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Patent US8071371 - Monoclonal antibody production by EBV transformation of B cells - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign in<nobr>Advanced Patent Search</nobr>PatentsA method for producing a clone of an immortalized human B memory lymphocyte, comprising the step of transforming human B memory lymphocytes using Epstein Barr Virus (EBV) in the presence of a polyclonal B cell activator. The method is particularly useful in a method for producing a clone of an immortalized...http://www.google.com/patents/US8071371?utm_source=gb-gplus-sharePatent US8071371 - Monoclonal antibody production by EBV transformation of B cellsAdvanced Patent SearchPublication numberUS8071371 B2Publication typeGrantApplication numberUS 11/719,835PCT numberPCT/IB2004/001071Publication dateDec 6, 2011Filing dateFeb 26, 2004Priority dateFeb 26, 2003Also published asEP2298804A2, EP2298804A3, US20100021470, US20120027768, US20140004123Publication number11719835, 719835, PCT/2004/1071, PCT/IB/2004/001071, PCT/IB/2004/01071, PCT/IB/4/001071, PCT/IB/4/01071, PCT/IB2004/001071, PCT/IB2004/01071, PCT/IB2004001071, PCT/IB200401071, PCT/IB4/001071, PCT/IB4/01071, PCT/IB4001071, PCT/IB401071, US 8071371 B2, US 8071371B2, US-B2-8071371, US8071371 B2, US8071371B2InventorsAntonio LanzavecchiaOriginal AssigneeHumabs LlcExport CitationBiBTeX, EndNote, RefManPatent Citations (27), Non-Patent Citations (65), Classifications (34), Legal Events (1) External Links: USPTO, USPTO Assignment, EspacenetMonoclonal antibody production by EBV transformation of B cellsUS 8071371 B2Abstract A method for producing a clone of an immortalized human B memory lymphocyte, comprising the step of transforming human B memory lymphocytes using Epstein Barr Virus (EBV) in the presence of a polyclonal B cell activator. The method is particularly useful in a method for producing a clone of an immortalized human B memory lymphocyte capable of producing a human monoclonal antibody with a desired antigen specificity, comprising the steps of: (i) selecting and isolating a human memory B lymphocyte subpopulation; (ii) transforming the subpopulation with Epstein Barr Virus (EBV) in the presence of a polyclonal B cell activator; (iii) screening the culture supernatant for antigen specificity; and (iv) isolating an immortalized human B memory lymphocyte clone capable of producing a human monoclonal antibody having the desired antigen specificity.
1. A method for producing immortalized memory B lymphocytes comprising contacting memory B lymphocytes with Epstein Barr virus (�EBV�) and an agonist of a Pattern Recognition Receptor that is expressed on memory B cells.
5. The method of any one of claim 1, 2, 3 or 4, wherein said Pattern Recognition Receptor is a Toll Like Receptor (�TLR�) that is expressed on memory B cells.
TECHNICAL FIELD This invention relates to monoclonal antibodies, to a method for preparing immortalised memory B cells, to a method for preparing immortalised memory B cells capable of producing a monoclonal antibody with a desired antigen specificity and to the use of antibodies produced by said immortalised memory B cells. The invention is particularly useful for preparing human monoclonal antibodies. In one embodiment, the invention relates to a method for preparing immortalised human memory B cells capable of producing antibodies specific for an infectious agent, more particularly where the infectious agent is the severe acute respiratory syndrome (SARS) virus. Further embodiments are described below.
BACKGROUND ART The success in generating murine monoclonal antibodies rests on the efficient and selective fusion of antigen-stimulated B cell blasts with a murine myeloma cell line followed by selection of stable antibody producing hybrids (Kohler & Milstein 1975). FR2817875 describes a modified version of this protocol where prior to immortalisation, B lymphocytes are induced to differentiate by a non-specific activating system and a cytokine. The B cell blasts may be taken from the spleen or lymph nodes. However, the difficulty in obtaining antigen-stimulated B cell blasts and the lack of suitable fusion partners has hampered this approach in the human system.
Another reason why the EBV method has become obsolete is that alternative approaches for making human or human-like monoclonal antibodies became available through genetic engineering. These include the humanization of murine antibodies, the isolation of antibodies from libraries of different complexity and the production of hybridomas using the classical method in mice transgenic for human Ig loci (the �xeno-mouse�). The literature on these alternative approaches is not reviewed here since is not directly relevant to the present invention. However, it is worth considering some limitations of these methods. Humanization of murine monoclonal antibodies is a laborious and incomplete procedure. Random antibody libraries represent an unbiased repertoire and can therefore be used to select antibody specificities against highly conserved antigens, but lead to antibodies of low affinity. Libraries selected from antigen primed B cells are enriched for a particular specificity, but do not preserve the original VH-VL pairing and generally lead to antibodies that have lower affinity for the antigen than those present in the original antibody repertoire. The impact of this technology has been limited. In contrast the xeno-mouse can be efficiently immunized against an antigen of choice (especially if this is a human antigen), but this system shares with the classical murine hybridoma technology the limitation that the antibodies are selected in a species other than human. Therefore these methods are not suitable to produce antibodies with the characteristics of those produced in the course of a physiological human immune response. This applies to the antibody response to human pathogens including HIV, the four Plasmodium species that cause malaria in humans (P. falciparum, P. vivax, P. malariae and P. ovale), human hepatitis B and C viruses, Measles virus, Ebola virus etc. (for an exhaustive list see Fields et al. 1996). It also applies to antibody responses to environmental allergens generated in allergic patients, to tumour antigens generated in tumour bearing patients and to self antigens in patients with autoimmune diseases.
DISCLOSURE OF THE INVENTION While the present invention is illustrated by embodiments where human monoclonal antibodies are produced, the techniques described herein are not so limited. The present invention can be used for any species for which it is desired to produce monoclonal antibodies efficiently.
(i) transforming a population of cells comprising or consisting of human memory B lymphocytes with Epstein Barr Virus (EBV) in the presence of a polyclonal B cell activator, (ii) screening the culture supernatant for specificity for the SARS virus, and (iii) isolating an immortalised human B memory lymphocyte clone capable of producing a human monoclonal antibody having specificity for the SARS virus. In this specification, the term �antibody having specificity for the SARS virus� means that an antibody molecule binds to the coronavirus that is the causative agent of SARS with a greater affinity compared to its binding affinity for other viruses.
In this specification, the term �polyclonal activator� means a molecule or compound or a combination thereof that activates B lymphocytes irrespective of their antigenic specificity. A range of different molecules may be used as the polyclonal activator.
Unmethylated DNA oligonucleotides (CpG) are TLR-9 agonists. They stimulate dendritic cell maturation and activate B cell proliferation and differentiation polyclonally, i.e. irrespective of the antibody specificity (Krieg et al. 1995; Krieg 2002). The biological effect of CpG is dependent on specific sequences and chemical modifications (Krieg 2002). CpG oligonucleotides can be used as polyclonal activators, and examples of suitable activators are CpGs such as CpG 2006 (5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO: 1); Hartmann et al. 2000) and other oligonucleotide sequences that trigger TLR-9. By �CpG� we mean sequences of unmethylated DNA oligonucleotides. More particularly, the term �CpG� includes single-stranded DNA molecules of between 5 and 100 nucleotides in length (e.g. 10-80, 20-70, 30-60 nucleotides) that include one or more instances (e.g. 2, 4, 6, 8, 10 or more) of the dinucleotide CG sequence, with the C in the dinucleotide(s) being unmethylated.
In relation to any particular pathogen, a �neutralising antibody� is one that can neutralise the ability of that pathogen to initiate and/or perpetuate an infection in a host. The invention provides a neutralising monoclonal human antibody, wherein the antibody recognises an antigen from a pathogen selected from: human immunodeficiency virus; hepatitis A virus; hepatitis B virus; hepatitis C virus; herpes simplex virus type 1 or type 2; SARS coronavirus; measles virus; mumps virus; rubella virus; rabies virus; ebola virus; influenza virus; papillomavirus; vaccinia virus; varicella-zoster virus; variola virus; polio virus; rhino virus; respiratory syncytial virus; P. falciparum; P. vivax; P. malariae; P. ovale; Corynebacterium diphtheriae; Clostridium tetani; Clostridium botilinum; Bordetella pertussis; Haemophilus influenzae; Neisseria meningitidis, serogroup A, B, C, W135 and/or Y; Streptococcus pneumoniae; Streptococcus agalactiae; Streptococcus pyogenes; Staphylococcus aureus; Bacillus anthracis; Moraxella catarrhalis; Chlaymidia trachomatis; Chlamydia pneumoniae; Yersinia pestis; Francisella tularensis; Salmonella species; Vibrio cholerae; toxic E. coli; a human endogenous retrovirus; etc.
Fragments of the monoclonal antibodies of the invention can be obtained from the monoclonal antibodies so produced by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Antibody �fragments� include Fab, F(ab′)2 and Fv fragments. The invention also encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of a monoclonal antibody of the invention e.g. the invention includes a scFv comprising the CDRs from an antibody of the invention.
The use of monoclonal antibodies as the active ingredient of pharmaceuticals is now widespread, including the products HERCEPTIN� (trastuzumab), RITUXAN� (rituximab), CAMPATH� (alemtuzumab), REMICADE� (infliximab), REOPRO� (abciximab), MYLOTARG� (gemtuzumab ozogamicin), ZEVALIN� (ibritumomab tiuxetan), Omalizumab, SYNAGIS� (palivizumab), ZENAPAX� (daclizumab), etc. These include antibodies that recognise human self-antigens (e.g. HERCEPTIN� (trastuzumab) recognises the Her2 marker) and antibodies that recognise pathogenic antigens (e.g. SYNAGIS� (palivizumab) recognises an antigen from respiratory syncytial virus).
Pharmaceutical compositions will include an effective amount of one or more antibodies of the invention and/or one or more transformed B cells of the invention i.e. an amount that is sufficient to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic effect. Therapeutic effects also include reduction in physical symptoms. The precise effective amount for any particular subject will depend upon their size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. The effective amount for a given situation is determined by routine experimentation and is within the judgment of a clinician. For purposes of the present invention, an effective dose will generally be from about 0.01 mg/kg to about 50 mg/kg, or about 0.05 mg/kg to about 10 mg/kg of the compositions of the present invention in the individual to which it is administered. Known antibody-based pharmaceuticals provide guidance in this respect e.g. HERCEPTIN� (trastuzumab) is administered by intravenous infusion of a 21 mg/ml solution, with an initial loading dose of 4 mg/kg body weight and a weekly maintenance dose of 2 mg/kg body weight; RITUXAN� (rituximab) is administered weekly at 375 mg/m2; etc.
Compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition, like SYNAGIS� (palivizumab) and HERCEPTIN� (trastuzumab), for reconsitution with sterile water containing a preservative). The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavoured). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient. For example, a lyophilised antibody can be provided in kit form with sterile water or a sterile buffer.
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows results of the ELISA using SARS virus-infected Vero cells lysed in 3% SDS as antigen. Shown are the OD values of serum (1/5000 dilution), supernatants of polyclonal cultures and of independent B cell clones (1/2 dilution).
MODES FOR CARRYING OUT THE INVENTION The present invention permits the cloning of human memory B lymphocytes with very high efficiency and achieves this by the combination of two stimuli, namely: EBV, that immortalizes human B cells with low efficiency and a polyclonal B cell activator that enhances the efficiency of EBV-immortalization.
Example 1 Cloning of B Cells Human memory B cells (CD19+ CD27+ IgM− IgD−) were isolated from healthy donors by cell sorting using methods well known in the art. Different numbers of cells were seeded in replicate cultures in 96 well microplates in the presence of irradiated mononuclear cells (5�105/ml) and either E13V (supernatant of B95-8 cells) alone or EBV in combination with CpG 2006 (2.5 μg/ml) and recombinant IL-2 (1000 U/ml). After 15 days the percentage of cultures containing growing cells was scored. Frequencies were determined by limiting dilution assays. Cultures were scored for growing cells. Cloning efficiency using four different sources of B cells were as follows:
Example 2 Production of Antibodies with a Desired Specificity In a further experiment it was demonstrated that the immortalisation can be used to exploit immunological memory to produce human monoclonal antibodies of the desired specificity.
Example 3 Immortalised Memory B Cells that Express Antibodies Specific for SARS Coronavirus Blood samples were obtained from two patients with a clinical record of SARS. Both patients had serum anti-SARS antibodies as detected by two assays: (i) a neutralization assay which detects neutralizing antibodies directed against surface proteins of the SAGS virus, likely the spike protein and (ii) an ELISA assay, that detects antibodies binding to any denatured protein of the SARS virus.
Example 4 Screening SARS Virus Convalescent Patients for Antibodies Peripheral blood was obtained from a patient at different times after acute infection with SARS virus (2, 4 and 6 months after infection). PBMC were isolated by gradient centrifugation. IgG+ memory B cells were isolated by an improved method that avoids triggering of the B cell receptor. Total B cells were isolated from PBMC using CD22 microbeads (Miltenyi), which were found to be even better than using CD19 microbeads. The cells were stained with antibodies to human IgM, IgD and IgA and negative cells carrying surface IgG were isolated by cell sorting. B cells were pulsed with EBV (50% supernatant of B-95-8 cells) for 8 hours and then plated at 10 cells/well in 96 well U-bottom microplates in complete RPMI medium supplemented with 10% FCS, 2.51 g/ml CpG 2006 and irradiated PBMC (2�105/ml). In this experiment IL-2 was not used. After 2 weeks the culture supernatants were screened for the presence of specific antibodies using the three assays described below. Positive cultures were cloned by limiting dilution in the presence of CpG 2006 and irradiated PBMC as above. Positive clones were expanded and the antibody produced was purified from culture supernatants by affinity chromatography on protein A columns (Amersham).
Example 5 Kinetics and Frequencies of Specific Memory B Cells IgG+ memory B lymphocytes from the 2-month, 4-month and 6-month post-SARS sera were immortalized with EBV under conditions where the number of B cells per culture was limiting, as described above (10 B cells per well). This strategy allows analysis of the product of only a few memory B cells per culture, thus ensuring that the specific antibody detected in positive cultures is monoclonal and, at the same time, increasing the probability of isolating a clone producing the desired antibody by limiting dilution. After two weeks of culture in the presence of EBV, CpG 2006 and irradiated feeder cells the culture supernatants were screened for the presence of specific IgG antibodies using ELISA or staining of spike transfectants. The frequency of cultures screening positive in the SARS virus ELISA assay or staining SARS virus spike transfectants were as follows:
Example 6 Isolation of Monoclonal Antibodies to SARS Virus The results shown above prove that it is possible to interrogate the human B cell memory repertoire with a variety of assays to identify cultures producing an antibody of the desired specificity. In these experiments, 29 of 38 attempts (76%) at cloning positive cultures led to the isolation of one or more clones producing antibodies of the selected specificity. The EBV clones were stable and monoclonal antibodies were recovered in the culture supernatant at concentrations of 10-20 μg/ml. Of these 29, 21 were positive in the ELISA assay and 8 were both positive in the spike staining assay and were able to neutralize SARS virus.
Out of the 8 independent clones staining spike transfectants and neutralizing SARS virus, one (S3.1, IgG1κ) was selected for in vivo neutralization assays. The monoclonal antibody from this clone was purified from the culture supernatant and tested for its capacity to stain spike-transfected cells and to neutralize SARS virus (FIG. 4). S3.1 neutralized 75 TCID50 SARS virus at concentrations of �300 ng/ml, and was up to 300 fold more potent than convalescent serum. Furthermore S3.1 neutralized the Frankfurt and Urbani isolates with the same efficiency (data not shown), and decorated the spikes of SARS-CoV as detected by immunoelectron microscopy (FIG. 5).
Example 7 S3.1 Neutralizes SARS Infection in an Animal Model The in vivo neutralizing activity of the S3.1 monoclonal antibody was tested in a mouse model of acute SARS infection. Purified antibody was transferred to na�ve mice by intraperitoneal injection to determine whether antibody alone could prevent replication of SARS virus in the respiratory tract.
Example 8 R-848 R-848 is an agonist of TLR7 and TLR8. This compound was compared with CpG 2006 in terms of efficiency of EBV-induced immortalization of human B cells. Memory B cells were isolated from healthy donors using anti-CD19 or anti-CD22 magnetic microbeads followed by negative depletion of cells carrying IgM, IgD and IgA (or IgG). 48 replicate cultures were set up in 96 well U-bottomed microplates by limiting dilution at 30, 10 and 3 B cells per well in complete medium in the presence of irradiated mononuclear cells, EBV (20% supernatant from B95-8 cells) and in the presence or absence of 2.5 μg/ml CpG 2006 or 2.5 μg/ml R-848. The frequency of cultures positive for cell growth and Ig production was measured after 14 days and the efficiency of transformation was calculated. Results were as follows:
Example 9 Isolation of High Affinity Antibodies Neutralizing SARS-CoV A new series of monoclonal antibodies with SARS-CoV neutralizing capacity was produced as described above from immortalized memory B cells isolated from a convalescent patient six months after infection. Serial dilutions of supernatants from the B cell clones were tested for their antigen specificity (NP, matrix (M) or spike proteins), and their capacity to neutralize the cytopathic effect of SARS CoV (Frankfurt isolate) on Vero cells. The concentration of monoclonal IgG was measured by ELISA in the same culture supernatants. Neutralizing titers were expressed as the final concentration of IgG (ng IgG per ml) in tissue culture capable of completely neutralizing the virus (mean values of at least three tests). Results were as follows:
Thus the invention is routinely able to provide antibodies that can neutralize the virus at concentrations lower than 10−9 M and even down to 10−10 M (MW of human IgG is �150 kDa, and so 150 ng/ml is �10−9 M).
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(1996).65Vollmer, Jorg, Immunopharmacology of CpG Oligodeoxynucleotides and Ribavirin, Antimicrobial Agents and Chemotherapy, 2004, 2314-2317, vol. 48, No. 6.Classifications U.S. Classification435/326, 435/375, 435/346International ClassificationC12Q, C12P21/08, C07K16/12, A61P31/14, C12N5/02, C12N5/0781, C07K16/10, G01N33/49, C12N5/00, A61K39/395, C07K16/20, C12N5/24, C07K16/00Cooperative ClassificationC07K16/1282, C12N5/0635, C07K2317/21, C12N2510/04, A61K2039/505, C07K2316/96, C07K16/10, C07K16/20, C07K16/1027, C12P21/005, C12N2510/02, C07K16/00European ClassificationC07K16/10F, C07K16/12B16, C12N5/06B11B, C07K16/10, C07K16/00, C07K16/20Legal EventsDateCodeEventDescriptionMay 22, 2008ASAssignmentOwner name: INSTITUTE FOR RESEARCH IN BIOMEDICINE, SWITZERLANDFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:LANZAVECCHIA, ANTONIO;REEL/FRAME:020985/0227Effective date: 20070515RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google