage of IL-2, as measured in the supernatants using ELISA; with 100% being the value obtained with Rituxan® (at 2,500 ng/mL anti-CD20 antibody). Mean of 12 patients.
FIG. 11: Production of IL-2 induced by the CAT-13.6E12 murine antibody in the presence of Jurkat-CD16 cells (FcγRIIIA).
FIG. 12: Schematic representation of the heavy chain and light chain pRSV-HL-EMAB603 expression vector used for the production of antibody EMAB603.



EXAMPLES
Example 1: Construction of Expression Vectors for Anti-CD20 Chimeric Antibodies EMAB6 and EMAB603
A. Determination of the Sequence of the Variable Regions of the CAT-13.6E12 Murine Antibody
Total RNA from murine hybridoma CAT-13.6E12 cells (supplier: DSMZ, ref. ACC 474), which produces an IgG2a, κ-type immunoglobulin, was isolated (RNAeasy kit, Qiagen ref. 74104). After reverse transcription, the variable domains of the light (Vκ) and heavy (VH) chains of the CAT-13.6E12 antibody were amplified using the 5′RACE technique (Rapid Amplification of cDNA Ends) (GeneRacer kit, Invitrogen ref. L1500-01). The primers used for the two steps were the following:
1. Reverse Transcription Primers
a. Murine Kappa Specific Antisense Primer (SEQ ID No. 1)









5′-ACT GCC ATC AAT CTT CCA CTT GAC-3′






b. Murine G2a Specific Antisense Primer (SEQ ID No. 2)










5′-CTG AGG GTG TAG AGG TCA GAC TG-3′






2. 5′RACE PCR Primers

a. Murine Kappa Specific Antisense Primer (SEQ ID No. 3)










5′-TTGTTCAAGAAGCACACGACTGAGGCAC-3′






b. Murine G2a Specific Antisense Primer (SEQ ID No. 4)










5′-GAGTTCCAGGTCAAGGTCACTGGCTCAG-3′






The resulting VH and Vκ PCR products were cloned into vector pCR4Blunt-TOPO (Zero blunt TOPO PCR cloning kit, Invitrogen, ref. K2875-20) and sequenced. The nucleotide sequence of the Vκ region of the murine CAT-13.6E12 antibody is shown as sequence SEQ ID No. 5 and the deduced peptide sequence is sequence SEQ ID No. 6. The Vκ gene belongs to the Vκ4 class [Kabat et al. (1991) “Sequences of Proteins of Immunological Interest”. NIH Publication 91-3242].
The nucleotide sequence of the VH region of CAT-13.6E12 is sequence SEQ ID No. 7 and the deduced peptide sequence is sequence SEQ ID No. 8. The VH gene belongs to the VH1 class [Kabat et al. (1991) “Sequences of Proteins of Immunological Interest”. NIH Publication 91-3242].
B. Construction of Heavy and Light Chain Expression Vectors for Chimeric Antibodies EMAB6 and EMAB603
1. Light Chain Kappa Vector
1.1. Light Chain Vector for Antibody EMAB6
The Vκ sequence cloned into the pCR4Blunt-TOPO sequencing vector was amplified using the following cloning primers:
a) Vκ Sense Primer (SEQ ID No. 9)







5′-CTCAGTACTAGTGCCGCCACCATGGATTTTCAAGTGCAGATTTTC


 


AG-3′






The underlined sequence corresponds to the SpeI restriction site, the sequence in bold lettering corresponds to a Kozak consensus sequence, the ATG initiator is in italics.
b) Vκ Antisense Primer (SEQ ID No. 10)







5′-TGAAGACACTTGGTGCAGCCACAGTCCGGTTTATTTCCAGCCTGG


 


T-3′






This primer joins the murine Vκ sequences (in italics) to the human constant region (Cκ) (in bold). The underlined sequence corresponds to the DraIII restriction site.
The resulting Vκ PCR product contains the sequence which codes for the natural signal peptide of the CAT-13.6E12 murine antibody. This Vκ PCR product was then cloned between the SpeI and DraIII sites of the light chain chimerisation vector (FIG. 1), which corresponds to sequence SEQ ID No. 11, at 5′ in the human constant region Cκ, the nucleic acid sequence of which is sequence SEQ ID No. 21 and the deduced peptide sequence of which is sequence SEQ ID No. 22. The human Cκ sequence of this chimerisation vector had been modified beforehand by silent mutagenesis in order to create a DraIII restriction site to allow cloning of murine Vκ sequences to take place. This chimerisation vector contains an RSV promoter and a bGH (bovine Growth Hormone) polyadenylation sequence together with the dhfr (dihydrofolate reductase) selection gene.
The light chain sequence of the chimeric EMAB6 antibody encoded by this vector is shown as SEQ ID No. 13 for the nucleotide sequence and corresponds to the deduced peptide sequence SEQ ID No. 14.
1.2. Light Chain Vector for Antibody EMAB603
The protocol is the same as for the light chain vector for the EMAB6 antibody (see Example 1, B-1.1), apart from the Vκ antisense primer which is:
b′) Vκ Antisense Primer (SEQ ID No. 29)









5′-TGAAGACACTTGGTGCAGCCACAGTCCG TTTATTTCCA



 



GCCTGGT-3′






This primer joins the murine Vκ sequences (in italics) to the human constant region (Cκ) (in bold). The underlined sequence corresponds to the DraIII restriction site.
This primer also introduces the mutation AAC→AAA (framed nucleotide in the antisense primer sequence SEQ ID No. 29), which corresponds to mutation N106K (see nucleotide sequence and deduced peptide sequence SEQ ID No. 25 and SEQ ID No. 26) relative to the natural Vκ sequence of CAT-13.6E12 (see. SEQ ID No. 5 and SEQ ID No. 6).
The light chain sequence of the chimeric EMAB603 antibody encoded by this vector is shown as SEQ ID No. 27 for the nucleotide sequence and corresponds to the deduced peptide sequence SEQ ID No. 28.
2. Heavy Chain Vector
A similar approach was applied to the chimerisation of the heavy chains of the EMAB6 and EMAB603 antibodies.
The VH sequence cloned into the pCR4Blunt-TOPO vector was first of all amplified using the following cloning primers:
a) VH Sense Primer (SEQ ID No. 15)







5′-CTCAGTACTAGTGCCGCCACCATGGGATTCAGCAGGATCTTTCT


 


C-3′






The underlined sequence corresponds to the restriction site SpeI, the sequence in bold lettering corresponds to a Kozak consensus sequence, the ATG initiator is in italics.
b) VH Antisense Primer (SEQ ID No. 16)







5′-GACCGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACTGAGGTT


 


CC-3′






This primer joins the murine VH sequences (in italics) to the human G1 constant region (in bold). The underlined sequence corresponds to the ApaI restriction site.
The amplified VH fragment contains the sequence which codes for the natural signal peptide of the CAT-13.6E12 murine antibody. This VH PCR product was then cloned between the SpeI and ApaI sites in the heavy chain chimerisation vector (FIG. 3) which corresponds to sequence SEQ ID No. 17, at 5′ of the γ1 human constant region, the nucleic acid sequence of which is sequence SEQ ID No. 23 and the deduced peptide sequence of which is sequence SEQ ID No. 24. This chimerisation vector contains an RSV promoter and a bGH (bovine Growth Hormone) polyadenylation sequence as well as the neo selection gene.
The heavy chain sequences of the chimeric EMAB6 and EMAB603 antibodies encoded by this vector are shown as SEQ ID No. 19 for the nucleotide sequence and as SEQ ID No. 20 for the deduced peptide sequence.
3. Final Expression Vectors
3.1. EMAB6 Antibody Expression Vectors
For the expression of the EMAB6 antibody, the RSV promoter of the kappa light chain chimerisation vector (see Example 1, B-1.1) was replaced with the human EF-1 alpha promoter. The final light chain pEF-EMAB6-K expression vector is shown in FIG. 2 and corresponds to sequence SEQ ID No. 12.
The light chain sequence of the chimeric EMAB6 antibody encoded by this vector is shown as SEQ ID No. 13 for the nucleotide sequence and corresponds to the deduced peptide sequence SEQ ID No. 14.
For the expression of the EMAB6 antibody, the RSV promoter of the heavy chain chimerisation vector (see Example 1, B-2) was replaced with the human EF-1 alpha promoter. The thus-obtained final heavy chain pEF-EMAB6-H expression vector is shown in FIG. 4 and corresponds to sequence SEQ ID No. 18.
3.2. EMAB603 Antibody Expression Vector
A unique expression vector containing both heavy chain and light chain transcription units of the anti-CD20 EMAB603 antibody was constructed from two light and heavy chain chimerisation vectors (see Example 1, B-1.2 and B2 respectively) by sub-cloning into the XhoI site of the heavy chain vector, a BglII-PvuII fragment of the light chain vector containing the light chain transcription unit and the dhfr gene. This pRSV-HL-EMAB603 expression vector includes two selection genes, i.e. neo (neo-phosphotransferase II) and dhfr (dihydrofolate reductase), together with two heavy chain and light chain transcription units under the control of an RSV promoter (FIG. 12).
Example 2: Production of Cell Lines Derived from the YB2/0 Line Producing Anti-CD20 Chimeric EMAB6 and EMAB603 Antibodies
The rat YB2/0 cell line (ATCC # CRL-1662) was cultivated in EMS medium (Invitrogen, ref. 041-95181M) containing 5% foetal calf serum (JRH Biosciences, ref. 12107). For transfection, 5 million cells were electroporated (Biorad electroporator, model 1652077) in Optimix medium (Equibio, ref. EKITE 1) with 25 μg of light chain vector pEF-EMAB6-K (FIG. 2), linearised with AatII, and 27 μg of heavy chain vector pEF-EMAB6-H (FIG. 4), linearised with ScaI, for the expression of the EMAB6 antibody, or with vector pRSV-HL-EMAB603, for the expression of the EMAB603 antibody. The electroporation conditions applied were 230 Volts and 960 microFarads in a 0.5-mL cuvette. Each electroporation cuvette was then distributed over 5 P96 plates at a density of 5,000 cells/well.
Placement in a selective RPMI medium (Invitrogen, ref 21875-034) containing 5% dialysed serum (Invitrogen, ref. 10603-017), 500 μg/mL G418 (Invitrogen, ref. 10131-027) and 25 nM methotrexate (Sigma, ref. M8407), was carried out 3 days after transfection.
The supernatants from the resistant transfection wells were screened for the presence of chimeric immunoglobulin (Ig) by applying an ELISA assay specific to the human Ig sequences.
The 10 transfectants producing the largest amount of antibody were amplified on P24 plates and their supernatants re-assayed using ELISA to estimate their productivity and select, by limited dilution (40 cells/plate), the best three producers for cloning.
After cloning, the R509.6A4 clone (R509-33903/046-6H1(1)6A4, productivity: 17 μg/106 cells), hereafter referred to as “R509”, as well as the R603 clone were selected for the production of the chimeric EMAB6 and EMAB603 antibodies respectively and progressively acclimated to the CD Hybridoma production medium (Invitrogen, ref. 11279-023).
The production of the chimeric EMAB6 and EMAB603 antibodies was achieved by expanding, in CD Hybridoma medium, the acclimated culture obtained by dilution to 3×105 cells/mL in 75-cm2 and 175-cm2 vials and then dilution to 4.5×105 cells/mL in roller flasks. Once the maximum volume (1 L) was achieved, culture was continued until the cell viability was only 20%. After production, the chimeric EMAB6 and EMAB603 antibodies were purified using protein-A affinity chromatography (HPLC estimated purity <95%) and checked by polyacrylamide gel electrophoresis.
Example 3: Characterisation of the Functional Activity of Chimeric Antibodies EMAB6 and EMAB603
A. Specificity
Specificity of the antigen recognition of the chimeric EMAB6 antibody was evaluated by studying the competition with the murine antibody CAT-13.6E12 (CAT13) for binding the CD20 antigen expressed by Raji cells.
For that purpose, the EMAB6 antibody (10 μL at 0.5 to 50 μg/mL) was incubated at 4° C. with a fixed quantity of CAT-13.6E12 murine antibody (10 μL at 5 μg/mL) for 20 minutes in the presence of Raji cells (50 μL at 4×106 cells/mL). After washing, a mouse anti-IgG antibody coupled to phycoerythrin (PE) was added to the Raji cells so as to specifically detect the binding of the CAT-13.6E12 murine antibody. The Median Fluorescence Intensities (MFIs) obtained in the presence of various concentrations of EMAB6 are converted to percentages, with 100% corresponding to binding to CAT-13.6E12 cells in the absence of the EMAB6 antibody.
An inhibition curve is thus obtained for binding of the CAT-13.6E12 (CAT13) antibody to Raji cells in the presence of increasing concentrations of EMAB6 (FIG. 5).
This study demonstrates that the chimerisation process has not adversely affected the specificity of the EMAB6 antibody, which does compete with the parental CAT-13.6E12 murine antibody for binding to CD20 expressed on the surface of Raji cells.
The antigen recognition specificity of the EMAB603 antibody is comparable with that of the EMAB6 antibody.
B. Complement-Dependent Cytotoxic Activity
Complement-dependent cytotoxic activity of the EMAB6 and EMAB603 antibodies was examined with Raji cells in the presence of young rabbit serum as a source of complement; the anti-CD20 chimeric antibody Rituxan® was included in one test, for comparison.
For this test, the Raji cells were adjusted to 6×105 cells/mL in IMDM (Iscove's Modified Dulbecco's Medium) 5% FCS (Foetal Calf Serum). The antibodies were diluted with IMDM+0.5% FCS. The reaction mixture was made up of 50 μL antibody, 50 μL young rabbit serum (1/10 IMDM+0.5% FCS dilution of Cedarlane CL 3441 reagent), 50 μL target cells and 50 μL IMDM+0.5% FCS medium. The final antibody concentrations were 5,000, 1,250, 250 and 50 ng/mL. A control without antibodies was included in the test. After 1 hr incubation at 37° C. in a 5% CO2 atmosphere, the plates were centrifuged and the levels of intracellular LDH released into the supernatant estimated using a specific reagent (Cytotoxicity Detection Kit 1 644 793).
The percentage lysis was estimated using a calibration range obtained using various dilutions of target cells lysed using triton X100 (2%) corresponding to 100, 50, 25, and 0% lysis respectively.
The results shown in FIG. 6(A) demonstrate that EMAB6 and Rituxan® both induce complement-dependent lysis of the Raji cells. Nevertheless, EMAB6 complement activity appeared to be slightly less than that of Rituxan®. This difference is greater at the low concentrations of antibody used in this test. Thus for concentrations of 50 and 250 ng/mL, the activity of EMAB6 is of the order of 45% of that of Rituxan®. This difference becomes smaller as the antibody concentration is increased, with the % complement-dependent cytotoxic activity of the EMAB6 antibody representing 92% of that of Rituxan® at the highest concentration tested, i.e. 5,000 ng/mL.
This lower complement-dependent cytotoxic activity of the AMAB6 antibody compared to that of Rituxan® may be regarded as an advantage, since it limits the potential in vivo toxicity of EMAB6 compared to Rituxan®, associated with the activation of the conventional complement pathway, which leads to the production of various molecules with undesirable inflammatory, allergic and vascular activities.
The complement activity of the EMAB603 antibody is shown in FIG. 6(B).
C. ADCC Activity
The cytotoxicity of the chimeric EMAB6 antibody was evaluated in the presence of Raji cells or B lymphocytes from patients with CLL. The anti-CD20 chimeric antibody Rituxan® was included in the tests for comparison.
The calcein-labelling ADCC measurement technique used was as follows:
NK cells were isolated from PBMCs using the separation on magnetic beads (MACS) technique from Myltenyi. The cells were washed and re-suspended in IMDM+5% FCS (45×105 cells/mL). The effector cells and target cells were used in a ratio of 15/1. The Raji cells or the PBMCs (Peripheral Blood Mononuclear Cells) from patients with B-CLL obtained after Ficoll treatment (>95% B cells) were labelled beforehand with calcein (1 mL cells at 3×106 cells/mL in IMDM+5% FCS+20 μL calcein (20 μM), 20 min incubation at 37° C. and then washing with HBSS (Hank's Buffered Saline Solution)) and adjusted to 3×105 cells/mL in IMDM+5% FCS. The antibodies were diluted with IMDM+0.5% FCS (final concentrations: 500; 50; 5; 0.5; 0.05 and 0.005 ng/mL).
The reaction mixture was made up of 50 μL antibody, 50 μL effector cells, 50 μL target cells and 50 μL IMDM medium in a P96 microtitration plate. Two negative controls were used:

    
    
        Lysis without NK: NK effector cells were replaced with IMDM+5% FCS.
        Lysis without antibodies (Ab): antibodies were replaced with IMDM+5% FCS.
    
    


After 4 hrs incubation at 37° C. in a 5% CO2 atmosphere, the plates were centrifuged and the fluorescence associated with the supernatant was measured using a fluorimeter (excitation: 485 nm, emission: 535 nm).
The percentage lysis was estimated using a calibration range obtained using various dilutions of target cells lysed using Triton X100 (2%), corresponding to 100, 50, 25, and 0% lysis respectively.
The results were first calculated using the following formula:

% lysis=(% lysis with Antibody and NK)−(% lysis without Antibody)−(% lysis without NK)

and then expressed as relative percentages, with 100% being the value obtained at the highest concentration of Rituxan®.

The results obtained for the EMAB6 antibody on the Raji line cells shown in FIG. 7(A) demonstrate that, irrespective of the concentration being tested, the cytotoxicity induced by the EMAB6 antibody is greater than that induced by Rituxan®. This difference is particularly large at low antibody concentrations. Thus, at 0.5 ng/mL, the lysis percentages were 96% and 4% for EMAB6 and Rituxan® respectively. By increasing the dose 500-fold (250 ng/mL), the difference is still appreciable since the relative percentages of ADCC are 164% and 100% for EMAB6 and Rituxan® respectively. When the EC50s were calculated (antibody concentration corresponding to 50% of the E Max, the maximum effectiveness obtained at the highest antibody concentration and at the plateau) by graphical estimation (in ng/mL) and assuming that Rituxan® and EMAB6 attain the same E Max, the Rituxan® EC50/EMAB6 EC50 ratio in this test was then equal to 300.
The cytotoxicity of the chimeric EMAB603 antibody was evaluated in the presence of Raji cells using the same procedure as for the EMAB6 antibodies. Its activity was comparable with that of the EMAB6 antibody (see FIG. 7(B)).
With the lymphocytes from patients with B-CLL, the results obtained, shown in FIG. 8, demonstrate that, irrespective of the concentration being tested, the cytotoxicity induced by the EMAB6 antibody is greater than that induced by Rituxan®. As already observed with the Raji cells, this difference is particularly large at low antibody concentrations. A concentration of 0.5 ng/mL EMAB6 induces the same percentage lysis as 500 ng/mL Rituxan®, i.e. a concentration ratio of 1,000. At 5 ng/mL, the lysis percentages are 269% and 9% for EMAB6 and Rituxan® respectively. At the maximum dose tested (500 ng/mL), the difference is still very large since the relative percentages of ADCC are 350% and 100% for EMAB6 and Rituxan® respectively. An interesting result corresponds to the concentrations which give rise to 50% lysis. In this test, the Rituxan® EC50/EMAB6 EC50 ratio was estimated as 10,000 (graphical estimate in ng/mL for EC50 assuming that Rituxan® and EMAB6 attain the same E Max).
In these tests, the cytotoxic activities of EMAB6 and EMAB603 are therefore much greater than that of Rituxan®.
D. Activation of CD16 (IL-2 Secretion)
The activation of CD16 (FcγRIIIA) induced by the chimeric EMAB6 antibody was determined in the presence of Raji cells or B lymphocytes from patients with CLL. This test evaluated the ability of the antibody to bind to CD16 (FcγRIIIA) receptor expressed on the Jurkat-CD16 cells and to induce the secretion of IL-2. The anti-CD20 chimeric antibody Rituxan® is included in the tests for comparison.
Measurement of CD16 activation was carried out in the following manner on the Jurkat-CD16 cell line in the presence of Raji cells or B lymphocytes from patients with CLL.
Mixture in 96-well plate: 50 μL antibody solution (dilution to 10,000, 1,000, 100 and 10 ng/mL with IMDM+5% FCS for B lymphocytes from patients with B-CLL and 10,000, 2,000, 1,000, 200, 100, 50 and 25 ng/mL for Raji cells), 50 μL PMA (Phorbol Myristate Acetate, diluted to 40 ng/mL with IMDM+5% FCS), 50 μL Raji or PBMCs from patients with B-CLL obtained after Ficoll treatment (>95% B cells) diluted to 6×105/mL with IMDM+5% FCS, and 50 μL Jurkat-CD16 cells (20×106/mL in IMDM+5% FCS). Controls without antibodies were included in all tests. After incubation overnight at 37° C., the plates were centrifuged and the IL-2 contained in the supernatants estimated using a commercial kit (Quantikine from R/D). The OD readings were made at 450 nm.
The results were initially expressed as IL-2 levels as a function of the antibody concentration (from 0 to 250 ng/mL final concentration), then as relative percentages, where 100% is the value obtained with Rituxan® at the highest test concentration.
The results obtained with the Raji line cells shown in FIG. 9(A) demonstrate that, in the presence of EMAB6 and Rituxan®, the Jurkat-CD16 cells secrete IL-2, which indicates cell activation via binding of the Fc portion of the antibodies to CD16. The EMAB6 antibody, however, has an inductive activity which is much stronger than the Rituxan® antibody. Thus, at 6.25 ng/mL, the IL-2 percentages were 112% and 21% for EMAB6 and Rituxan® respectively. At 50 ng/mL, the difference is still large, with the percentages of IL-2 being 112% and 65% respectively. This difference decreases as concentration increases, with the respective percentages of IL-2 for EMAB6 and Rituxan® being 124% and 100% at 2,500 ng/mL. In this test, the Rituxan® EC50/EMAB6 EC50 ratio is estimated at 15 (graphical estimate in ng/mL for EC50 assuming that Rituxan® and EMAB6 attain the same E Max).
These results confirm the ADCC results, both being CD16-dependant. They demonstrate that the binding to CD16 (FcγRIIIA) by the Fc portion of the EMAB6 antibody is followed by a strong cellular activation which leads to the induction of effector functions.
The activation of CD16 (FcγRIIIA) induced by the chimeric EMAB603 antibody in the presence of Raji cells is comparable with that induced by the EMAB6 antibody.
With lymphocytes from patients with B-CLL, the results obtained shown in FIG. 10 demonstrate that in the presence of anti-CD20 Rituxan® and EMAB6, the Jurkat-CD16 cells secrete IL-2, which indicates cell activation via binding of the Fc portion of the antibodies to CD16. The EMAB6 antibody, however, has an inductive ability which is much greater than the Rituxan® antibody. In fact, the IL-2 secretion induction activity of Rituxan® is close to the base line at concentrations of 2.5 and 25 ng/mL, whereas that of the EMAB6 antibody is significant. Thus at 25 ng/mL, the IL-2 percentages were 132% and 34% for EMAB6 and Rituxan® respectively. At the highest concentration (2,500 ng/mL), the IL-2 percentages were 148% and 100% respectively. The Rituxan® EC50/EMAB6 EC50 ratio in this test is greater than 100: it is estimated at 300 (graphical estimate in ng/mL for EC50 assuming that Rituxan® and EMAB6 attain the same E Max).
In conclusion, all the tests carried out on Raji cells demonstrate that the EMAB6 and EMAB603 antibodies, unlike Rituxan®, are highly cytotoxic and induce the activation of cells which express CD16 (FcγRIIIA), especially at low antibody concentrations. On the contrary, under the same conditions, the complement-dependent cytotoxic activity of EMAB6 decreases by about 50% compared to that of Rituxan®.
These results are confirmed by the studies carried out using cells isolated from patients with B-CLL, suggesting that the EMAB6 antibody is much more cytotoxic than Rituxan® towards B lymphocytes from patients with B-CLL. The differences between the two antibodies are more marked with lymphocytes from patients with B-CLL than with the Raji cells, which demonstrates the significant therapeutic interest of EMAB6 compared to Rituxan® for this condition.
The reason of this increased difference may be, amongst other, the lower antigen expression of CD20 on B lymphocytes from patients with B-CLL compared to Raji cells.
By analogy with Raji cells, it may be suggested that the complement-dependent cytotoxic activity of the EMAB6 antibody towards lymphocytes from patients with B-CLL must be less than that induced by Rituxan®, thus exhibiting the advantage of being less toxic in vivo as a result of the undesirable effects associated with a strong activation of the conventional complement pathway.
Example 4: Analysis of EMAB6 and EMAB603 Glycans by HPCE-LIF
The N-glycan structure of the heavy chains of the EMAB6 and EMAB603 antibodies was analysed using HPCE-LIF. The N-glycan structure of the heavy chain of Rituxan® was also analysed for comparison.
For that purpose, anti-CD20 monoclonal antibodies were desalted on a Sephadex G-25 column (HiTrap Desalting, Amersham Biosciences), evaporated and re-suspended in the hydrolysis buffer of PNGase F (Glyko) in the presence of 50 mM β-mercaptoethanol. After 16 hrs incubation at 37° C., the protein fraction was precipitated by adding absolute ethanol and the supernatant, which contained the N-glycans, was evaporated. The resulting oligosaccharides were either directly labelled using a fluorochrome: APTS (1-amino-pyrene-3,6,8-trisulphonate), or subjected to the action of specific exoglycosidases before labelling with APTS. The resulting labelled oligosaccharides were injected onto an N-CHO capillary, separated and quantified by capillary electrophoresis with laser-induced fluorescence detection (HPCE-LIF).
The estimation of the fucose level was carried out either by the addition of the isolated fucosylated forms, or more specifically after the simultaneous action of neuraminidase, β-galactosidase and N-acetylhexosaminidase, which resulted in 2 peaks corresponding to the fucosylated or non-fucosylated pentasaccharide [GlcNac2-Man3] being obtained on the electrophoretogram:







TABLE 1







Analysis of anti-CD20 EMAB603 and Rituxan ® N-glycans












Anti-CD20
% Fucose
% Galactose
Fuc/Gal
















EMAB603
15
37
0.4



Rituxan ®
93
57
1.63










The fucose level, expressed as %, was calculated using the following formula:




  
    Fucose
    ⁢
    
      
    
    ⁢
    level
  
  =
  
    
      
        fucosylated
        ⁢
        
          
        
        [
        
          GlcNac
          ⁢
          
            
          
          ⁢
          2
          ⁢
          
            -
          
          ⁢
          Man
          ⁢
          
            
          
          ⁢
          3
        
        ]
      
      ×
      100
    
    
      [
      
        
          GlcNac
          ⁢
          
            
          
          ⁢
          2
          ⁢
          
            -
          
          ⁢
          Man
          ⁢
          
            
          
          ⁢
          3
        
        +
        
          fucosylated
          ⁢
          
            
          
          ⁢
          GlcNac
          ⁢
          
            
          
          ⁢
          2
          ⁢
          
            -
          
          ⁢
          Man
          ⁢
          
            
          
          ⁢
          3
        
      
      ]
    
  




The galactose level, expressed as %, was calculated by adding the percentages of the oligosaccharide forms containing galactose obtained after the action of neuraminidase and fucosidase. The formula used is as follows:

Galactose level=(G1+G1B)+2x(G2+G2B)

The fucose/galactose ratio is obtained by dividing the fucose level by the galactose level, calculated as described above.
From this analysis (see Table 1), it appears that the EMAB6 and EMAB603 antibodies are little fucosylated (% fucose<25%) compared to Rituxan® (% fucose=93%). In addition, the Fuc/Gal ratio (fucose/galactose ratio) for EMAB6 and EMAB603 is low (Fuc/Gal ratio<0.6), unlike the antibodies expressed in CHO cells such as Rituxan® (Fuc/Gal ratio=1.63).

## Claims

1. A method for the treatment of a disease in which the target cells are cells which express CD20, which comprises administering to a patient an effective amount of a monoclonal antibody directed against the CD20 antigen, wherein each of the light chains thereof has the murine-human chimeric amino acid sequence SEQ ID No. 28, and each of the heavy chains thereof has the murine-human chimeric amino acid sequence SEQ ID No. 20.

2. The method of claim 1, wherein the disease in which the target cells are cells which express CD20 is an immune dysfunction disease involving B lymphoid cells.

3. The method of claim 2, wherein the immune dysfunction disease involving B lymphoid cells is selected from auto-immune diseases.

4. The method of claim 1, wherein the disease in which the target cells are cells which express CD20 is chronic graft-versus-host disease.

5. The method of claim 1, wherein the disease in which the target cells are cells which express CD20 is organ transplant rejection.

6. The method of claim 5, wherein the organ transplant rejection is kidney transplant rejection.

7. The method of claim 1, wherein each of the light chains thereof is encoded by murine-human chimeric nucleic acid sequence SEQ ID No. 27, and each of the heavy chains thereof is encoded by murine-human chimeric nucleic acid sequence SEQ ID No. 19.

8. The method of claim 1, wherein the antibody is produced by a rat hybridoma cell line.

9. The method of claim 8, wherein the antibody is produced in the rat hybridoma cell line YB2/3HL.P2.G11.16Ag.20, registered at the American Type Culture Collection under ATCC number CRL-1662.

10. The method of claim 9, wherein the antibody is the EMAB603 antibody produced by clone R603, registered under registration number CNCM I-3529 at the Collection Nationale de Cultures de Microorganismes (CNCM).

11. The method of claim 1, wherein the antibody is produced in a CHO line.

12. A method for the treatment of a disease in which the target cells are cells which express CD20, which comprises administering to a patient an effective amount of a monoclonal antibody directed against the CD20 antigen and one or more further antibodies, wherein each of the light chains of the monoclonal antibody directed against the CD20 antigen has the murine-human chimeric amino acid sequence SEQ ID No. 28, and each of the heavy chains of the monoclonal antibody directed against the CD20 antigen has the murine-human chimeric amino acid sequence SEQ ID No. 20.

13. The method of claim 12, wherein the disease in which the target cells are cells which express CD20 is an immune dysfunction disease involving B lymphoid cells.

14. The method of claim 13, wherein the immune dysfunction disease involving B lymphoid cells is selected from auto-immune diseases.

15. The method of claim 12, wherein the disease in which the target cells are cells which express CD20 is chronic graft-versus-host disease.

16. The method of claim 12, wherein the disease in which the target cells are cells which express CD20 is organ transplant rejection.

17. The method of claim 16, wherein the organ transplant rejection is kidney transplant rejection.