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WO1993004187A1 - Anticorps monoclonal specifique a un antigene sur des cellules dendritiques - Google Patents

Anticorps monoclonal specifique a un antigene sur des cellules dendritiques Download PDF

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Publication number
WO1993004187A1
WO1993004187A1 PCT/GB1992/001506 GB9201506W WO9304187A1 WO 1993004187 A1 WO1993004187 A1 WO 1993004187A1 GB 9201506 W GB9201506 W GB 9201506W WO 9304187 A1 WO9304187 A1 WO 9304187A1
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cells
mab
cell
dendritic
mrc
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PCT/GB1992/001506
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English (en)
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Mary Brenan
Michael Joseph Puklavec
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Medical Research Council
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily

Definitions

  • the present invention relates to ligands for dendritic cells.
  • the present invention relates to ligands for dendritic cells which are antibodies especially monoclonal antibodies (iriAbs), and to derivatives, fragments and functional equivalents of these antibodies.
  • Lymphoid dendritic cells are a heterogeneous group of cells with- dendritic morphology found in lymphoid and non-lymphoid tissues (reviewed in 1). Dendritic cells originate from bone marrow, but the lineage of dendritic cells is unresolved (reviewed in 2) . Attention has been focused on dendritic cells following the observation that these cells are the most potent accessory cells identified at inducing primary T-cell responses in vitro (3). The immunoregulatory role of dendritic " cells in vivo is implicated by their presence in the T-dependent areas of ⁇ secondary lymphoid organs permitting T-cell activation (4,5) and in the thymic medulla (6).
  • the present application discloses a methodology for making ligands e.g. mAbs which are highly specific for dendritic cells and which also bind a subpopulation of CD3 + cells, and provides a mAb produced by carrying out the disclosed methodology.
  • the antibody and cell line producing this antibody are both designated MRC OX-62.
  • the MRC OX-62 cell line secretes a mouse IgGl mAb, highly restricted to rat dendritic cells and a subpopulation of CD3 + cells as determined by immunocytochemistry, flow cytofluorography and in vitro proliferative responses.
  • the MRC OX-62 mAb immunoprecipitates four bands from radioiodinated dendritic cells under both non-reducing and reducing conditions: a major band of approximately 150 kD apparent Mr, a minor band of approximately 120 kD apparent Mr, and two lower bands ⁇ 30 kD apparent Mr.
  • the 150 kD apparent Mr band is the only band detected by the MRC OX-62 mAb on Western blots.
  • the MRC OX-62 mAb In lymphoid organs, the MRC OX-62 mAb predominantly labels populations of cells with dendritic morphology in the thymic medulla, T-cell areas and red pulp of the spleen, the subcapsular sinus, T-cell areas and medulla of the lymph node, inter-follicular areas and epithelial regions of the Peyer' s patch and veiled cells in lymph. In non-lymphoid organs, the MRC OX-62 mAb labels populations mostly coinciding with known dendritic cell distribution. Enrichment of dendritic cells assessed by potent stimulator activity in the primary allogenic MLR can be effectively achieved by magnetic sorting after labelling cells with the MRC OX-62 mAb.
  • the present invention provides the hybridoma MRC OX-62, mAbs produced by said hybridoma and derivatives, functional equivalents and fragments of these antibodies.
  • hybridoma MRC OX-62 has been deposited under the terms of the Budapest Treaty at ECACC on 18 June 1991 under accession No. 91061805. It will be understood by those skilled in the art that the hybridoma may be subject to genetic mutation or other changes, while still retaining its ability to produce monoclonal antibody of the same specificity. The present invention therefore encompasses mutants, other derivatives and descendants of the hybridoma MRC OX-62.
  • a monoclonal antibody can be subjected to the techniques of recombinant DNA technology to produce derivative other antibodies or chimeric molecules which retain the specificity of the original monoclonal antibody.
  • Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions ( CDR' s ) , of the monoclonal antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin, for example to convert the mouse-derived monoclonal antibody into one having largely human immunoglobulin characteristics (see EP 184187A, GB 2188638A).
  • EP-A-O 120 694 (Boss et al/Celltech) describes the cloning and expression of chimeric antibodies.
  • the variable domains from one immunoglobulin are fused to constant domains from another immunoglobulin.
  • the variable domains are derived from an immunoglobulin from one species, say a mouse or a rat, and the constant domains are derived from an immunoglobulin from a different species, perhaps a human.
  • This technology is now very well known in the art.
  • Another possibility is to attach just the variable region of the monoclonal antibody to another non- immunoglobulin molecule, to produce a derivative chimeric molecule (see WO 86/01533, Neuberger and Rabbits/Celltech) .
  • a further possibility would be to produce a chimeric immunoglobulin having different specificity in its different variable regions, one of which is that the monoclonal antibody of the present invention (see EP 68763A).
  • Yet another possibility would be to produce a mutation in the DNA encoding the monoclonal antibody, so as to alter certain of its characteristics without changing its essential specificity. This can be done by site-directed mutagenesis or other techniques known in the art.
  • the famous Winter patent application EP-A-0 239 400 describes how it is possible to make an altered, derivative, antibody by replacing the complementarity determining regions (CDRs) of a variable region of an immunoglobulin with the CDRs from an immunoglobulin of different specificity, using recombinant DNA techniques, so called "CDR-grafting” .
  • CDRs complementarity determining regions
  • This enables antigens-binding specificity of one antibody (in the present case it might be MRC OX-62 mAb or an antibody with the same binding specificity or an antibody which is cross- reactive with MRC OX-62 mAb) to be transferred to another antibody.
  • This enables "humanisation" of antibodies.
  • a "humanised” antibody with the CDRs of a rat antibody specific for an antigen of interest might well be less likely to be recognised as foreign by the immune system of a human. It follows that a "humanised” antibody with the same binding specificity as MRC OX-62 mAb or cross-reactive with it (see later), might well be of particular use in human therapy and/or diagnostic methods.
  • the uses of an antibody according to the present invention are discussed later in the text. All such uses of the monoclonal antibody are encompassed by the present invention.
  • MRC OX-62 mAb allows persons skilled in the art to obtain binding partners e.g. antigens or epitopes which bind to it. These partners may be rat dendritic cells originally utilised as the antigen, the novel subpopulation of CD3 + cells identified using MRC OX-62 mAb, or ho ologue cell populations from other species, or parts e.g. epitopes of said cells.
  • binding partners e.g. antigens or epitopes which bind to it.
  • These partners may be rat dendritic cells originally utilised as the antigen, the novel subpopulation of CD3 + cells identified using MRC OX-62 mAb, or ho ologue cell populations from other species, or parts e.g. epitopes of said cells.
  • the ability to obtain these binding partners by use of MRC OX-62 mAb will overcome the prior art problem relating to the difficulty in obtaining large numbers of dendritic cells for immunization. Therefore, the present invention also
  • binding partners obtained by contacting a sample suspected of containing a binding partner with MRC OX-62 mAb, are provided.
  • the binding partner may comprise part or all of a 150 kD protein derived from dendritic cells.
  • the binding partners obtained by use of the MRC OX- 62 mAb may also be used to produce further ligands e.g. antibodies (or molecules having antibody-like binding function e.g. fragments, derivatives and synthetic analogues of antibodies) other than the MRC OX-62 mAb. Therefore, also provided are ligands e.g. mAbs which are able to bind with a binding partner which is able to bind with the MRC OX-62 mAb. Such ligands (“cross- reactive ligands”) e.g. mAbs may recognize different epitopes, or the same epitope as recognized by MRC OX-62 mAb on said binding partner.
  • the present invention also provides derivatives, functional, equivalents (e.g. a molecule having an antibody-like binding specificity) and fragments of said cross-reactive ligands, perhaps produced using one or more of the techniques of recombinant DNA technology referred to and discussed above. Also included are single domain ligands (dAbs) as described in WO 90/05144 (Winter et al/MRC).
  • dAbs single domain ligands
  • a first round of immunoaffinity uses the MRC OX-62 mAb to take out from a sample the antigen binding partner, which may then be used in the column to select from a heterogeneous population of ligands, those ligands which are cross-reactive with the MRC OX-62 mAb, ie which bind the same binding partner.
  • a binding partner isolated using the MRC OX-62 mAb maybe used to select cross-reactive ligands from a repertoire or heterogenous population of antibodies generated by a whole variety of means.
  • One way is to select monoclonal antibodies and cell lines producing them by the standard hybridoma techniques.
  • Also provided by the present invention are immortalised cells e.g. hybridomas producing said cross-reactive ligands.
  • the present invention also provides processes for the preparation of immortalised cell lines (e.g. hybridomas ) , and ligands produced by these immortalised cell lines which comprise immunising a mammal with an antigenic binding partner which binds with said MRC OX- 62 mAb; immortalising antibody-producing cells from said mammal; selecting immortalised cells producing a ligand which binds with said binding partner; and cloning said selected cells.
  • immortalised cell lines e.g. hybridomas
  • ligands produced by these immortalised cell lines which comprise immunising a mammal with an antigenic binding partner which binds with said MRC OX- 62 mAb; immortalising antibody-producing cells from said mammal; selecting immortalised cells producing a ligand which binds with said binding partner; and cloning said selected cells.
  • the antibody-producing cells may be spleen cells.
  • the immortalisation step may be carried out by fusing said spleen cells with myeloma cells according to techniques known in the art.
  • Another way of selecting ligands which are cross- reactive with the MRC OX-62 mAb is to use the methods for producing members of specific binding pairs disclosed in WO 92/01047 (Cambridge Antibody Technology Limited and MRC/McCafferty et al ) . This discloses expression of polypeptide chain components of a genetically diverse population of specific binding pair members, such as antibodies, fused to a component of a secreted replicable genetic display package (RGDP), such as a bacteriophage,. which thereby displays the polypeptide on the surface.
  • RGDP secreted replicable genetic display package
  • Very large repertoires of displayed antibodies may be generated, and screened by means of antigen binding to obtain one or more antibodies of interest, along with their encoding DNA.
  • DNA encoding for a polypeptide displayed on the surface of an RGDP is contained within that RDGP and may therefore be easily isolated and cloned for expression.
  • the antibody repertoire screen may of course be derived from a human source.
  • the present invention also encompasses primary nucleotide sequences which encode the ligands e.g. mAbs as defined above, together with fragments of these primary sequences and secondary nucleotide sequences comprising derivatives, mutations and hybridising partners of said primary nucleotide sequences.
  • the present invention includes vectors (cloning and expression vectors ) incorporating said nucleotide sequences, transformed cells incorporating said vectors and expression products produced by use of a recombinant system utilising any such vectors or transformed cells.
  • the present invention also includes methods for expressing a ligand e.g. a mAb, derivative, functional equivalent or fragment thereof, which comprises using a nucleotide sequence, vector or transformed cell as defined above.
  • MRC OX-62 mAb will bind to an epitope on rat dendritic cells.
  • This epitope may then be purified, for instance utilising an immunoaffinity column (as discussed), and partially or wholly sequenced, for instance using repeated rounds of Edman degradation.
  • An analysis of the sequence allows the production of a nucleotide sequence (e.g. the construction of a synthetic nucleotide sequence) which encodes at least part or all of the epitope.
  • the nucleotide sequence can then be used as a probe to screen rat dendritic cells for hybridising mRNA species. Conversion of the hybridising mRNA into cDNA provides a rat cDNA probe which can be used to identify human homologues of the rat binding partner for MRC OX-62. Two routes are possible and other routes will be apparent to those skilled in the art:
  • PCR polymerase chain receptor
  • the conditions for the polymerase chain receptor (PCR) might involve trials at a number of low initial temperatures to accommodate any mismatches (Gould, S.J., Subramani, S. and Scheffler, I.E. (1989) Proc. Natl. Acad. Sci. USA 86, 1934-1938) but otherwise under standard conditions.
  • the PCR products can then be analysed by sizing on agarose or other gels, transfer to nitrocellulose or similar membranes and probed with the rat cDNA under non- stringent conditions for cross species hybridization.
  • PCR products corresponding to human homologue will be sequenced by standard methods. (These products could also be used as probes as in ( 1 ) for screening cDNA or genomic libraries).
  • a cDNA for the human homologue allows the testing of various human cell populations for expression of the human ' homologue either by PCR or by Northern blot analysis.
  • Production of a human homologue using for example prokaryotic or eukaryotic expression systems will provide material for the production of ligands e.g. polyclonal or monoclonal antibodies or other proteins with an epitope-binding function.
  • the MRC OX-62 mAb, derivatives, fragments and functional equivalents may provide means for manipulating antigen presentation in the tissues. Coupling a peptide onto dendritic cells by means of the antibody may permit more effective presentation of that peptide as antigen for T-cell mediated immunity. Such an adjurant effect would be useful in vaccination strategies.
  • the mAbs, derivatives, functional equivalents and fragments thereof as defined above have a number of useful applications based upon their specificity to dendritic cells and a novel subpopulation of CD3 + cells.
  • OX-62 can be used for the pretreatment of tissue and organ grafts and for the management of grant rejection.
  • the present invention encompasses a preparation for the treatment of foreign tissue which comprises a ligand e.g. an antibody as defined above or a fragment, derivative or functional equivalent thereof in a perfusion medium.
  • a ligand e.g. an antibody as defined above or a fragment, derivative or functional equivalent thereof in a perfusion medium.
  • the foreign tissue may be any tissue which would be seen by a recipient as not originating from said recipient.
  • the foreign tissue may be xenogeneic or allogenic.
  • the present invention also includes pharmaceutical preparations for the inactivation and/or depletion of dentritic cells from cancers or tissues adversely affected by autoimmune disease, which comprise a ligand e.g. an antibody, or a fragment, derivative or functional equivalent thereof, as herein provided, together with one or more excipients.
  • a ligand e.g. an antibody, or a fragment, derivative or functional equivalent thereof, as herein provided, together with one or more excipients.
  • the ligands e.g. antibodies or fragments, derivatives or functional equivalents thereof may also be used to diagnose the accumulation of dendritic cells which is indicative of an abnormal disease state. Therefore, the present invention also includes a diagnostic reagent which comprises a ligand e.g. an antibody, or a fragment, derivative or functional equivalent thereof, as herein provided, together with one or more exipients; a diagnostic kit comprising a diagnostic reagent and a diagnostic method which comprises contacting a ligand e.g. antibody, fragment, derivative or functional equivalent thereof as herein provided with a clinical tissue sample and detecting the binding of said ligand to said sample.
  • a diagnostic reagent which comprises a ligand e.g. an antibody, or a fragment, derivative or functional equivalent thereof, as herein provided, together with one or more exipients
  • a diagnostic kit comprising a diagnostic reagent and a diagnostic method which comprises contacting a ligand e.g. antibody, fragment, derivative or functional equivalent
  • the ligands as provided above and/or binding partners for these ligands can be used for the targeting of antigens. Therefore the present invention encompasses an antigen targeting method which comprises using a ligand and/or binding partner as herein provided to target an antigen.
  • the present invention also provides materials comprising a ligand and/or binding partner as herein provided for use in such a method.
  • OX-62 mAb Binding of OX-62 mAb to PVG rat lymphoid tissues. Cryostat sections ( 5 ⁇ m) of PVG thy us, spleen, cervical lymph node and Peyer's patch labelled using the indirect immunoperoxidase method with OX-62 mAb and isotype- matched negative control OX-21 mAb.
  • OX-62 mAb Binding of the OX-62 mAb to different isolated PVG cell populations. Cells were labelled with saturating levels of OX-62 mAb (-) and OX-21 mAb ( — ) followed by a second incubation with RAM-FITC. Bound mAb was measured by flow cytometry.
  • A TDL
  • B MLNX TDL
  • C MLNX TDL gradient enriched for dendritic cells (50% enrichment)
  • D PBL
  • E bond marrow cells
  • F thymocytes
  • G splenocytes
  • H lymph node cells
  • I resident peritoneal exudate cells
  • J thioglycollate elicited peritoneal exudate cells
  • K LPS blasts
  • L Con A blasts.
  • mice Balb/c (H-2 d ) and DBA/2 (H-2 d ) inbred mice were obtained from the Sir William Dunn School of Pathology, Oxford. FI hybrids between these two strains were bred at the MRC Cellular Immunology Unit, Oxford.
  • Rats: PVG (RT1 C ) and AO (RT1 U ) specific pathogen- free inbred rats were obtained from the MRC Cellular Immunology Unit, Oxford.
  • Suitable animals may be obtained from standard suppliers.
  • MLNX Mesenteric lymphadenectomized rats were allowed to recover for a minimum of 6 wks before thoracic duct cannulation.
  • Cell Populations Thoracic duct leukocytes (DL) were obtained by thoracic duct cannulation of normal and MLNX rats (22) . Cells were collected overnight into ice cold glucose saline (23) containing 20U/ml heparin. Density gradient enriched dendritic cells were prepared by centrifugation of MLNX T.DL over NycoprepTM 1.068 (Nycomed, Oslo, Norway).
  • Splenocyte ⁇ , lymph node cells and thymocytes were obtained by removing appropriate organs and teasing into single cell suspensions in PBS/0.25% BSA.
  • PBL were prepared from blood obtained by cardiac puncture and separated over Isopaque-Ficoll.
  • Bone marrow cells were obtained by flushing the marrow cavity of the femur with PBS.
  • Resident peritoneal exudate cells were obtained from the peritoneal cavity of freshly killed rats. Elicited peritoneal exudate cells were obtained from the peritoneal cavity 4 d after i.p. injection of 10 mis of thioglycollate broth.
  • Con A blasts were obtained following 3 d culture of lymph node cells at 10 6 /ml in RPMI 1640 supplemented with 5% heat inactivated FCS, 2mM glutamine, ImM sodium pyruvate, 2.5 x 10 ⁇ 5 M 2- mercaptoethanol and antibiotics (supplemented RPMI) and 5 ⁇ g/ml Con A. Con A blasts were separated over Isopaque-Ficoll and washed in -methyl-D-mannoside (20 mg/ml ) . LPS blasts were obtained following 2 d culture of lymph node cells at 10°/ml in supplemented RPMI 1640 and 10 ⁇ g/ml LPS.
  • Monoclonal antibodies OX-62 was produced by immunizing a Balb/c mouse with rat PVG density gradient enriched dendritic cells obtained from the cannulated thoracic duct of MLNX rats.
  • the immunization procedure comprised of a combination of i.v. and i.p. injections with 5-10 x 10" cells at monthly intervals. 5 d after the last injection the splenocytes were fused with NSO myeloma cells according to the method of Galfre & Milstein (24). After growth of the hybrid cell lines supernatants were screened for specificity by immunocytochemistry (see below). Selected hybridomas were cloned twice by limiting dilution.
  • Ascites fluid was prepared in (Balb/c X DBA/2)F1 mice pretreated with pristane.
  • the subclass of the mAbs were determined by an anti-mouse monoclonal isotyping kit (Serotec, Kidlington, Oxon. England.).
  • mAbs used were SN3 (mouse anti-squid Sgp 1) (25), OX-6 (mouse anti-rat MHC class II: IgGl ) (26), 0X- 8 (mouse anti-rat CD8: IgGl) (27), OX-12 (mouse anti-rat IgG kappa chain: IgG2a) (28), OX-17 (mouse anti-rat MHC class II: IgGl) (29).
  • OX-21 (mouse anti-human C3b inactivatorc IgGl) (3); OX-30 (mouse anti-rat CD45: IgG2a) (31) and OX-42 (mouse anti-rat CD18/CDllb: IgG2a) (32).
  • MAbs were used as tissue culture supernatants and purified IgG from ascites fluid (33).
  • Other antibodies used were rabbit anti-mouse IgG mAb peroxidase conjugated (RAM-peroxidase) (Dako, High Wycombe, Bucks. England.), and affinity purified rabbit F(ab')2 anti-mouse IgG mAb (Serotec, Kidlington, Oxon.
  • Cryostat sections Thymuses, spleens, cervical lymph nodes and Peyer's patches were removed from 8-12 wk PVG rats embedded in Tissue-TekTM OCT (Miles,
  • Isolated cells Cytospin preparations were made onto glass slides using a cytocentifruge, air dried and stored dessicated at -70°C.
  • cryostat sect ions , ep i dermal sheets and i s ol ated ce l l preparations were fixed in ethanol, washed and incubated with mAb tissue culture supernatant, washed and incubated with RAM-peroxidase. Peroxidase was visualized using 3.3 ' -diaminobenzidine tetrahydrocholoride. Slides were lightly counterstained with Harris' haematoxylin, dehydrated and mounted in DPX. Epidermal sheets were stained in wells and incubated with mAb tissue culture supernatants overnight and with RAM-peroxidase for 90 min. Flow cytofluorography
  • the magnetic separation was carried out at 40°C. Labelled cells were added to the appropriately sized column which had been preincubated with PBS/0.25%BSA/10 mM NaN 3 washed and placed in the magnetic field. Unlabelled cells were collected in the first 10-15 ml volume. The column was washed to remove unlabelled, weakly bound and dead cells. Magnetically labelled cells were eluted from the column outside the magnetic field using a 50 ml syringe filled with PBS/0.25%BSA/10 mM NaN 3 . In some experiments enriched and depleted populations were further depleted of contaminating cells by a second magnetic sort. The efficiency of depletion and enrichment was determined by flow cytofluorography using a FACScan. MLR
  • Cell surface radioiodination and immunoprecipitation 10 7 density gradient enriched cells were surface labelled by an H 2 0 2 1actoperoxidase-catalysed radioiodination method (38). Labelled cells were solubilized in 500 ⁇ l lysis buffer comprising 10 mM tris pH 7.5-8 150 mM NaCl, 5 mM Na 2 EDTA, 1% Nonidet P-40, ImM PMSF and 5 mM iodoacetamide for 30 min at 40°C.
  • Immunoprecipitated samples were washed in the following buffers (1) 10 mM tris-HCI pH 8.0 containing 500 mM NaCl, 0.5% NP-40 and 0.05% SDS (b) 10 mM tris-HCI pH 8.0 containing 150 mM NaCl, 0.5% Np-40, 0.5% sodium deoxycholate and 0.05% SDS (c) 10 mM tris-HCI pH 8.0 containing 0.05% SDS. Immunoprecipitates were released from the beads by boiling for 4 min in SDS-PAGE sample buffer. Samples and molecular weight markers (RainbowTM protein molecular weight markers. Amersham, Bucks, England.
  • Membranes were placed in PBS containing 5% dried non-fat milk powder (MarvelTM) for 24 h at 40°C. Ascites fluid containing the appropriate mAb was added to give a final concentration of 1-50 ⁇ g/ml and the membranes incubated for 2 h at 40°C with rocking. The membranes were washed three, times in PBS/0.05% TweenTM 20 before addition of 1 25I-RAM (specific activity approximately 1.5 ⁇ Ci/ ⁇ g IgG at a dilution of 10 5 cpm/ml) in PBS/0.05% Tween 20/1% BSA. After 1 h at 40°C the membranes were washed three times in PBS/0.05% Tween 20 and exposed to HyperfilmTM at -70°C.
  • Ascites fluid containing the appropriate mAb was added to give a final concentration of 1-50 ⁇ g/ml and the membranes incubated for 2 h at 40°C with rocking. The membranes were washed three, times
  • OX-62 mouse anti-rat dendritic cell IgGl mAb
  • Cannulated MLNX rats were used as a source of dendritic cells for injection. Isolating dendritic cells from lymph compared with lymphoid organs circumvents lengthy isolation procedures and possible modification of surface phenotypes (43). 2. Large numbers of dendritic cells (5-10 x 10°) were injected per mouse and each mouse received at least three injections. 3.
  • OX-62 + cells were present in the medulla forming a diffuse network (Fig. IA). OX-62 + cells were also present in the lobular septae with occasional cells present in the cortex (Fig. IA) correlating with the large scattered cells observed in the cortex in bone marrow chimaeras (6). In the spleen QX-62 + cells were concentrated in the T-dependent areas particularly around the region of the central arteriole but also throughout the red pulp (Fig. 1C). Labelling of cells in the red pulp was reported for NLDC 145 (13) and N418 (18) mAbs which recognize mouse dendritic cells.
  • OX-62 + cells were present in the inter- follicular areas and in the epithelial dome regions but were absent from the follicles (Fig. 1G). Background staining with an isotype matched mAb OX-21 was negligible (Fig. IB, D, F and G). 0X-62 + cells represented a minor population of the OX-6 + and OX-42 + cells (data not shown). Distribution of the OX-62 Antigen in Nonlymphoid Organs
  • OX-62 + cells with dendritic morphology that coincided with known MHC class II dendritic cell distribution (48, 63) were present in the lamina intestinal of the small intestine (Fig. 2, A and B), interstitium of the lung, portal triads of the liver, glomeruli of the kidney, islets of Langerhans of the pancreas, and epithelium of the choroid plexus (data not shown). Lack of 0X-62 + cells in heart and skeletal muscle (data not shown) contrasts with previous studies using anti-MHC class II mAbs (48, 63 and 64).
  • the OX-62 mAb revealed populations of 0X-62 + MHC class II " cells in the epithelium of the small intestine and in the epidermis of the skin. In the epithelium of the small intestine, only granular MHC class II staining has been reported (Fig. 2B; and 47), but this staining was not associated with the OX-62 + intraepithelial cells (Fig. 2A). In epidermal sheets, OX-62 mAb labelling primarily revealed cells with marked dendritic morphology comprising the cell body and nonoverlapping dendritic processes (Fig. 2C) in contrast to OX-6 mAb labelling, which primarily revealed cells with less prominent dendrites (Fig. 2D).
  • Double immunofluorescence showed that most if not all the OX- 62 "1" and MHC class II + populations were nonoverlapping (Fig. 2, E, F, and G).
  • the OX-62 + MHC class II " cells in the small intestine and the skin were CD3 + (data not shown) and probably represent gamma/delta T cells (65- 67).
  • the identification of nonoverlapping OX-62 + MHC class II” and OX-62 " MHC class II + populations in the skin with a similar distribution raises the question as to which cells migrate into the afferent lymphatics giving rise to the OX-62 + MHC class II + veiled cells.
  • Previous work has indicated that MHC class II + Langerhans cells (68) are the probably precursors (50, 69-71).
  • the presence of dendritic cells in autoimmune diseases has been documented (51 and reviewed in 52) therefore the distribution of OX-62 + cells in cryostat sections from rats with autoimmune diabetes and experimental allergic encephalomyelitis were examined.
  • the OX-62 mAb labelled increased numbers of OX-62 + cells in the islets of Langerhans with leukocyte infiltrates in the pancreas of rats with autoimmune diabetes when compared to control non-diabetic rats (data not shown).
  • increased OX-62 mAb labelling of cells in lesions in the spinal cord and brain of rats with experimental allergic encephalomyelitis was found in contrast to normal rats where OX-62 + cells are present in the choroid. plexus (data not shown). It could be that the increased labelling represents upregulation of the OX-62 antigen or migration of OX-62 + cells into the lesions, but what has taken place has not been established. Expression of the OX-62 antigen by different cell types
  • Fig. 3 shows FACScan profiles of different isolated types following labelling with the OX-62 mAb.
  • Resident peritoneal macrophages Fig. 31
  • exudate peritoneal macrophages Fig. 3J
  • LPS blasts Fig. 3K
  • Con A blasts Fig. 3L
  • Heterogeneous populations such as spleen, thymus and lymph node cells known to contain dendritic cells in low numbers by immunocytochemistry (Fig. IA, C and E) gave staining with the OX-62 mAb at a level barely distinguishable from background (thymocytes Fig. 3F, splenocytes Fig. 3G and lymph node cells Fig. 3H).
  • Dendritic cells can either be obtained in suspension in lymph or from lymphoid organs with or without enzyme digestion and adherence. Dendritic cells represent approximately 1% or less of these populations therefore further enrichment is dependent either on negative selection using relevant mAbs and/or non-specific separation using density gradients (reviewed in 60) . Enrichment of dendritic cells by labelling cells with the N418 mAb followed by cell sorting using flow cytofluorography has been reported (61) but the low recoveries inherent in cell sorting using flow cytofluorography (30% or less) combined with the low frequency of labelled cells in the presort population limits the number of dendritic cells obtainable.
  • the ability of the OX-62 mAb to enrich and deplete for OX-62 labelled cells was assessed phenotypically by flow cytofluorography using a FACScan after magnetic cell sorting and functionally using magnetically sorted cells as stimulators in the primary allogeneic MLR.
  • the OX-62 mAb in combination with magnetic sorting gave- 80% enrichment and recoveries were approximately 90%.
  • the potency of the cell populations separated after labelling with the OX-62 mAb and used as stimulators in the primary allogeneic MLR is shown in Fig. 4.
  • the OX-62 enriched cells were potent stimulators with 1.22 x 10 ⁇ cells per culture providing the same stimulus as 15.5 x 10 ⁇ unseparated cells.
  • the depleted cells had 12% of the activity of the unseparated cells indicating that the OX-62 + cells were the major stimulating cell in the MLR. It can be argued that almost all the stimulating activity is from OX-62 "1" cells since the activity of the depleted cells is in accord with the level of OX-62 + cell contamination.
  • the OX-62 mAb in combination with magnetic sorting represents a useful method for enriching for dendritic cells in large numbers with greater than 80% purity more quickly than by cell sorting using flow cytofluorography. To obtain purer populations of dendritic cells magnetically sorted cells could be sorted by flow cytofluorography.
  • the anti-CD4 mAb W3/25 used as a positive control inhibited in the concentration range from 1.25 ng/ml to 2.5 ⁇ g/ml (data not shown) .
  • 0X-42 and OX-30 which are expressed on dendritic cells failed to preclear the bands immunoprecipitated by the OX-62 mAb (data not shown). .
  • the 150 kD apparent Mr band was the only band detected by the OX-62 mAb on Western blots (Fig. 5B).
  • the biochemical properties of the OX-62 binding partner antigen are characteristic of an integrin.
  • Certain members of the integrin family under reducing conditions immunoprecipitate four bands representing the alpha and beta subunits (higher apparent Mr bands) and alternatively spliced cleavage products of the alpha subunit (lower apparent Mr bands) (reviewed in 60).
  • the strong 150 kD band present under non-reducing and reducing conditions is alpha-subunit like and the weaker 120 kD band, which migrated faster under non-reducing conditions, is beta-subunit like.
  • This 120 kD band could be present as a result of co-precipitation with the alpha-subunit (recognised by the OX-62 antibody), due to noncovalent association of the alpha and beta subunits.
  • the presence of the two bands of less than 30 kD under non-reducing (fig. 5A (1)) and reducing (fig. 5A(2)) conditions, perhaps representing cleaved fragments, is not the same as previously described integrin alpha-subunits.
  • MRC OX-62 is a mAb highly restricted to dendritic cells in lymphoid and non-lymphoid organs, and to a novel subpopulation of CD3+ cells with dendritic morphology.
  • the hybridoma MRC OX-62 has been deposited in accordance with the rules and regulations of the Budapest Treaty with the European Collection of Animal Cell Cultures (ECACC, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, UK) on 18 June 1991 under the accession number 91061805.
  • Lymphoid dendritic cells are potent stimulators of the primary mixed leukocyte reaction in mice. Proc. Natl. Acad. Sci. 75:5132.
  • VLA proteins in the integrin family structures, functions, and their role on leukocytes. Ann. Rev. Immunol. 8:365.

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Abstract

On décrit des ligands qui lient un partenaire de liaison limité à des cellules dendritiques, ainsi qu'une nouvelle sous-population de cellules CD3+. Un hybridome, MRC OX-62, qui produit des anticorps présentant cette spécificité de liaison, a été déposé sous le numéro d'entrée ECACC 91061808. Des techniques permettant d'obtenir d'autres ligands, mutants, dérivés et équivalents fonctionnels sont décrits, ainsi que leurs utilisations. L'anticorps précipite immunologiquement les antigènes de cellules dendritiques à Mr apparent de 150 KD environ (bande principale), à Mr apparent de 120 KD (bande mineure) et à deux bandes inférieures à Mr apparent de moins de 30 KD.
PCT/GB1992/001506 1991-08-14 1992-08-14 Anticorps monoclonal specifique a un antigene sur des cellules dendritiques WO1993004187A1 (fr)

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WO1995012409A1 (fr) * 1993-11-04 1995-05-11 Canterbury Health Limited Anticorps specifiques des cellules dendritiques et leurs procedes de preparation
US6004807A (en) * 1992-03-30 1999-12-21 Schering Corporation In vitro generation of human dendritic cells
WO2000000592A1 (fr) * 1998-06-27 2000-01-06 Yong Soo Bae Anticorps monoclonal 3-6-a specifique a la surface des cellules dendritiques parmi les leucocytes du sang peripherique
WO1999058678A3 (fr) * 1998-05-11 2000-02-10 Micromet Gmbh Anticorps des cellules dendritiques et populations de cellules dendritiques humaines et utilisation de ceux-ci
EP1028754A4 (fr) * 1997-11-12 2002-11-27 Univ Pennsylvania Prevention d'une immunoreactivite par depletion ou inhibition de cellules presentant l'antigene
US7560534B2 (en) 2000-05-08 2009-07-14 Celldex Research Corporation Molecular conjugates comprising human monoclonal antibodies to dendritic cells
US7563876B2 (en) 2000-05-08 2009-07-21 Celldex Therapeutics, Inc. Human monoclonal antibodies to dendritic cells
US8236318B2 (en) 2007-11-07 2012-08-07 Celldex Therapeutics Inc. Antibodies that bind human dendritic and epithelial cell 205 (DEC-205)
US9243064B2 (en) 2003-01-31 2016-01-26 Celldex Therapeutics Inc. Antibody vaccine conjugates and uses therefor
US9259459B2 (en) 2003-01-31 2016-02-16 Celldex Therapeutics Inc. Antibody vaccine conjugates and uses therefor
WO2022074206A1 (fr) 2020-10-08 2022-04-14 Affimed Gmbh Lieurs trispécifiques
WO2023007023A1 (fr) 2021-07-30 2023-02-02 Affimed Gmbh Corps duplex

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6004807A (en) * 1992-03-30 1999-12-21 Schering Corporation In vitro generation of human dendritic cells
WO1995012409A1 (fr) * 1993-11-04 1995-05-11 Canterbury Health Limited Anticorps specifiques des cellules dendritiques et leurs procedes de preparation
US5876917A (en) * 1993-11-04 1999-03-02 Canterbury Health Limited Dendritic cell-specific antibodies and method for their preparation
EP1028754A4 (fr) * 1997-11-12 2002-11-27 Univ Pennsylvania Prevention d'une immunoreactivite par depletion ou inhibition de cellules presentant l'antigene
US7288255B1 (en) 1997-11-12 2007-10-30 Yale University Prevention of immunoreactivity by depleting or inhibiting antigen presenting cells
WO1999058678A3 (fr) * 1998-05-11 2000-02-10 Micromet Gmbh Anticorps des cellules dendritiques et populations de cellules dendritiques humaines et utilisation de ceux-ci
WO2000000592A1 (fr) * 1998-06-27 2000-01-06 Yong Soo Bae Anticorps monoclonal 3-6-a specifique a la surface des cellules dendritiques parmi les leucocytes du sang peripherique
US7563876B2 (en) 2000-05-08 2009-07-21 Celldex Therapeutics, Inc. Human monoclonal antibodies to dendritic cells
US7560534B2 (en) 2000-05-08 2009-07-14 Celldex Research Corporation Molecular conjugates comprising human monoclonal antibodies to dendritic cells
US8142790B2 (en) 2000-05-08 2012-03-27 Celldex Research Corporation Methods of using molecular conjugates comprising monoclonal antibodies to dendritic cells
US9095626B2 (en) 2000-05-08 2015-08-04 Celldex Therapeutics, Inc. Monoclonal antibodies to dendritic cells
US9243064B2 (en) 2003-01-31 2016-01-26 Celldex Therapeutics Inc. Antibody vaccine conjugates and uses therefor
US9259459B2 (en) 2003-01-31 2016-02-16 Celldex Therapeutics Inc. Antibody vaccine conjugates and uses therefor
US8236318B2 (en) 2007-11-07 2012-08-07 Celldex Therapeutics Inc. Antibodies that bind human dendritic and epithelial cell 205 (DEC-205)
US8362214B2 (en) 2007-11-07 2013-01-29 Celldex Therapeutics Inc. Antibodies that bind human dendritic and epithelial cell 205 (DEC-205)
US8586720B2 (en) 2007-11-07 2013-11-19 Celldex Therapeutics Inc. Antibodies that bind human dendritic and epithelial cell 205 (DEC-205)
US9624300B2 (en) 2007-11-07 2017-04-18 Celldex Therapeutics Inc. Antibodies that bind human dendritic and epithelial cell 205 (DEC-205)
WO2022074206A1 (fr) 2020-10-08 2022-04-14 Affimed Gmbh Lieurs trispécifiques
WO2023007023A1 (fr) 2021-07-30 2023-02-02 Affimed Gmbh Corps duplex

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