AU2012203957B2 - Anti-D monoclonal antibodies - Google Patents

Abstract

The invention concerns a method for obtaining and selecting monoclonal antibodies by an ADDC-type test, said antibodies capable of activating type III Fcy receptors and having a 5 particular glycan structure. The inventive anti-D antibodies can be used for preventing Rhesus isoimmunisation in Rh negative persons, in particular for haemolytic disease in a new-born baby or for uses such as idiopathic thrombocytopenic purpure (ITP).

Description

P/00/01i1 Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Anti-D monoclonal antibodies The following statement is a full description of this invention, including the best method of performing it known to us: la ANTI-D MONOCLONAL ANTIBODIES This application is a divisional application of Australian patent application no. 2010257417, which is a divisional of 2007202060, which is a divisional of 2001254858, the entire 5 disclosures of which are incorporated herein by reference. The present invention relates to a method for obtaining and selecting monoclonal antibodies using an assay of the ADCC type, said antibodies being capable of activating Fcy type III receptors. The invention is also directed toward monoclonal 10 antibodies having a particular glycan structure, the cells producing said antibodies, the methods for preparing the producer cells, and also the pharmaceutical compositions or the diagnostic tests comprising said antibodies. The anti-D antibodies according to the invention can be used for preventing Rhesus isoimmunisation 15 of Rh-negative individuals, in particular haemolytic disease of the newborn (HDN), or in applications such as Idiopathic Thrombocytopenic Purpura (ITP). Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that 20 this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art. Passive immunotherapy using polyclonal antibodies has been carried 25 out since the 1970s. However, the production of polyclonal immunoglobulins poses several problems: The immunisation of volunteers was discontinued in France in 1997 because of the ethical problems that such acts present. In France, as in Europe, the number of immunised donors is too low to 30 ensure a sufficient supply of certain antibodies, to such an lb extent that it provides necessary to import hyperimmunised plasma from the United States for example. Thus, the immunoglobulin shortage does not make it possible to envisage antenatal administration for preventing HDN. 5 Various studies have resulted in the production of human monoclonal antibodies for the purpose of replacing the polyclonal antibodies obtained from fractionating plasmas from voluntary donors. Monoclonal antibodies have several advantages: they can 10 -2 be obtained in large amounts at reasonable costs, each batch of antibodies is homogeneous and the quality of the various batches is reproducible since they are produced by the same cell line which is cryopreserved 5 in liquid nitrogen. It is possible to ensure the safety of the product with regard to an absence of viral contamination. Several publications describe the production of cell 10 lines producing human anti-Rh D monoclonal antibodies of IgG class, from B cells of immunized donors. Boylston et al. 1980; Koskimies 1980; Crawford et al. 1983; Doyle et al. 1985; Goossens et al. 1987; Kurnpel et al. 1989(a) and McCann-Carter et al. 1993 describe 15 the production of B lymphocyte lines transformed with the EBV virus. Melamed et al. 1985; Thompson et al. 1986 and McCann-Carter et al. 1993 relate to heterohybridomas resulting from B lymphocyte (trans formed with EBV) X murine myeloma fusion. Goossens 20 et al., 1987 relates to heterohybrids resulting from B lymphocyte (transformed with EBV) X human mveloma fusion. Bron et al., 1984 and Foung et al., 1987 describe heterohybrids resulting from B lymphocyte (transformed with EBV) X human-mouse heteromyeloma 25 fusion and, finally, Edelman et al., 1997 relates to insect cells transfected with the gene encoding an anti-Rh(D) using the baculovirus system. Among the patents and patent applications relating to 30 such monoclonal antibodies and the lines secreting them, mention may be made of: EP 576093 (AETS (FR), Biotest Pharma GmbH (Germany) Composition for prophylaxis of the haemolytic disease of the new-born comprises two human monoclonal 35 antibodies of sub-class IgG1 and IgG3, which are active against the Rhesus D antigen), RU 2094462, WO 85/02413 (Board of Trustees of the Leland Stanford Jr. University, Human Monoclonal Antibody against Rh (D) antigen and its uses) , GB 86-10106 (Central Blood - 3 Laboratories Authority, Production of heterohybridomas for manufacture of human monoclonal antibodies to Rhesus D antigen), EP 0 251 440 (Central Blood Laboratories Authority, Human Anti-Rhesus D Producing 5 Heterohybridomas), WO 89/02442, WO 89/02600 and WO 89/024443 (Central Blood Laboratories Authority, Human Anti-Ph (D) Monoclonal Antibodies), WO 8607740 (Institut Pasteur, Protein Performance SA, Paris, FR, Production of a recombinant monoclonal antibody from a 10 human anti-rhesus D monoclonal antibody, production thereof in insect cells and uses thereof), JP 88-50710 (International Reagents Corp., Japan, Reagents for Determination of Blood Group Substance Rh (D) Factor), JP 83-248865 (-Mitsubishi Chemical Industries Co., Ltd., 15 Japan, Preparation of Monoclonal Antibody to Rh (D) positive Antigen); CA 82-406033 (Queens University at Kingston, Human Monoclonal Antibodies) and GB 8226513 (University College London, Human Monoclonal Antibody against Rhesus D Antigen). 20 While the use of monoclonal antibodies has many advantages compared to the use of pools of polyclonal antibodies, it may, on the other hand, prove to be difficult to obtain an effective monoclonal antibody. 25 In fact, it has been found, in the context of the invention, that the Fcy fragment of the immunoglobulin obtained must have very particular properties in order to be able to interact with and activate the receptors of effector cells (macrophage, TH lymphocyte and NK) 30 The biological activity of certain G immunoglobulins is dependent on the structure of the oligosaccharides present on the molecule, and in particular on its Fc component. IgG molecules of all human and murine 35 subclasses have an N-oligosaccharide attached to the

CH

2 domain of each heavy chain (at residue Asn 297 for human IgGs). The influence of this glycan-containing residue on the ability of the antibody to interact with effector molecules (Fc receptors and complement) has - 4 been demonstrated. inhibiting glycosylation of a human IgG1, by culturing in the presence of tunicamycin, causes, for example, a 50-fold decrease in the affinity of this antibody for the FcyRI receptor present on 5 monocytes and macrophages (Leatherbarrow et al., 1985). Binding to the FcyRIII receptor is also affected by the loss of carbohydrates on IgG, since it has been described that a nonglycosylated IgG3 is incapable of inducing iysis of the ADCC type via the FcyRIII 10 receptor of NK cells (Lund et al., 1990). However, beyond the necessary presence of these glycan containing residues, it is more precisely the heterogeneity of their structure which may result in 15 differences in the ability to initiate effector functions. Galactosylation profiles which are variable depending on individuals (human serum IgGls) have been observed. These differences probably reflect differences in the activity of galactosyltransferases 20 and other enzymes between the cellular clones of these individuals (Jefferis et al., 1990). Although this normal heterogeneity of post-translational processes generates various glycoforms (even in the case of monoclonal antibodies) , it may lead to atypical 25 structures associated with certain pathological conditions, such as rheumatoid arthritis or Crohn's disease, for which a considerable proportion of agalactosylated residues have been demonstrated (Parekh et al., 1985). 30 The glycosylation profile of the purified molecule is the consequence of multiple effects, some parameters of which have already been studied. The protein backbone of IgGs, and in particular amino acids in contact with the terminal N-acetylglucosamine (GlcNAc) and galactose 35 residues of the mannose o-l,06 arm (aa 246 and 258 of IgGs), may explain the existence of preferential structures (galactosylation), as shown in the study carried out on murine and chimeric Igc-s of different isotypes (Lund et al., 1993).

- 5 The differences observed also reveal specificities related to the species and to the cell type used for producing the molecule. Thus, the conventional 5 structure of the N-glycans of human IgGs reveals a significant proportion of bi-antennary types with a GlcNAc residue in the bisecting position, this being a structure which is absent in antibodies produced by murine cells. Similarly, the sialic acid residues 10 synthesized by the CHO (Chinese Hamster Ovary) line are exclusively of the a-2,3 type, whereas they are of the ax-2,3 and (X-2,6 type with murine and human cells (Yu Ip et al., 1994) . Immunoglobulin production in expression systems other than those derived from mammals may 15 introduce much more important modifications, such as the presence of xylose residues produced by insect cells or plants (Ma et al., 1995). Other factors, such as the cell culture conditions 20 (including the composition of the culture medium, the cell density, the pH, the oxygenation), appear to have an effect on glycosyltransferase activity in the cell and, consequently, on the glycan structure of the molecule (Monica et al., 1993; Kumpel et al., 1994 b). 25 Now, in the context of the present invention, it has been found that a structure of the bi-antennary type, with short chains, a low degree of sialylation, and nonintercalated terminal mannoses and/or terminal 30 GlcNAcs, is the common denominator for glycan structures which confer strong ADCC activity on monoclonal antibodies. A method for preparing such antibodies capable of activating effector cells via EcyRIII, in particular anti-Rh(D) antibodies, has also 35 been developed. Blood group antigens are classified in several systems depending on the nature of the membrane-bound molecules expressed at the surface of red blood cells. The Rhesus - 6 (Rh) system comprises 5 molecules or antigens: D, C, c, E and e (ISSITT, 1988) The D antigen is the most important of these molecules because it is the most immunogenic, i.e. it can induce the production of 5 anti-D antibodies if Rh-D-positive red blood cells are transfused into Rh-negative individuals. The D antigen is normally expressed in 85% of Caucasian individuals, these people are termed "Rh--positive"; 25% 10 of these individuals are therefore Rh-negative, i.e. their red blood cells do not exhibit any D antigen. D antigen expression exhibits certain variants which may be linked either to a weak antigenic density, reference is then made to weak D antigens, or to a 15 different or partial antigenicity, reference is then made to partial D antigens. The weak D characteristic is characterized in that it is a normal antigen, but the number of sites thereof per red blood cell is decreased more or less considerably; this characteris 20 tic is transmissible according to Mendelian laws. Partial D phenotypes have been discovered in Rh-D positive individuals who have anti-D serum antibodies; these partial D antigens can therefore be characterized as having only part of the mosaic. Studies carried out 25 with polyclonal and monoclonal antibodies have made it possible to define 7 categories of partial D antigens with at least 8 epitopes constituting the D antigen being described (LOMAS et al., 1989; TIPETT 1988). 30 The importance of anti-Rh D antibodies became apparent with the discovery of the mechanisms leading to hemolytic disease of the newborn (HDN) . This corres ponds to the various pathological conditions observed in some fetuses or in some newborn babies when there is 35 a feto-maternal blood group incompatibility which is responsible for the formation of maternal anti-Rh D antibodies capable of crossing the placental barrier. In fact, fetal Rh-positive red blood cells passing into an Rh-negative mother can lead to the formation of -7 anti-D antibodies. After immunization of the Rh-negative mother, the IgG class anti-D antibodies are capable of crossing the 5 placental barrier and of binding to the fetal Rh positive red blood cells. This binding leads to the activation of immunocompetent cells via their surface Fc receptors, thus inducing hemolysis of the sensitized fetal red blood cells. Depending on the strength of the 10 reaction, several degrees of seriousness of HDN can be observed. An HDN diagnosis can be carried out before and after birth. Prenatal diagnosis is based on the development 15 of the anti-D antibody level in the mother using several immunohematological techniques. Post-partum diagnosis may be carried out using an umbilical cord blood sample, analyzing the following parameters: determining the blood groups of the fetus and of the 20 father; searching for anti-D antibodies; assaying the hemoglobin and the bilirubin. Prophylactic treatment for HDN is currently systematic ally given to all women with an Rh-negative blood group 25 who have given birth to an Rh-positive child, with injections of human anti-D immunoglobulin. The first real immunoprophylaxis trials began in 1964. For the prevention to be effective, the immunoglobulin must be injected before the immunization, i.e. within the 30 72 hours following the birth, and the antibody doses must be sufficient (10 gg of anti-D antibodies per 0.5 ml of Rh+ red blood cells). Several anti-D monoclonal antibodies have been the 35 subject of therapeutic assessment: BROSSARD/FNTS 1990 (not published); THOMSON/IBGRL 1990; KUMPEL/IBGRL 1994; BELKINA/Institute of hematology, Moscow, 1996; BIOTEST/LFB 1997 (not published). The clinical effectiveness of the antibodies in inducing clearance 8 of RH(D)-positive red blood cells was assessed in RH(D) -negative volunteers. A single IgGl antibody showed an effectiveness equivalent to that of anti-D polyclonal immunoglobulins, but only in some 5 patients (KUMPEL et al., 1995). The invention proposes to provide monoclonal antibodies which reply to the abovementioned problems, i.e. antibodies selected using an assay of the ADCC type specific for the antibody and/or the antibodies having a glycan structure required for obtaining [0 good effectiveness. Description As used herein, except where the context requires otherwise the term 'comprise' and variations of the term, such as 'comprising', 'comprises' and 'comprised', are not intended to [5 exclude other additives, components, integers or steps. The present invention relates to a method for preparing a monoclonal antibody capable of activating effector cells expressing FcyRIII, characterised in that it comprises the following steps: 20 a) purifying monoclonal antibodies obtained from various clones originating from cell lines selected from hybridomas, in particular heterhybridomas, and animal or human cell lines transfected with a vector comprising the gene encoding said antibody; 25 b) adding each antibody obtained in step a) to a different reaction mixture comprising: - the target cells for said antibodies, - effector cells comprising cells expressing the FCyRIII, 8a - polyvalent IgGs; c) determining the percentage lysis of the target cells and selecting the monoclonal antibodies which activate the effector cells causing significant lysis of the target cells 5 (FCyRIII-type ADCC activity) . The clones may originate from heterohybrid cell lines obtained by fusion of human B lymphocytes (originating from immunised individuals) with murine, human or ~0 heterohybrid myeloma cells, in particular the K6H6-B5 myeloma (ATCC No. CRL 1823); or else from animal or human cell lines transfected with a vector containing the gene encoding a human IgG immunoglobulin, said 5 lines possibly being selected in particular from the CHO-K, CHO-LeclO, CHO Lec-1, CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, YB2/0, BHK, K6H6, NSO, SP2/0-Ag 14 and P3X63Ag8.653 lines. 10 The polyvalent IgGs are used to inhibit the mechanism of lysis of the effector cells via FcyRIII. In this method, the antibodies which exhibit an FcyRIII-type ADCC level greater than 60%, 70%, 80%, or preferably greater than 90%, are selected. 15 The target cells can be red blood cells treated with papain. In this case, the following are deposited per well: - 100 l of purified monoclonal antibodies at approximately 200 ng/ml, 20 - 25 gl of papain-treated red blood cells, i.e. approximately 1 x 106 cells, - 25 gl of effector cells, i.e. approximately 2 x 106 cells, - and 50 gl of polyvalent IgGs, in particular of 25 TEGELINE Th (LFB, France), at a concentration of between 1 and 20 mg/ml. It is thus possible to compare the amount of target cell lysis to two positive controls consisting of a chemical compound such as NH6Cl and a reference 30 antibody active in vivo, and to a negative control consisting of an antibody inactive in vivo. It is also possible to use polyclonal antibodies of commercial origin as positive controls and a monoclonal 35 antibody incapable of inducing clearance in vivo as a negative control. Advantageously, this method makes it possible to prepare anti-Ph(D) monoclonal antibodies as indicated - 10 above. Rhesus D red blood cells are then used as target cells. The invention is therefore based on developing an assay 5 for biological activity in vitro, in which the activities measured correlate with the biological activity in vivo of the monoclonal or polyclonal antibodies already evaluated from the clinical point of view with regard to their potentiality in inducing 10 clearance of Rh(D)-positive red blood cells in Rh(D) negative volunteers. This assay makes it possible to evaluate the antibody-dependent lytic activity = ADCC (antibody-dependent cellular cytotoxicity) induced essentially by the Fcy type III receptors (CD16) , the 15 Fcy type I receptors (CD61) being saturated by the addition of human IgG immunoglobulins (in the form of therapeutic polyvalent IgGs) . The FcyRIII specificity of this ADCC assay was confirmed by inhibition in the presence of an anti-FcyRIII monoclonal antibody (see 20 figure 6) . Mononuclear cells from healthy individuals are used as effector cells in an effector/target (E/T) ratio close to physiological conditions in vivo. Under these conditions, the lytic activities of the poly clonal immunoglobulins and of the anti-D monoclonal 25 antibodies ineffective in vivo (antibody DF5, Goossens et al., 1987, and the antibodies AD1+AD3, FR 92/07893 LFB/Biotest and FOG-1, GB 2189506) are, respectively, strong and weak. 30 The selection of the antibodies described in the present invention was therefore carried out by evaluating their biological activity in this ADCC-type assay (see example 1). 35 In another aspect, the invention relates to the anti bodies which can be obtained using the method described above, said antibodies exhibiting FcyRIII-type ADCC levels greater than 60%, 70%, 80%, or preferably greater than 90%, relative to the reference polyclonal.

11 The monoclonal antibodies of the invention, directed against a given antigen, activate effector cells expressing FCyRIII, causing lysis greater than 60%, 70%, 80%, preferably greater than 90%, of the lysis caused by polyclonal antibodies directed against said 5 antigen. Advantageously, said monoclonal antibodies are directed against rhesus D. They may preferably be produced by clones derived from the Vero (ATCC No. CCL 81), YB2/0 (ATCC No. CRL 1662) or CHO Lec-1 (ATCC No. CRL 1735) lines and may belong to the IgG1 or IgG3 class. 10 The invention also relates to antibodies which have a particular glycan structure conferring FCyRIII-dependent effector activity. Such antibodies can be obtained using a method explained above and have, on their FCy glycosylation site (Asn 297), glycan structures of the bi-antennary type, with short chains and a low 15 degree of sialylaton. Preferably, their glycan structure exhibits nonintercalated terminal mannoses and/or terminal GlcNAcs. Such antibodies are more particularly selected from the forms: ~~X-W GO GOF GI GlF GlcNAc Mannose Galactose Fucose 11a Thus, the invention is directed toward a monoclonal antibody characterised in that it has, on its FCy glycosylation site (Asn 297), glycan structures of the bi-antennary type, with short chains, a low degree of sialylation, and nonintercalated mannoses 5 and G1cNAcs - 12 with a terminal point of attachment. Said antibodies, directed against a given antigen, activate effector cells expressing FcyRIII, causing lysis greater than 60%, 70%, 80%, preferably greater than 90%, of the 5 lysis caused by polyclonal antibodies directed against said antigen. More particularly, the invention relates to antibodies and compositions comprising said antibodies as defined 10 above, in which the sialic acid content is less than 25%, 20%, 15% or 10%, preferably 5%, 4%, 3% or 2%. Similarly, the invention relates to antibodies and com positions comprising said antibodies as defined above, 15 in which the fucose content is less than 65%, 60%, 50%, 40% or 30%. Preferably, the fucose content is between 20% and 45%, or else between 25% and 40%. A particularly effective composition according to the 20 invention comprises, for example, a content greater than 60%, preferably greater than 80%, for the GO + G1 + GOF + GlF forms, it being understood that the GOF + GlF forms are less than 50%, preferably less than 30%. 25 - 13 Table 1: Quantification (%) of the oligosaccharide structures of the various anti-RhD antibodies Antibodies active by FcRyIII ADCC Antibodies inactive by ?cRyIT ADCC R297 R270 F60 D31 F5 Struc- HPCE- HPCE- HPLCs HPCE- HPLCs HPCE- HPCE- HPLCs ture LIF LIF LF LIF LIF Fucos- 34.3 45.9 37.2 47.7 46.6 Sial- 1.0 2.2 4.1 9.6 ylated 19.6 G2S2FB -2-8 G2S2F 0.0 0.0 n.d. 4.2 0.0 11.3 11.9 4.1 G2S1FB 1 6.1 G2S1F 1.0 1.0 n.d. 2.7 2.5 21.4 30.5 28 G2S1 0.0- 1.2 n.d. 3.0 0.0 0 0 GS 1 B 6.2 G1F 1.7 G2F 3.9 5.0 3.0 10.3 11.6 16.9 22.1 4.2 G2 12.1 6.1 3.3 7.0 13.3 2.0 0.0 0.0 G1FB 25. 62, 1 N2fl 2 16.1 21.5 12.4 0.0 0.0 0.0. L1.7 3.0 0.0. .9 0.5 An alternative for specifically targeting FcyRIII 5 consists in preparing antibodies of the "high mannose" type. In another aspect, the invention relates to a cell producing an antibody mentioned above. It may be a 10 hybridoma, in particular a heterohybridoma obtained with the fusion partner K6H6-B5 (ATCC No. CRL 1823); or an animal or human cell transfected with a vector comprising the gene encoding said antibody, in particular a cell derived from the Vero (ATCC No. 15 CCL 81), YB2/0 (ATCC No. CRL 1662) or CHO Lec-1 - 14 (ATCC No. CRL 1735) lines. These cells correspond to the cell lines selected using the method according to the invention, said cells producing antibodies which have the characteristics mentioned above. 5 A preferred antibody according to the invention shows considerable biological activity (greater than or equal to that of the anti-Rh(D) reference polyclonal anti body) in the ADCC assay using FcYRTIT-positive effector 10 cells. Its ability to activate FcyRIII receptors (after binding) is confirmed on in vitro models which demonstrate modification of intracellular calcium flux, phosphorylation of activation signal transduction 15 molecules, or release of chemical mediators. These properties are associated with a particular structure of the oligosaccharides of the N-glycosylation site of the Fc component of the antibody: presence of short chains, low degree of 20 galactosylation, little sialylation, may have non intercalated terminal mannoses and/or terminal GlcNAcs, for example. This antibody has therapeutic applications: prevention of HDN, treatment of ITP in Rh(D)-positive individuals, 25 and any other application to which the use of anti-D polyclonal inmmunoglobulins relates. A preferred antibody according to the invention may also have a specificity other than anti-Rh(D) (anti cancer cell for example) . It may have the properties 30 described above (functional activity dependent on a mechanism of binding to/activation of FcyRII receptors, particular structure of oligosaccharides) and may be used in immunotherapy for cancers or for any other pathological condition for which a curative or 35 peventive treatment rm-ay be carried out using a monoclonal antibody the mechanism of action of which corresponds to an activity which is functional via t-he ?cyRITI receptor.

- 15 Another aspect relates to a pharmaceutical composition comprising an antibody according to the invention and to the use of said antibody for producing a medicinal product. The invention also relates to a pharmaceutical composition s comprising monoclonal antibodies having, on the Fcy glycosylation site (Asn 297), glycan structures of the bi-antennary type, with short chains and a low degree of sialylation, and nonintercalated terminal mannoses and/or terminal N acetylglucosamines, wherein the glycan structures of the io antibodies in the composition have a content in forms GO+Gl+GOF+G1F greater than 60% and a fucose content less than 65%. The invention further relates to a pharmaceutical composition comprising monoclonal antibodies having, on the Fcy glycosylation 15 site (Asn 297), glycan structures of the bi-antennary type, with short chains and a low degree of sialylation, and nonintercalated terminal mannoses and/or terminal N acetylglucosamines, wherein the fucose content of the glycan structures of the antibodies in the composition is less than 20 30%. Preferably, the invention relates to the use of an anti-Rh(D) antibody described above, for producing a medicinal product intended for the prevention of Rhesus alloimmunization of Rh negative individuals. The method of action of the anti-D 25 immunoglobulins in vivo is specific binding of the antibodies to the D antigen of the Rh(D) -positive red blood cells, followed by elimination of these red blood cells from the circulation essentially in the spleen. This clearance is associated with a dynamic mechanism of suppression of primary immune response in 30 the individual, and therefore prevents the immunization.

- 16 Thus, an antibody of the invention may be used prophylactically for preventing alloimmunization of Rhesus-negative women immediately after the birth of a Rhesus-positive child, and or preventing, at the time of subsequent pregnancies, hemolytic 5 disease of the newborn (HDN); at the time of abortions or of extra-uterine pregnancies in a situation of Rhesus D incompatibility or else at the time of transplacental hemorrhages resulting from amniocentesis, from chorionic biopsies or from traumatic obstetric manipulations in a io situation of Rhesus D incompabitibility. In addition, an antibody of the invention may be used in the case of Rh-incompatible transfusions with blood or labile blood derivatives. The invention also relates to the use of an antibody of the is invention for producing a medicinal product intended for therapeutic use in Idiopathic Thrombocytopenic Purpura (ITP). The antibodies of the invention are also of use for producing a medicinal product intended for the - 17 treatment of cancers by immunotherapy or f or the treatment of infections caused by viral or bacterial pathogenic agents. 5 An additional aspect of the invention relates to the use of said antibodies in particular for diagnosis. The invention is therefore directed toward a kit comprising an antibody described above. 10 For the remainder of the descrition. refe c i made to the legends of the figures presented below. Legends 15 Figure 1: ADCC evaluation of F60 and T125 YB2/0 (R270) This figure represents the percentage lysis obtained as a function of the antibody concentration in the presence of 100 and 500 gg/well of TEGELINEM (LFB, France). A high percentage lysis is obtained for the 20 antibodies according to the invention F60 and T125. Figure 2: Anti-D binding to the receptor (FcyRIII) A high binding index is obtained for the antibodies according to the invention F60 and T125. Figure 3: Construction of the expression vector T125 25 H26 for expressing the H chain of T125. Figure 4: Construction of the expression vector T125~ K47 for expressing the L chain of T125 Figure 5: Construction of the expressiOn vector T12S IG24 for expressing the whole antibody T125 30 Figure 6: ADCC inhibition in the presence of anti PcRIIIT (CD016) The ADCC assay is established according to the oroceoure described in 53.3 in the presence of the comercial anti-CD16 3GB (TEBU) , the action of which is 35 to block the BFcRI1 receptors present on the ef:ector cells. The final concentration cf 3C8 is 5 gg/-weil (25 pg/ml) . A control is carried out in parallel in the absence of 3G8. Th n re andies t!j-- Le s t ed ar PoyD Win7 ho, the - 18 antLbody F60 (Pt L55. 99/47) obtained according to the method described in example I, and R297 (Pf 210 01/76) obtained according to the method described in example II. 5 Results: an inhibition is observed in the presence of 3G8, which demonstrates that the ADCC induced by the three antibodies tested is mainly FcRIII-dependent. A slightly stronger inhibition is observed in the presence of Poly-D WinRho (83% compared to 68% and 61% 10 inhibition for F60 and R297, respectively). This difference may be due to the presence, in the Poly-D, of non-anti-D hurran IgGs which will inhibit type I receptors (FCRI or CD64) and therefore act synergistically with the anti-CD16. 15 Figure 7: Characterization of the anti-D glycans by mass spectrometry (MS) Figure 8: Comparison of the MS spectra for R290 and DF5 Figure 9: Study of the glycosylation of the anti-D 20 D31DMM by MS EXA2vPLE 1: ESTABLISHING A HETEROHYBRID CELL LINE PRODUCING AN ANTI-RH(D) ANTIBODY 25 1- Production of lymphoblastoid and heterohybrid clones: 2.2 -Lymaphocybe source : The B lymphocyte donor is selected from anti-Rh(D) donors undergoing plasmapheresis, based on the activity 30 of his or her anti-Rh(D) serum antibodies in the ADCC activity assay described in §33. After a whole blood donation, in 1998, the "buffy coat" fraction (leukocyte concentrate) was recovered. s L - -C. fmora2ation ei ih3 2 lymehocytes r he dorcor The peripheral blood mononuclear cells are seoaratced -rom the other elements by centrifugation on Ficoll Plus (Pharmacia) . They are then diluted to 100 cell /ml -7tal o-ncin.no 20 v~i c, -.. I se ' PC \ - 19 co which 20% of culture supernatant of the B95.-8 line (ATCC-CRL1612), 0.1 99/M Of cyclosoorin A (Sandoz), 50 gg/ml of gentamvcin sulfate (Life Technologies) are added, and distributed into round-bottomed 96-well 5 plates or 24-well plates (P24 Greiner) . They are then placed in an incubator at 37 0 C, 7% CO 2 . After 3 weeks, the presence of anti-Ph(D) antibodies is sought by ADCC. A Each one of the 16 microwells of a positive P24 10 plate well is transferred into . new PA 4 a e enrichment is repeated after 10 to 15 days of culturing and each microwell is amplified in a P96 and then in a P24. A The positive P96 wells are taken up and 15 amplified in a flat-bottomed P24 (Nunc) . After a few days of culturing, the presence of anti-Rh(D) antibodies is sought by ADCC. 1.3-Enrichment by immunorosetting (.rR) 20 The cells derived from one or more P24 wells are enriched in specific cells by the formation and separation of rosettes with papain-treated Rh(D) positive red blood cells: one volume of red blood cells washed in 0.9% NaCl is incubated for 10 minutes at 37 0 C 25 with 1 volume of papain (Merck) solution at 1/1 000th (m/v), and then washed 3 times in 0.9% NaCl. The cells were then washed once in Hanks solution, suspended in FCS and mixed with the papain-treated red blood cells in a radio of 1 cell to 33 red blood cells. The mixture 30 is placed in a cone-bottomed centrifuged tube, centrifuged for 5 minutes at 80 g and incubated for one hour in melting ice. The mixture is then carefully agitated and Ficoll s deposited at the bottom of the tube for separation at 900 gfo- 20 minutes. The p.ile, 35 containing the rosettes is hemolyzed in a solut ion of NSC for 5 minutes and the cells are placed in culture again in a P24 containing irradiated human itononucl cells. After approximately I eek. he supernatants are e.valuated in CELA (paragrp--h 3.2) and ADCC as -20 che presence of anzi-Rh(D) antibodies having good acivity- A further cycle of enrichment is carried out if the percentage of cells forming rosetes significan -y increases compared to the preceding 5 cycle. .L4-Cloning of the lymphoblastoid cells: The IR-enriched cells are distributed at 5 and 0.5 cells per well in round-bottomed 96-well plates 10 containing irradiated human monoler cls After approximately 4 weeks of culturing, the super natants from the wells containing cell aggregates are evaluated by ADCC assay. 15 1.5-Heterofusion: The wells from cloning the EBV-transformed cells exhibiting an advantageous ADCC activity are amplified in culture and then fused with the heteromyeloma K6H6-B5 (ATCC CRL-1823) according to the standard PEG 20 technique. After fusion, the cells are distributed, in a proportion of 2 X 10- cells/well, into flat-bottomed P96s containing murine intraperitoneal macrophages and in a selective medium containing aminopterin and ouabain (Sigma). 25 After 3 to 4 weeks of culturing, the supernatants of the wells containing cell aggregates are evaluated by ADCC assay. 1.-Conngof the hetehybrdomas: 30 Cloning by limiting dilution is carried out at 4, 2 and 1 cell/well in flat-bottomed P96s After 2 weeks, the microscopi c appearance of the wells is examined in order to identify' the single clones. and the medium is then renewed. After approximately 2 weeks, the super 35 natants of the wells containing cell aggregates are evaluated by ADCC assay.

-21 2- H-istory of the clones selected 2,1-Clone producing an IgG1 EBV transformation of the. cells of donor dl3 madeic possible to select a well, designated T125 2A2, on which the following were successively carried out: 2 enrichments, 3 cycles of IR, and cloning at : cells/ well to give 2 clones: 1) T125 2A2 (5/l)A2 from which the DNA was extracted in 10 order to prepare the recombinant vector; 2) T125 (5/1)A2 which was fused with K6H6-B5 to give F60 2F6 and then, after 5 rounds of cloning, F60 2F6 (5) 4C4, a clone selected for constituting a cell stock prior to preparing libraries. 15 It is an IgG1 possessing a Kappa light chain Donor cells EBV transformation 20 T125 2A2 t Enrichment in P24 T125 2A2 (2)E6 3 cycles of immunorosetting 25 T125 2A2 (2)E6 RI(3) 2 round 0 T 1 round of cloning cloning T 125 2A2 (5/1)A2 T125 2A2 (5/1)IC5 30 DNA recombinant vector 60 2F6 5 rounds of F60 2F6 (5)4C4 cloning 2,2-Clone proUCing an IgG3 35 A line producing an ToG3 was prepared according to the same method as that used to prepare the antibody of IG isotype. The cells of origin origiat from a donation of whoe blood. from a designated donor, from whch the "buffy coat" fraction (leukocyte concentrate) A4') Cas recovered. 1;. sanIg3posssngaKap lgh han -22 Donor cells E.BV transformation T151 4HI1 2 cycles of irmunorose t ting T151 4-HI'RI (2) Fusion (x K6HC/R5) F41 1Dil 10 5 rounds of cloning 3- Methods for evaluating the anti-Rh(D) antibodies: 15 After purification by affinity chromatography on protein A sepharose (Pharmacia) and dialysis in 25 mM Tris buffer, 150 mIM NaCl, pH 7.4, the concentration of the antibody T125 is determined by the ELISA technique. The biological activity in vitro is then measured by 20 the ADCC technique. 3.1-Deterination of the igG level and of the isotypes by the ELISA technique: 0 Total IgGs 25 Coating: anti-IgG (Calbiochem) at 2 gg/ml in 0.05M carbonate buffer, pH 9.5, overnight at 4 0 C. Saturation: dilution buffer (PBS + 1% BSA + 0.05% Tween 20, pH 7.2), 1 h at ambient temperature. Washing (to be renewed at each step) : H2O + 150 mM NaCI + 0 -05% 30 -Tween 20. Dilution of the samples, in dilution buff to approximately 100 ng/ml and of the control range made up of LFB poly'valent human igGs prediluted tc. 100 ng/mi. Incubation for 2 h at ambient :emperature. Conjugate: anti-IgG (Diagnostic Pasteur) diuted to 3, 1/5 000, 2 hours at ambient temperature. Substrate; OPD at 0.J mg/ml (Sigma) in phosphate-citrate butfer containing sodium perborate (Sigma) , 10 minutes in the dark. Reacton stopped with IN HCl, a read a- /923 a AssayingOf Kpa hi - 23 Coatirg: arti.-Kappa (Caltag Lab) at 5 gg/ml in 0.05M carbonate buffer, pH 9.5, overnight at 4 0 C. Saturation: dlution buffer (PBS + 1% BSA + 0.05% Tween 20, pH '7.2), 1 h at ambient temperature. The washing (to be 5 renewed at each step): H 2 0 + 150 m-M NaCi + 0.05% Tween 20. Dilution of the samples, in dilution buffer, to approximately 100 ng/ml and of the control range made up of the LFB monoclonal antibody AD3Tl (Kappa/ gamma 3) prediluted to 100 ng/ml. Incubation for 2 h at 10 ambient temperature. Conjugate: biotinylated anti-Kappa (Pierce) diluted to 1/1 000 in the presence of streptavidin-peroxidase (Pierce) diluted to 1/1 500, 2 hours at ambient temperature. Substrate: OPD at 0.5 mg/ml (sigma) in phosphate-citrate buffer contain 15 ing sodium perborate (Sigma), 10 minutes in the dark. The reaction is stopped with 1N HCl, and read at 492 nm. 3.2-Specific assaying of anti-D by the CELA (Cellular 20 Enzyme Linked Assay) technique: This method is used for specifically assaying the anti-D antibodies in particular when this involves a culture supernatant at culturing stages at which other non-anti-D imrmunoglobulins are present in the solution 25 (early stages after EBV transformation). Principle: The anti-D antibody is incubated with Rhesus-positive red blood cells and then revealed with an alkaline phosphatase-labeled anti-human ig. 100 ul of R~h+ red blood cells at 10% diluted in Liss-l% 30 BSA dilution buffer. Dilution of the samples. in dilution buffer, to approximately 500 ng/ml and of the control range made up of a purified ronoclonal human anti-D IgG (DF5, LFB) prediluted to 500 ng/mi. Incubation for 45 i at ambient temperature. Washing 35 (to be renewed al. each step) : H20 + 150 nii NaCl . Conjugate: anti-IgG alkaline phosphatase (Jackson) diluted to 1/ 000 in PBS + 1% BSA, 1 h 30 at a.Obien temperature .Substrate: PNPP at 1. mg/ml (sigma) in diethanclamin? . t l gtlf pH 9.8. - rco- -24 stooed with 1N NaGH, and read at 405 nm. 3.3-ADCC technique The ADCC (ancibodv-dependent. cellular cVtotoxicitV) 5 cechique makes it possible to evaluate the ability of the (anti-D) antibodies to induce lysis of Rh-positive red blood cells, in the presence of effector cells (mononuclear cells or lymphocytes). Briefly, the red blood cells of an Rh-positive cell 10 concentrate are treated it paprain (I mmrnl 10 mi a: 37 0 C) and then washed in 0.9% NaCl. The effector cells are isolated from a pool of at least 3 buffy-coats, by centrifugation on Ficoll (Pharmacia), followed by a step of adhesion in the presence of 25% FCS, so as to 15 obtain a lymphocyte/monocyte ratio of the order of 9. The following are deposited, per well, into a microtitration plate (96 well): 100 A! of purified anti-D antibody at 200 ng/mi, 25 ul of Rh+ papain treated red blood cells (i.e. 1 x 106), 25 gl of 20 effector cells (i.e. 2 x 106) and 50 gl of polyvalent IgG (Tegeline, LFB, for example) at the usual concentrations of 10 and 2 mg/ml. The dilutions are made in IMDM containing 0.25% FCS. After overnight incubation at 37 0 C, the plates are centrifuged, and the 25 .hemoglobin released into the supernatant is then measured in the presence of a substrate specific for peroxidase activity (2,7-diaminofluorene, DAF). The results are expressed as percentage lysis, 100% corresponding co otal red blood cell lysis in NH4C1 30 (100% control) . and 0% to the reaction mixture without. antibody (0% control). Thle specific lsis is calculated as a percentage according to the following formula: (D samle -- ( 0% control ) x 100 3 % kDCC OD 100% control - OD C% control The results given in figure I show the activity of antibodypoucd by the heerybri cop -25 chose of be reference ancibodies: -e anti-Rh(D) oolvclonal antibodies POLY-D LFB 51 and WinRho W03 (Cangene) = positive controls - the monoclonal antibody DF5 (inactive in vivo on 5 clearance of Ph(D)-positive red blood cells (BROSSAPD/FNTS, 1990, not published)) negative control - the IgGis purified (separated from the IgG3s) from the polyclonal WinRhO W03. 10 Two concentrations of human IgGs (Tegeln) TiPP. r used to show that inhibition of activity of the negative control is linked to the binding of competing IgGs to the Fcy type I receptors. 15 3.4-FcYRIII(CD16) -binding technique: This assay makes it possible to assess the binding of the anti-Rh(D) antibodies of IgGl isotype to FcyRIII, and in particular to differentiate IgG3 antibodies. Given the low affinity of this receptor for monomeric 20 IgGs, prior binding of the antibodies to the D antigen is necessary. Principle: The antibody to be tested (anti-D) is added to membranes of Rh+ red blood cells coated with- a microtitration plate, followed by transfected Jurkat 25 cells expressing the FcyRIII receptor at their surface. After centrifugation, the "Rh+ membrane/anti-D/CD16 Jurkat" interaction is visualized by a homogeneous plating of the CD16 Jurkats in the well. In the absence of interaction, the cells are, on the contrary, grouped 30 at the center of the well. The intensity of the reaction is expressed as numbers of +. Method: 1) Incubation for i h at 37 0 C of the anti-D antibody (50 gl at 1 p.g/rml in IMDM) on a Capture R plate (Immunochim), and then washes in wate + 0 .9% 35 NaCl.Additionf CD16 Jurkat (2 x 100 cells/mI) i % FCS. Incubation for 20 mi at 37 0 C and then cenrifugatiol and 2valuation of cell adhesion (against a conrol range) 2) vl o o t _ D to h cap-,-- .

-26 plates by an ELISAk.-type technique using andi-human IgC peroxidase at 1/5 000 (Sanofi Diagnostics Pasteur) after having lysed the CD16 Jurkat cells with C .2m Tris-HCl, 6M urea, pH 5.3-5.5. OPD revelation and then 5 reading of optical density (0.D.) at 492 nm. Expression of results: an arbitrary value of 0 to 3 is allotted as a function of the binding and of the plating of the CD16 Jurkat cells. These values are allotted at each OD interval defined (increments of 10 0.1). The following are plotted: * either a curve: adhesion of the Jurkat cells (Y) as a function of the amount of anti-D bound to the red blood cell membranes x). * or a histogram of the "binding indices" corres 15 ponding, for each antibody, to the sum of each Jurkat cell binding value (0 to 3) allotted per OD interval (over a portion common to all the antibodies tested). An example of a histogram is given in figure 2. The anti-Rh(D) antibodies of IgG1 isotype (F60 and T125 20 YB2/0) show a binding index close to that of the polyclonal IgGls (WinRho) , whereas the negative control antibodies DF5 and AD1 do not bind. Similarly, the antibody of IgG3 isotype (F41) exhibits a good binding index, slightly less than that of the IgG3s purified 25 from the polyclonal Winrho and greater than that of the antibody AD3 (other IgG3 tested and ineffective in clinical trial, in a mixture with AD1 (Biotest/LFB, 1997, not published). 30 Example 2: PRODUCTION OF A RECOMBINAT ANT'-D A1TLIODY (Ab) 1 solution and amplification of the cDNAs encoing' e hea d lght chAins of the The total RNAs were extracted. from an anti-D Ab p-roducing clone (IgG 1/Kappa) obtaIned by EV transtformationi: 1125 A2 (/ k2 (see paragraph 2 -27 exanole 1). The corresponding cDNAs were synthesized by reverse transcription of the total RNAs using oligo dT orimers. 5 1.2-Amplification of the variable region of the heavy chain of T125-A2: V9/T125-A2 sequence The VH/T125-A2 sequence is obtained by amplification of the T125-A2 cDNAs using the following primers: - primer A2VH5, located 5' of the leader region of the 10 VH gene of T -A2 introduces consensus leader sequence (in bold) deduced from leader sequences already published and associated with VH genes belonging to the same VH3-30 family as the VH gene of T125-A2; this sequence also comprises an Eco RI 15 restriction site (in italics) and a Kozak sequence (underlined): A2VH5 (SEQ ID No. 1): 5'-CTCTCCGAATTCGCCGCCACCATGGAGTTTGGGCTGAGCTGGGT-3' - antisense primer GSP2ANP, located 5' of the constant 20 region (CH) of T125-A2: GSP2ANP (SEQ ID No. 2): 5'-GGAAGTAGTCCTTGACCACGCAG-3'. 1.3-Amplification of the constant region of T125-A2: CH/T25-A2 sequence 25 The CH/T125-A2 sequence is obtained by amplification of the T125-A2 cDNAs using the following primers: - primer G1, located 5' of the CH region of T125-A2: Gl (SEQ ID No. 3): 5'-CCCTCCACCAAGGG CCCATCGGTC-3' The first G base of the CH sequence is here 30 replaced with a C (underlined) in order to recreate, after cloning, an Eco RI site (see paragraph 2.1.1). - antisense primer H3'Xba, located 3' of the CH of T4125-A2, introduces an Xba I sie (underlined) 3' of 35 the ampliied sequence: H3'Xba (SEQ ID No. 4): 5' -GAGAGGTCTAGACTATTTACCCGGAGACACGAACG-3 -28 1.4-Ampiification of the Kappa light chain: K/T125-A2 sequence The entire Kappa chain of T125 -A2 (K/T125-A2 secruence) is amplified from the T125-A2 cDNAs using the following 5 primers: - primer A2VK3, located 5' of the leader region of the VK gene of T125-A2, introduces a consensus sequence (in bold) deduced from the sequence of several leader regions of VK VH genes belonging to the same 10 VKl subgroup as the VK gen of TG ?5A2 - C sequence also comprises an Eco RI restriction site (in italics) and a Kozak sequence (underlined): A2VK3 (SEQ ID No. 5): 5' -CCTACCGAATTCGCCGCCACCATGGACATGAGGGTCCCCGCTCA- 3 ' 15 - antisense primer KSE1, located 3' of Kappa, introduces an Eco RI site (underlined): KSE1 (SEQ ID No. 6): 5' -GGTGGTGAATTCCTAACACTCTCCCCTGTTGAAGCTCTT-3'. Fig. 1 gives a diagrammatic illustration of the 20 strategies for amplifying the heavy and light chains of T125-A2. 2- Construction of expression vectors 25 2.1-Vector for expressing the heavy chain of T125-A2: T125-H26 The construction of T125-H26 is summarized in fig. 2. it-- is carried out in two stages: first of all, con scruction of the intermediate vector V51-CH/T125-A2 by 30 insertion of he constant region of T125-A2 into the expression vector VS derived from pCI-neo (fig. 3) and chen cloning of the variable region into \j51- CH /T 125 -AS 2.1.1 Cl1on;irg of ,he constanregion of T- 25-A2 The amplified CH/T 125-A2 sequence is inserted, after phosphorylation, at the Eco RI site of the vector V51 ( . 3). The ligation is performed after prior 'reat mer o the Eco RI sticky ends of V51 with the Kleno polymvrerase in~ coder to make them"lu-ndd -29 The Drimer G1 used for amplify ing CH/T125-A2 makes it possible to recreate, after its insertion into V51, an Eco RI site 5' of CH/T125-A2. 2.1.2 Cloning cf the variable region of T125-A2 The V-HI/T125-A2 sequence obtained by amplification is digested with Eco RI and Apa I and then inserted at the Eco RI and Apa I sites of the vector V51-Gl/T125-A2. 2.2-T125-A2 light chain vector: T125-K47 10 The construction of T125-K47 is given in fig. 4. The K/T125-A2 sequence obtained by PCR is digested with Eco RI and inserted at the Eco RI site of the expression vector V47 derived from pCI-neo (fig. 5). 15 2.3-Tl25-A2 heavy and light chain vector: T125-IG24 The construction of T125-IG24 is illustrated diagram matically in fig. 6. This vector, which contains the two transcription units for the heavy and Kappa chains of T125-A2, is obtained by inserting the Sal I-Xho I 20 fragment of T125-K47, containing the transcription unit for K/T125-A2, at the Xho I and Sal I sites of T125-H26. Thus, the heavy and light chains of T125-A2 are expressed under the control of the CMV promoter; other 25 promoters may be used: RSV, IgG heavy chain promoter, MMLV LTR, HIV, -actin, etc. 2._-T125-A2 heavy and light chain specific lea de vector: T 25 -iaS 4 30 A second vector for expressing T125-A2 is also constructed, in whi ch the consensus leader sequence of che Kappa chain is replaced with the real sequence of the leader region of T125-A2 determined beforehand by sequencing Products from "PCR 5'-PACE'' (Rapid 35 Xf>folif ication of cDNA 5' nds) The construction of this T125-LS4 vector is described in i . It is carried out in two stages: first of all, constLruccion o a new vector for expressirg the TKd asserbl cf -30 te final expression vector, TI 1 25-LS4, containing the two neavy chain and modified liaht chain transcripion uni* ts. 2.4.1 Construction of the vector T125-KLS18 5 The 5' portion of the Kappa consensus leader sequence o: the vector Tl25-K47 is replaced with the specific leader sequence of T125 (KLS/T125-A2) during a step of amplification of the K/T125-A2 sequence carried out using the following primers: 10 - nrimer A2VK9.. modifies the 5,' prtin of th- la region (in bold) and introduces an Eco RI site (underlined) and also a Kozak sequence (in italics) A2VK9: 5' -CCTACCGAATTCGCCGCCACC'ATGAGGGTCCCCGCTCAGCTC-3' - primer KSE1 (described in paragraph 1.4) 15 The vector T125-KLS18 is then obtained by replacing the Eco RI fragment of T125-K47, containing the K/T125-A2 sequence of origin, with the new sequence KLS/T125-A2 digested via Eco RI. 2.4.2 Construction of the final vector T125-LS4 20 The Sal I-Kho I fragment of T125-KLS18, containing the modified KLS/T125-A2 sequence, is inserted into T125 H26 at the Xho I and Sal I sites. 3- Production Of: anti-D Abs in the YB2/0 Iine 25 31--Without gene amplification The two expression vectors T125-IG24 and T125-LS4 were used to transfect cells of the YB2/0 line (rat myeloma, ATCC line No. 1662) . After transfection by eleccro 30 poration and selection of transformants in the presence of G418 (neo selection), several clones were isolated. The production of recombinant anti-D Abs is approximately 0.2 vg/106 cells/24 h (value obtained for clone 3B2 f 270) The ADCC activity of this 35 recombinant Ab is greater than or e-ual to tha f he poly-D controls (figure 1) . The Abs produced using the wo expression vectors are not significantly difeent in terms of 1evsl of production or of ADCC acti v -31 3.2-With- gene amplification The gene amplification system used is based on the selection of transformants resistant to methotrexate (MTX) It requires the prior introduction of 5 transcription unit encoding the DHFR (dihydroFolate reductase) enzyme into The vector for expressing the recombinant Ab (SHITARI et al., 1994). 3.2.1 Construction of the expression vector T125 dhfr 13 10 The scheme shown in fig. 8 describes the conS trc of the vector for expressing T125-A2, containing the murine dhfr gene. A first vector (V64) was constructed from a vector derived from pCI-neo, V43 (fig. 9), by replacing, 3' of 15 the SV40 promoter and 5' of a synthetic polyadenylation sequence, the neo gene (Hind III--Csp 45 1 fragment) with the cDNA of the murine dhfr gene (obtained by amplification from the plasmid pMT2) . This vector is then modified so as to create a Cla I site 5' of the 20 dhfr transcription unit. The Cla I fragment containing the dhfr transcription unit is then inserted at the Cla I site of T125-LS4. 3.2.2 Selection in the presence of MTX * 1st strategy: 25 YB2/0 cells transfected by electroporation with the vector T125-dhfr13 are selected in the presence of G418. The recombinant Ab-producing transforrnants are then subjected to selection in the presence of increasing doses of MTX (from 25 nM to 25 gM) . The 30 progression o the recombinant Ab production, reflecting the gene amplification process, is followed during the bMTX selection steps. The MTX-resistan. transformants are then cloned by limiting dilution. The 1lve and the stability of the recombinant 35 porduction are evaluated for each clone obtained. The ani antibody productivity after gene amolificatio-n is approximately 13 (+/- 7) 'g/105 cells/24 h. nd srategy: -32 T125-dh.r13 are select-ed in the presence of G4.38 The best recombinant Ab-producing transformants are cloned by limiting dilution before selection in the presence of increasing doses of MTX. The progression of the S production by each clone, reflecting the aene amplification process, is followed during the MTX selection steps. The level and the stability of the recombinant Ab production are evaluated for each MTX resistant clone obtained. 10 4- Evaluation of the activity of the T125 antibody expressed in YB2/0 After purification by affinity chromatography on 15 protein A Sepharose (Pharmacia) and dialysis into 25 mM Tris buffer, 150 mM NaCl, pH 7.4, the concentration of the T125 antibody is determined by the ELISA technique. The biological activity in vitro is then measured by the ADCC assay described above. The results are given 20 in figure 1. EXAMPLE 3: DEMONSTRATION OF THE RELATIONSHIP BETWEEN GLYCAN STRUCTURE AND FcyRIII-DEPENDENT ACTIVITY: 25 1- Cell culture in the presence of deoxymannojiirmycin (D101) Several studies describe the effect of enzymatic inhibitors on the glycosylation of irmunoglobulins and on their biological activity. an increase in ADCC 30 activity is reported by ROTHM'AN et al., 1989, this being an increase which cannot be attributed to an enhancement of the affinity of the antibody for its target. The modification of glycosylation caused by adding DI consists of inhib it i.n of the '-,2 35 mannosidase I present in le Golgi. 12 leads to the production of a greater proportion of polymannosylated, nonfucosylated structures. Various anti-.Rh(D) antibody-oducing l ine s weree brgt inFo contac i D -33 activity of the monoclonal antibodies produced was evaluated in the form of culture supernatants or after purilfication. The cells (he:erohvbrid or lymphoblastoid cells) are 5 seeded at between 1 and 3 x 105 cell/ml, and cultured in IDM culture medium. (Life Te hnologies) with 10% of FCS and in the presence of 20 gg/ml of DM (Sigma, Boehringer) . After having renewed the medium 3 times, the culture supernatants are assayed by human IgG ELISA 10 and then by ADCC. Table 2: Effect of culturing in the presence of DMM on the ADCC activity of various anti-Rh(D)s ADCC activity as % of the Minimum dose activity of poly-D LFB51 of DM4 Samples Cu.lture Culture in the necessary wi thout DMM presence of DMM gg/M F60 109 113 NT D31 19 871 10 DF5 26 62 20 T125 RI(3) 3 72 20 T15CO' 0 105 5 NT - not tested 15 Culturing in the presence of deoxymannojirimycin (DMI1) brings a significant improvement to the A.DCC results for the antibodies previously weakly active, produced by: 20 a human-mouse hybridoma D31 a human lymohoblastoid line DF5 a cransfected nurine line T125 in CHO 0The addition DM214 may iake it possible to restore 2the ADCC activity of an antibody derived from the cloid T125 = T125 RI(3) (described in example 1) and which h±as lost thisctivity through sustained cult uing.

-34 hecerohvbridoma FGG (the production of which is described in example 1) is not modified by culturing in the presence of DIM 5 2- Production of recombinant anti-D antibodies by various cell lines: 2.1-Preparation of an expression vector for the anti body DF5: The nucleotide sequence of the antibody DF5, a negative 10 control in the ADCC assay. is use o transfection of this antibody into some lines, in parallel to transfection of the antibody T125. The sequences encoding the Ab DF5 are isolated and amplified according to the same techniq-ues used for the 15 recombinant Ab T125-A2. The corresponding cDNAs are first of all synthesized from total RIJA extracted from the anti-D Ab- (IgG Cl/Lambda)-producing~ clone 2MDF5 obtained by EBV transformation. 20 o Amplification of the heavy and light chains is then carried out from these cDNAs using the primers presented below. a Amplification of the variable region of the heavy chain of DF5 (VH/DF5 sequence): 25 - primer DF5VH1, located 5' of the leader region (in bold) of the V~-I gene of DF5 (sequence published: L. Chouchane et al.); this primer also comprises an Eco RI restriction site (in italics) and a Kozak sequence (underlined) 30 DF5VH1 (SEO ID No. 8): 5 'CTCTCCGAATTCCCGCCACCATGGACT~GGACCTGGGTCCTTTGT 3' - antisense primer GSP2-ANP, located 5' of the consLant region (CU) already described. in pa-agraph 1.2 (ample 2) 35 -A lificacion of the constan- region CU of DF5 (CH/DFS se-uence) : ;Drimers C1 and H3'Xba already described in paragraph 1.3 (example 2) krAmplificoation of the Lambda light chain of YF- -35 - primer D5VLBDL, located 5- of the leader region of the VL gene oF DF5, introduces a consensus sequence (in bold) deduced from the secruence of several leader regions of VL genes belonging :o the same VL 1 subgroup 5 as the VL gene of 2MDF5; this sequence also comprises an Eco RI restriction site (in italics) and a Kozak sequence (underlined) DFSVLBD1 (SEQ ID No. 9): 5' CCTACCGAATTCGCCGCCACCATGGCCTGGTCTCCTCTCCTCCTCAC- 3 ' 10 - antisense primer LSEl, located 3' of Lambda, intr duces an Eco R site (underlined): LSE1 (SEQ ID No. 10): 5' -GAGGAGGAATTCACTATGAACATTCTGTAGGGGCCACTGTCTT-3'. e The construction of the vectors for expressing the 15 heavy chain (DF5-H31), light chain (DFS-L10) and heavy and light chains (DF5-IGl) of the Ab DF5 is carried out according to a construction scheme similar to vectors expressing the Ab T125-A2. All the leader sequences of origin (introduced in the 20 amplification primers) are conserved in these various vectors. 2.2-Transfection of various cell lines with the antibodies T125 and DF5 25 The three expression vectors T125-IG24, T125-LS4 and DF5-IgG1 are used to transfect cells of various lines: Stable or transient transfections are performed by electroporation or using a transfection reagent.

-36 Table 3: Cell lines used for the trans fczion of anti RI(D) antibodies __________ _______ ______ 7.7Cel -- e 3. CHO-Kl ATCC CCL 61 Chinese hamster ovary (epithelium like) CHO-Lec10 Fenouiliet et al- I Chinese hamster ovary 1996, Virology, (epithelium like) 218, 224-231 Jurkat ATCC TIB-152 Human T lymphocyte (T eukezmia) Molt-4 ATCC CRL 1582 Human T lymphocyte (acute lymphoblastic leukemia) WIL2-NS ATCC CRL 8155 EBV-transformed human B lymphocyte Vero ATCC CCL 81 African green monkey kidney I (fibroblast like) COS-7 ATCC CRL 1651 SV40-transformed African I green monkey kidney (fibroblast like) 293-HEK ATCC CRL 1573 Primary human embryonic kidney transformed with defective adenovirus 5 DNA YB2/0 i ATCC CRL 1662 Nonsecreting rat myeloma BHK-21 ATCC CCL 10 Newborn hamster kidney (fibroblast like) K6H6-B5 ATCC CRL 1823 Nonsecreting human-mouse heteromyeloma NSO ECACC 85110503 Nonsecreting mouse myeloRa (lymphoblast like) SP2/0-g 14 ECACC 85072401 Nonsecreting mouse x mouse hvbridoma CHO Lec-1 ATCC CRL 1735 Chinese hamster ovary CHO dhfr ECACC 9406007 Chinese hamster ovary CHO Pro-5 ATCC CRL 1731 Chinese hamster ovary P3X63 ATCC CRL 1580 Nonsecreting mouse myeloma Ag8.653 Afte0 selection of the transformants in the presence of 418J (neo selct ion) several clones were isolate..

-37 monoclonal antibody as a function of the expressing cell has been described by CROWE et al. (1992), with the CHO, NSO and YB2/0 cell lines. 5 The results obtained here confirm the importance of the expressing cell line with respect -o the functional characteristics of the antibody to be produced. Arnong the cells tested, only the Vero, YB2/0 and CHO Lec-1 lines make it possible to express recombinant anti 10 Rh(D) monolnl nibde .'ithstronglytic activity in the ADCC assay (see example 1 and table 3). Table 3: ADCC activity of the antibodies DFS and T125 obtained by transfection into various cell lines. The 15 results are expressed as percentage of the activity of the reference polyclonal antibody: Poly-D LFB 51 Transfected cell lines CHO- CHO- Wil-2 Jurkat Vero Molt-4 COS-7 293- YB2/0 K1 Lec10 HEK T125 7 22 3 6 90 0 13 161114 +/-8 +/-6 +/-5 +/-8 +/-21 n=1 +/-2 +/-13 +/--28 n=13n n=12 n=7 n5 n=4 n=1 5 DF5 NT 51 NT NT 72 NT 21 12 94 +/-19 + -17_ n=3 n=5 n=4 n=12 n=15 TLransfected cell li1Les CHO- SP2/0- CHO I CHO P3X6 3A SO BHFV K6H6-E5 1 Leci A Pro-- deft g8 .633 13 106 0 9 3 13 3> T 2125~ +/-8 +/-.-5 +/-60 +/-0 +/-8 +/ +/~8 n=!f n= n=4 n r~ -38 3- Study of the glycazz structures Characterization of the glycan structures of zhe anti Rh'-D antibody was carried out- on four purified produces 5 having an ADCC activity (F60, and three recorbinant proteins derived from T125) in comparison with two purified products inactive or very weakly active in the ADCC assay according to the invention (D31 and DF5) In practice, the oligosaccharides are separated from 10 theIJC ptein b C 4 dy -pecIf nymtic deglycs'yla'in wth PNGase F at Asn 297. The oligosaccharides thus released are labeled with a fluorophore, separated and identified by various complementary techniques which allow: 15 - fine characterization of the glycan structures by matrix-assisted laser desorption ionization (MALDI) mass spectrometry by comparison of the experimental masses with the theoretical masses. - determination of the degree of sialylation by ion 20 exchange HPLC (GlycoSep C) - separation and quantification of the oligo sacharride forms according to hydrophilicity criteria by normal-phase HPLC (Glycosep N) - separation and quantification of the 25 oligosaccharides by high performance capillary electrophoresis-laser induced fluorescence (HPCE-LIF). ) Chairterization of the glycans of active forms 30 The various active forms studio ed are F60 and three recorbinant antibodies, R 290; R 297 and R 270, derived fror T125 and produced in YB2/0. ine characerization the glycan structures by mass soectrometry (:igure 7) shows 'that these forms are all of the 35 bi-antennary type. In the case of R 270, the major form is of the acaactosylated.. nonfucosylted ype (GO.. exp. mass 1459.37 Da, fig. 1) . Three oher s Lruc ures are identified: agalactosylaed. fucosyle (GOF a 60*-5 .4Da -,- -ooaatslae nnuosltd(-a -39 1621.26 Da) and monogalactosvlated, fucosylated (GIF ac 1767.43 Da) in minor amount. These same four structures are c-aracteristic of R 290, F 60 and R 297 (figure 1) These four antibodies which are active in ADCC are also 5 characterized by the absence of oligosaccharides having a bisecting N-acetvlglucosamine residue. Ouantification of the glycan structures by the various techniques of HPLC and HPCE-LIF (table 1) confirms the presence of the four forms identified by mass: GO, GOF, 10 Gl and GiF. The degree of sialylation is very iow, in particular for the recombinant products, from 1 to 9.4%, which is confirmed by the similarity of the mass spectra obtained before and after enzvmatic desialylation. The degree of fucosylation ranges from 15 34 to 59%. 2) Inactive forms The various inactive forms studied are D31 and DF5. 20 Quantification of the glycan structures by the various chromatographic and capillary electrophoresis techniques (table 1) reveals, for these two antibodies, a degree of sialylation close to 50%, and a degree of fucosvlation of 88 and 100% for D31 and DF5, 25 respectively. These degrees of sialvlation and fucosylation are much higher than those obtained from the active forms. CharacterizaJion of the glycan structures shows thac 30 the major form is, for the two antibodies, of t-h bi-anltennary,. monosialylated, digalactosylated; fucos vlated tve (G2SlF, table 1) . The characcerization by mass spectrometry of D31 (figure 7) reveals that te neutral forms are mainly of the monogalactsylated, c osylated typ (GlF at 1767.43 Da) and digalactos yated, fucosylated t'pe (G2F at 1929.66 Da) '''' D~ is ch~etris b K- 40 GLcNAc residue. In particular, the mass analysis (figure 8) reveals the presence of a major neutral form of the monogalactosylated, fucosylated, bisecting, intercalated GlcNAc type (GlFB at 1851.03 Da). On the other hand, these structural 5 forms are undetectable or present in trace amounts on the active antibodies studied. The ADCC activity of D31 after the action of DMM increases from 10% to 60%. The glycan structures of DMM D31 differ from those of D31 by the presence of oligomannose forms (Man 5, Man 6 and Man 7) L0 (see figure 9). 3) Conclusion The various active antibodies are modified on Asn 297 with N glycosylations of the bi-antennary and/or oligomannoside type. For the bi-antennary forms, this involves short structures with a [5 very low degree of sialylation, a low degree of fucosylation, a low degree of galactosylation and no intercalated GlcNAc.

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mrunol Methods 101: 193-200 (1987). Issitt, P.D. Genetics of the Ph Blood Group System: 10 Some Current Concepts. Med. Lab. Sci. 45: 395-404 (1988). jefferis, R, Lund, J., Mizutani, H., Nakagawa, H., Kawazoe, Y., Arata, Y. and Takahashi, N. A comparative 15 study of the N-linked oligosaccharides structure of human IgG Subclass proteins. Biochem. J., 268: 529-537 (1990) Koskimies, S. Human Lymphoblastoid Cell Line Producing 20 Specific Antibody against Rh-Antigen D. Scand. Immunol. 11: 73-77 (1980). Kumpel, B.M., Goodrick, M.J., Pamphilon, D.H., Fraser, I.D., Poole G.D., Morse, C., Standen, G.R., 25 Chapman, G.E., Thomas, D.P. and Anstee, D.J. Human Rh D monoclonal antibodies (BP.AD-3 and BRAD-5) Cause Accelerated Clearance of Rh D + Red blood Cells and suppression of Rh D Immunization in Rh D Volunteers. Blood, Vol. 86, No. 5, 1701-1709 (1995) 30 Kumpel, B.M., Poole, G.D. and _rdley, B.A. Human Monoclonal Anli-D Antibodies. I. Their Production, Serology, Quantitation and Potential Use as Blood Grouping Reagents. Brit- J. Haemat . '- - i 17 Kumpel, B.M., Rademacher, T.W-, Rock, williams, P.J., Wilson. T.B.M. GalacatosylationI o1 huma gat-D producd by BB-rnfre j 3 - - 43 blastoid cell lines Is dependent on culture method and a=Fects 1Fc receptor mediated functional activity. Hum. Antibodies and Hybridomas, 5: 143-151 (1994). 5 Leatherbarrow, R.J., Rademacher, T.W., Dwek, R.A., Woof, J. , Clark, A., Burton. D.R.. Richardson, N. and Feinstein, A. Effector functions of monoclonal aglycosylated mouse IgG2a; binding and activation of complement component CI and itneraction with human Fc 10 receptor. Molec. Immun. 22, 407-415 (1985). Lomas, C., Tippett, P., Thompson, K.M., Melamed, M.D. and Hughes-Jones, N.C. Demonstration. of seven epitopes on the Rh antigen D using human monoclonal anti-D 15 antibodies and red cells from D categories. Vox Sang. 57: 261-264 (1989). Lund, J., Takahaski, N., Nakagawa, H., Goodall, M, Bentley, T., Hindley, S.A., Tyler, R. and Jefferis, R. 20 Control of IgG/Fc glycosylation: a comparison of oligosaccharides from chimeric human/mouse and mouse subclass immunoglobulin G5. Molec. Immun. 30, No. 8, 741-748 (1993). 25 Lund, J., Tanaka, T., Takahashi, N., Sarmay, G., Arata, Y. and Jefferis, R. A protein structural change in aglycosylated IgG3 correlates with loss of hu FcRI and Hu FcyRIII binding and/or activation. Molec. Immun. 27, 1145-1153 (1990). Ma, J.K. and Hein, M.B. Imtmunoherapeutic potential. of antibodies produced in plants. Trends Biotechnol. 13, 522-527 (1995) M_ Cann-Carter, M.C.. Bruc, M. Shaw.. E..4 Thorpe, S.J., Sweeney, G. Armstrong, S.S. and james, K. The production and evaluation of two human monoclonal anti-D antibodies. Transf. Med. 3: 107.-194 - 44 Melamed. M.D., Gordon, J.. Ley, S.J., Edgar, D. and Hughes-JOnes, N.C. Senescence of a -human lymphoblastoid clone producing anti-Rhesus (D) Eur. J. I-mmunol. 115 5 242-746 (1985). Parekh, R.B., Dwek, R.A., Sutton, B.J., Fernanes, D.L.. Leung, A., Stanworth, D., Rademacher, T. W., Mizuochi, T., Taniguchi, T., Matsuta. K., Takeuchi, F. 10 Nagano, Y., Miyamoto, T. and Kobata, A. Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature, 316: 452-457 (1985). 15 Rothman, R.J., Perussia, B., Herlyn, D. and Warren, L. Antibody-dependent cytotoxicity mediated by natural killer cells is enhanced by castanospermin e-induced alterations of IgG glycosyiation. Mol. Immunol. 26(12) 1113-1123 (1989). 20 Shitara K., Nakamura K., Tokutake-Tanaka Y., Fukushima M., and Hanai N. A new vector for the high level expression of chimeric antibodies to myeloma cells. J. Immunol. Methods 167: 271-278 (1994). 25 Thompson, K.M., -iough, D.W., Maddison, P.J., Mclamed, M.D. and Hughes-Jones, i.C. Production of -human monoclonal IgG and IgM antibodies with anti-D (rhesus) specific- using heterohybridomas. Immunology 30 53: 157-160 (196). hormson, A., Contreras, M., Gorick, E., Kumpel, ., Chapman, G.E., Lane, R.S., Teesdale, P. Hughes-Jones, N.C. and Mollison, P.L. Clearance of Rh D-positive red cels with monocional anti-D. Lancet 336: 1147-1150 (1990) . Tippett. P. Sub-divisions of the Rh(D) anige. Ci. Lab. SciI45 : 88 9 (19 ) .R - 45 Ware, R.E. and Zirrmerman, S.A. Anti-D: Mechanisrms o action. Seminars in Hematology, vol. 35, No. 1, suo 1: 14 -22 (1998). Yu, .P.C., Miller, W.J., Silberklang, M., Mark, G.E., Ellis, R.W., Huang, L., Glushka, j., Van Halbeek, H., Zhu, J. and Alhadeff, J.A. Structural characterization of the N-Glycans of a humanized anti-CD1 8 muri ne 10 immunoglobulin G. Arch. Biochem. Biophys. 308, 387-399 (1994) . Zupanska, B., Thompson, E., Brojer, E. and Merry, A.H. Phagocytosis of Erythrocytes Sensitized with -Know 15 Amounts of IgGl and IgG3 anti-Rh antibodies. Vox Sang. 53: 96-101 (1987).

Claims (29)

1. An isolated clone, wherein said isolated clone produces monoclonal antibodies having on their Fcy glycosylation sites (Asn 297) bi-antennary glycan structures, 5 wherein the glycan structures of the monoclonal antibodies have a low degree of sialylation, wherein the glycan structures of the monoclonal antibodies have a fucose content less than 65%, and wherein the glycan structures of the monoclonal antibodies 0 have a content greater than 60% for the GO+Gl+GOF+GlF forms, as herein defined.

2. The isolated clone of claim 1, wherein the fucose content is less than 30%.

3. The isolated clone of claim 1, wherein the fucose content is [5 between 20% and 45%.

4. The isolated clone of claim 3, wherein the fucose content is between 25% and 40%.

5. The isolated clone of any one of claims 1 to 4, wherein the purified monoclonal antibodies are directed against an antigen, 20 and activate effector cells expressing Fcy type III receptors, causing a lysis of target cells presenting the antigen greater than 60% of a lysis caused by polyclonal antibodies directed against the antigen.

6. The isolated clone of claim 5, wherein the antigen is rhesus 25 D.

7. The isolated clone of any one of claims 1 to 6, wherein the purified monoclonal antibodies are directed against an antigen, and activate effector cells expressing Fcy type III receptors, 47 causing a lysis of target cells presenting the antigen greater than 90% of a lysis caused by polyclonal antibodies directed against the antigen.

8. The isolated clone of claim 7, wherein the antigen is rhesus 5 D.

9. The isolated clone of any one of claims 1 to 8, wherein the purified monoclonal antibodies are IgG1 antibodies.

10. The isolated clone of any one of claims 1 to 8, wherein the purified monoclonal antibodies are IgG3 antibodies. 10

11. The isolated clone of any one of claims 1 to 10, wherein said glycan structures of the purified monoclonal antibodies have a sialic acid content of less than 25%.

12. The isolated clone of any one of claims 1 to 11, wherein said isolated clone is derived from cell line CHO-K, CHO-Lec10, 15 CHO Lec-1, CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, YB2/0, BHK, K6H6, NSO, SP2/0-Ag 14, or P3X63Ag8 .653.

13. The isolated clone of claim 12, wherein said isolated clone is derived from Vero, YB2/0, or CHO Lec-1. 20

14. The isolated clone of claim 13, wherein said isolated clone is derived from YB2/0.

15. An isolated clone, wherein said isolated clone produces monoclonal antibodies having on their Fcy glycosylation sites (Asn 297) bi-antennary glycan structures, 25 wherein the glycan structures of the monoclonal antibodies have a low degree of sialylation, 48 wherein the glycan structures of the purified monoclonal antibodies have a content less than 50% for the GOF+G1F forms, and wherein the glycan structures of the monoclonal antibodies 5 have a content greater than 60% for the GO+Gl+GOF+GlF forms, as herein defined.

16. The isolated clone of claim 15, wherein the glycan structures of the purified monoclonal antibodies have a content less than 30% for the GOF+G1F forms. 10

17. The isolated clone of claim 15 or claim 16, wherein said glycan structures of the purified monoclonal antibodies have a content greater than 80% for the G0+G+GOF+GlF forms.

18. The isolated clone of any one of claims 15 to 17, wherein the monoclonal antibodies are directed against an antigen, and 15 activate effector cells expressing Fc type III receptors, causing a lysis of target cells presenting the antigen greater than 60% of a lysis caused by polyclonal antibodies directed against the antigen.

19. The isolated clone of claim 18, wherein the monoclonal 20 antibodies cause a lysis of target cells presenting the antigen greater than 70% of a lysis caused by polyclonal antibodies directed against the antigen.

20. The isolated clone of claim 19, wherein the monoclonal antibodies cause a lysis of target cells presenting the antigen 25 greater than 80% of a lysis caused by polyclonal antibodies directed against the antigen.

21. The isolated clone of claim 20, wherein the monoclonal antibodies cause a lysis of target cells presenting the antigen greater than 90% of a lysis caused by polyclonal antibodies 30 directed against the antigen. 49

22. The isolated clone of any one of claims 15 to 21, wherein said monoclonal antibodies are IgG1 antibodies.

23. The isolated clone of any one of claims 15 to 21, wherein said monoclonal antibodies are IgG3 antibodies. 5

24. The isolated clone of any one of claims 15 to 23, wherein said monoclonal antibodies are anti Rh(D) antibodies.

25. The isolated clone of any one of claims 15 to 24, wherein the sialic acid content of the glycan structures of the purified monoclonal antibodies is less than 25%. 10

26. The isolated clone of any one of claims 15 to 25, wherein said isolated clone is derived from cell line CHO-K, CHO-LeclO, CHO Lec-1, CHO Pro-5, CHO dhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, YB2/0, BHK, K6H6, NSO, SP2/0-Ag 14, or P3X63Ag8.653. 15

27. The isolated clone of claim 26, wherein said isolated clone is derived from Vero, YB2/0, or CHO Lec-1.

28. The isolated clone of claim 27, wherein said isolated clone is derived from YB2/0.

29. The isolated clone according to claim 1 or 15, substantially 20 as hereinbefore described.