EP0787151A1 - Fragments d'anticorps monoclonaux possedant une activite immunodepressive - Google Patents

Fragments d'anticorps monoclonaux possedant une activite immunodepressive

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Publication number
EP0787151A1
EP0787151A1 EP95941017A EP95941017A EP0787151A1 EP 0787151 A1 EP0787151 A1 EP 0787151A1 EP 95941017 A EP95941017 A EP 95941017A EP 95941017 A EP95941017 A EP 95941017A EP 0787151 A1 EP0787151 A1 EP 0787151A1
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Prior art keywords
fab fragment
mab
fab
cells
hla
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German (de)
English (en)
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Zoltan Nagy
Damir Vidovic
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F Hoffmann La Roche AG
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F Hoffmann La Roche AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • MHC major histocompatibility complex
  • Th cells helper cells
  • mAb Monoclonal antibodies specific for class II MHC molecules have been shown to be ex-tremely potent selective inhibitors of Th cell responses in vitro (Ref. 2). Since their discovery, they have been considered as potential drugs for selective immuno ⁇ suppressive treatment of autoimmune disorders, such as rheumatoid arthritis.
  • Initial in vivo studies demonstrated the beneficial influence of these mAbs on Th-cell mediated hetero- and autoimmune responses (Refs. 3-6).
  • Fab monovalent mAb fragments
  • the Fab fragments of the invention are therefore potent class II MHC-specific immuno ⁇ suppressive compounds without cytotoxic side effects.
  • FIG. 1 Effects of a DR binding competitor peptide and a DR- specific mAb on the EBV transformed B cell line Priess.
  • Fig. 2 Time course of modulatory and cytotoxic effects of mAb L243 on LG2.
  • Fig. 4 Downregulation of HLA-DR expression on different APC populations after co-culture with DR specific mAb L243 and its fragments.
  • Fig. 5 Effects of increasing concentrations of DR specific Fab fragment on the EBV transformed B cell line LG2.
  • Fig. 6 Effects of prolonged co-culture of L243 and its fragments on LG2 cells.
  • Fig. 8 Selectivity of DR downregulation on resting B cells and monocytes/macrophages.
  • Fig. 10 Selectivity of DR downregulation on LG2 cells.
  • Fig. 12 Pan-class II downregulation on TS-10 cells by 1-1C4 Fab.
  • Fig. 13 Lack of TNF ⁇ secretion increase upon co-culture of LG2 and Priess cells with L243 and its fragments.
  • Fig. 14 Antibody concentration requirement for Th cell inhibition and DR downregulation.
  • Fig. 15 Effect of anti-DR mAb and Fab fragments on antigen presentation by fixed APC.
  • Fig. 16 Effect of antigen load on the potency of mAb, Fab, and peptide antagonists.
  • Fig. 17 Relative effects of Fab and peptide on antigen dose- response curves.
  • Fig. 18 Effect of class II antagonists on ongoing Th cell response.
  • HLA-DR Human Leukocyte Antigen of the type "DR”; a class II Major Histocompatibility Complex (“MHC”) molecule
  • MHC Major Histocompatibility Complex
  • the same mAbs also inhibit the activation of human Th cell clones that require antigen presentation by HLA-DR molecules for activation.
  • the inhibitory potency of such mAbs is several 100 to several 1000 fold higher than that of the currently available peptide antagonists (see Table 1, below).
  • This downregulation of the expression of HLA-DR and the inhibition of the activation of Th cells is a pharmacological activity which results in immunosuppression.
  • This immunosuppression would be useful in the treatment of autoimmune diseases, especially rheumatoid arthritis.
  • the present invention comprises a Fab fragment of an anti-HLA-DR mAb wherein said intact mAb is cytotoxic to antigen presenting cells and downregulates HLA-DR expression on the remaining antigen presenting cells. Such a mAb inhibits Th cell activation.
  • the mAbs from which the Fab fragments of the present invention are derived all bind to the first domain of HLA-DR.
  • SFR3-DR5 (rat IgG 2b , DRBl*110X-specif ⁇ c; ref. 13; ATCC Accession No. HB-151).
  • HLA-DR downregulating mAb, 1-1 C4 (mouse IgG2a > ⁇ -chain specific; ref. 14), which additionally downregulates HLA-DQ and -DP, was generated by conventional means as described in Example 22.
  • the Fab fragments of the above mAbs are encompassed by the present invention.
  • CCCL20 mouse IgG2 b , specific for three DRB 1 ( ⁇ -chain) allelic forms (DRB1*0101, DRB1*0401, DRB1 *0404); ref. 12) and 8D1, 9F1, 9F2, 10F12 (all four mouse IgGl), inhibit Th cell activation only very weakly or not at all.
  • the active mAbs are cytotoxic to B lymphoblastoid cells and to a small proportion of normal activated B cells.
  • the bivalent F(ab)'2 fragments of these mAbs mediate downregulation but are also cytotoxic.
  • the monovalent Fab fragments of these mAbs loose cytotoxicity, but surprisingly retain the downregulating property of the parent mAb.
  • the anti-HLA-DR mAbs used to obtain the Fab fragments of the invention may be produced by any conventional means, e.g., generally by the procedure first described by Kohler and Milstein.
  • monoclonal antibodies can be prepared by recovering antibody producing cells from such an immunized animal and immortalizing said cells obtained in conventional fashion like fusion with myeloma cells, e.g., PAI mouse myeloma cells, SP2/0- or SP2/0-Agl4-cells [ATCC No. CRL 1581 ; ATCC No.
  • CRL 8287 [for a general guideline for producing antibodies see, e.g., "Antibodies-A Laboratory Manual” , Harlow & Lee, ed. Cold Spring Harbor Laboratory Press (1988)]. Supematants of cultures of such hybridomas can then be screened for monoclonal antibodies (mAbs) by conventional procedures like radioimmuno- or enzymimmuno- or dot-immunobinding or immunofluoresence assays.
  • mAbs monoclonal antibodies
  • mAbs can be purified from hybridoma supematants by conventional chromatographic procedures like, for example, ion- exchange chromatography, affinity chromatography on protein A, anti-immunoglobulin-antibodies, or the antigen or a part thereof bound to a solid support, HPLC and the like.
  • chromatographic procedures like, for example, ion- exchange chromatography, affinity chromatography on protein A, anti-immunoglobulin-antibodies, or the antigen or a part thereof bound to a solid support, HPLC and the like.
  • the production of a mAb useful in accordance with the invention is shown in Example 22.
  • hybridomas secreting the desired antibody can be injected intraperitoneally into mice which have been pretreated with, for example, pristane before injection. Up to around 100 mg of a mAb can be produced by the resulting ascites tumors in one mouse. mAbs can be purified from the ascites fluid produced by such tumors by the methods described above.
  • mAbs can be characterized according to their subclass by known methods, such as by Ouchterlony immunodiffusion. It is also known in the art that mAbs can be modified for various uses, or fragments thereof can be generated, which are still capable of binding antigen. Such fragments can be generated, for example, by enzymatic digestion of mAbs with papain, pepsin, or the like.
  • Immunogen for producing mAb which can be used in accordance with the invention is preferably HLA-DR ⁇ -chain [see e.g. WO92/10589; J. Biol. Chemistry 2£2, 8748-8758 (1987); sequence information can be obtained also from sequence data bases, for example like Genbank (Intelligenetics, California, USA), European Bioinformatics Institute (Hinxton Hall, Cambridge, GB), NBRF (Georgetown University, Medical Centre, Washington DC, USA) and Vecbase (University of Wisconsin, Biotechnology Centre, Medision, Wisconsin, USA); such sequences can than be used by methods known in the state of the art to produce the antigen for the preparation of the monoclonal antibodies for use in the present invention] which has been purified by conventional means, such as SDS-PAGE.
  • the identification of the above-described anti-HLA-DR mAbs which are cytotoxic to APC and also downregulate HLA-DR expression may be performed by any conventional means.
  • this identification of anti-HLA-DR mAb is performed in accordance with Example 3 where an Epstein-Barr Vims transformed human B-Lymphoblastic Cell Line (EBV-LCL) is used as a model of an APC (particularly, activated B cells). Cultures of at least 1 ml containing about 10 5 EBV-LCL cells per milliliter are preferably used.
  • EBV-LCL Epstein-Barr Vims transformed human B-Lymphoblastic Cell Line
  • cytotoxicity and downregulation are defined as follows: 1 ) the mAb is cytotoxic to antigen presenting cells if, under the above conditions, at least 25%, preferably 40%, of the EBV-LCL antigen presenting cells are killed by the intact mAb; and 2) the mAb downregulates HLA-DR expression on antigen presenting cells if, under the above conditions, it reduces the number of HLA-DR molecules on the surface of the EBV-LCL antigen presenting cells which remain alive by at least an average of 50%, .
  • the EBV-LCL antigen presenting cells are labeled to detect the dead cells and to measure the HLA-DR express ion of the remaining viable cells by conventional immunofluoresence.
  • the cells are analyzed using a flow cytometer (e.g., FACScan, Becton-Dickinson, San Jose, California), and the percent dead cells and reduction of HLA-DR expression on the remaining cells is calculated by conventional means, preferably using the software normally used with the flow cytometer (e.g., LYSIS II software with the FACScan cytometer).
  • a flow cytometer e.g., FACScan, Becton-Dickinson, San Jose, California
  • EBV-LCL used to screen for monoclonal antibodies having the desired properties are not critical. Any conventional EBV-LCL may be used in accordance with the invention.
  • EBV- LCL useful in accordance with the invention are Priess (ECACC (Salisbury, UK) Accession No. 86052111), LG2 (Istituto Nazionale Per La Ricerca Sul Cancro (Genova, Italy) Accession No. G201 12301), and TS-10 (ECACC (Salisbury, UK) Accession No. 8510291 1 ).
  • the Fab fragment of the above-described mAbs may be produced by any conventional means.
  • the Fab fragment may be produced by digestion of the parent mAb by pepsin and isolating the Fab fragments by means known in the art (e.g., Andrew and Titus, "Fragmentation of immunoglobulin G", Current Protocols in Immunology, Coligan et al., eds. (Greene & Wiley 1994)). Accordingly such a process and a Fab fragment whenever prepared by such a process are also an object of the present invention.
  • the Fab fragments of the invention are produced by a recombinant cell line which expresses a gene which encodes the desired Fab fragment.
  • Such recombinant cell lines may be produced by any conventional means.
  • the portion of the gene encoding the Fab fragment would be cloned by conventional means from the hybridoma which secretes the parent mAb of the Fab fragment.
  • the Fab fragment cDNA may then be incorporated by conventional means into an expression vector, which in turn, is used to transfect an appropriate cell line.
  • the preferred Fab fragments of the invention are "humanized" so that they are less antigenic when administered to humans than a Fab fragment that is produced directly from an animal, preferably murine, mAb.
  • the method by which humanized Fab fragments of the invention are produced is not critical. Any conventional means known in the art may be used.
  • any immunoglobulin such as a monoclonal antibody
  • the variable domain in turn, consists of six complementarity-determining regions ("CDR's") embedded in a framework region (three CDR's on each of the light chain and the heavy chain of the Ig). (Ref. 42). It is the CDR's which are responsible for the specificity of the mAb.
  • mAbs are of murine or other animal species origin
  • humanization of mAbs is performed essentially by replacing at least one, but preferably all six, of the CDR's of a human immunoglobulin with the corresponding CDR's of an animal mAb which has the desired specificity.
  • the human Ig serves as the framework for the animal CDR's.
  • the animal mAb, usually murine is described as the "donor” and the human Ig is described as the "acceptor.”
  • EP 0 620 276 discloses a hierarchy of particular substitutions which may be made to the acceptor Ig outside the engrafted donor CDR's in order to increase the specificity of the humanized mAb. Such substitutions are disclosed as obviating the need to select a human acceptor Ig whose variable region has a high degree of homology with the variable region of the donor mAb.
  • a specific protocol for humanization is provided in EP 0 620 276 at pages 8-9.
  • Methods for generating DNA sequences for the expression in a host cell of a humanized intact mAb useful for obtaining the Fab fragments of the invention, or for the expression of a humanized Fab fragment of the invention are known in the art and are not critical. Such methods include, e.g., site-directed mutagenesis, constructing the whole variable region using overlapping oligonucleotides which incorporate the animal CDR's on a human framework, and using PCR grafting. (WO 90/7861, EP 0 620 276, Ref. 43).
  • the invention may be further described as a Fab fragment comprising a immunoglobulin Fab fragment and six complementarity-determining regions which are contained within said immunoglobulin Fab fragment, wherein from one to six of said complementarity-determining regions are the complementarity- determining regions of a monoclonal antibody having the following properties:
  • the monoclonal antibody binds to the first domain of HLA-
  • the monoclonal antibody is cytotoxic to antigen presenting cells which express HLA-DR
  • the monoclonal antibody downregulates HLA-DR expression on the antigen presenting cells.
  • the preferred embodiment of the invention is a humanized Fab fragment wherein, in accordance with the above, the immunoglobulin Fab fragment is human and the monoclonal antibody having properties 1-3 is animal, preferably murine.
  • This humanized Fab fragment would be a human immunoglobulin Fab fragment in which from one to six of the CDR's contained therein had been replaced by the corresponding CDR's of the animal mAb.
  • the preferred Fab fragment of the invention is a Fab fragment comprising a human immunoglobulin Fab fragment and six complementarity-determining regions which are contained within said human immunoglobulin Fab fragment, wherein from one to six of said complementarity-determining regions are the complementarity-determining regions of a monoclonal antibody having properties 1-3, above. It is preferred that this humanized Fab fragment of the invention contain all six of the animal mAb CDR's.
  • the Fab fragment of the invention can be a Fab fragment of the monoclonal antibody, itself, which has properties 1-3, above. Such a Fab fragment would be useful as an intermediate for use in obtaining the animal CDR's that will be contained in the preferred humanized Fab fragment of the invention.
  • the preferred Fab fragments of the invention have properties similar to Fab fragments obtained from monoclonal antibodies LB3.1, L243, SFR3-DR5 and 1 -1C4, described above. Since the Fab fragments of the invention inhibit Th cell activation, they may be used in the treatment of various diseases in which activated Th cells are a source of disease damage or symptoms.
  • One such disease is rheumatoid arthritis. (Ref. 33). Downregulating HLA-DR on APC of a patient with rheumatoid arthritis, and thereby inhibiting Th cell activation in such a patient, would slow or stop the progression of the disease. Inhibiting Th cell activation in a patient with rheumatoid arthritis would also relieve symptoms, such as pain and inflammation, by decreasing or halting the release of mediators which are the cause of those symptoms.
  • the invention also comprises Fab fragment as described herein and their use as a therapeutically active agent, especially as an immunsuppressive agent and more specifically for the treatment of rheumatoid arthritis and a method of suppressing the immune response of a patient comprising administering a therapeutically effective amount of a Fab fragment of the invention to a patient in need of such treatment.
  • the invention further comprises a method of treating rheumatoid arthritis in a patient by administering a therapeutically effective amount of a Fab fragment of the invention to a patient in need of such treatment.
  • the amount of Fab fragment to be administered may be determined by any conventional means. Also, the administration of the Fab fragment of the invention may be performed by any conventional means.
  • Administration of a Fab fragment of the invention is preferably accomplished using a pharmaceutical composition of the invention (described below).
  • Administration is preferably performed parenterally (subcutaneously, intramuscularly, or intravenously), especially intravenously.
  • the dosage required to inhibit Th cell activation in a patient, and thereby treat rheumatoid arthritis, may be determined by any conventional means, e.g., by dose-limiting clinical trials. However, a dosage of ⁇ bout 1-10 mg/day i.v., especially about 3-7 mg/day, particularly about 5 mg/day, is preferred (Refs. 34, 35), preferably delivered as a bolus.
  • Treatment is preferably once daily for one week or less, however daily treatment may be continued for up to three weeks, if necessary.
  • the Fab fragments of the invention may be formulated as a fluid pharmaceutical composition, e.g. for parenteral administration comprising the Fab fragment of the invention dissolved in a conventional pharmaceutically acceptable fluid carrier material.
  • the composition may further comprise other pharmacologically active substances.
  • the composition contains about 0.5- 5 mg/ml of a Fab fragment of the invention, especially about 1-2 mg/ml.
  • the preferred fluid carrier is sterile, physiological saline.
  • Examples 1 -7 indicate that inhibition of Th cell activation by HLA-DR-specific mAb can operate by multiple actions: (a) elimination of available APC by direct cytotoxicity, (b) reduction of available HLA-DR molecules on the remaining viable APC by downregulation of cell surface expression, (c) hindrance of class II MHC-TcR interaction.
  • the Th cell inhibition may also occur by mAb blocking the peptide binding groove on DR molecule, rendering it inaccessible for antigen (30). It is therefore understandable why mAbs are superior Th cell inhibitors to the current peptide antagonists, the effect of the latter being exclusively based on blocking the antigen binding site of HLA-DR molecules.
  • cytotoxicity requires crosslinking
  • downregulation is also achieved with monovalent Fab fragments of the invention without the cytotoxicity of the parent mAb. This difference permits the separation of these two properties by antibody fragmentation.
  • the Fab fragments of the invention provide for the therapeutic use of these antibodies as immunosuppressants, without adverse effects.
  • Antibodies 8D1, 9F1 , 9F2 and 10F12 are pan-DR specific, whereas 1-1C4 can recognize all three human class II isotypes (DR, DP and DQ).
  • Transfectant M12.C3.25 was derived from a mouse class II- negative host line M12.C3 (15), and expresses MHC product composed of ⁇ l and ⁇ l domains derived from a human HLA- DRA*0101 /DRB 1 *0401 molecule, and ⁇ 2 and ⁇ 2 domains of a mouse I-E d protein, to which DR-specific reagents do not bind.
  • Transfectant CH27.105 was derived from a mouse class II-positive host line CH27 (16), and expresses the original mouse E ⁇ k chain associated with the chimeric human/mouse ⁇ chain described above. Standard indirect immunofluoresence staining was performed using indicated IgG2 and IgGl antibodies, in conjunction with protein A-FITC (Boehringer, Mannheim, Germany) and goat anti-mouse IgGi-FITC (Southern, Birmingham, Alabama), respectively. Samples were analyzed on a FACScan flow cytometer (Becton-Dickinson, San Jose, California). Results are shown in Table 2 (presentation is as in Table 1).
  • the DR expression and viability of antigen presenting cells (APC) pre-cultured with either a DR-binding competitor peptide aXA (17), or DR-specific mAb was examined at respective concentrations that, in the case of aXA and two mAb (LB3.1, 1- 1C4), blocked antigen presentation to Th cells.
  • APC an Epstein- Barr vims transformed human B-lymphoblastic cell line (EBV-LCL) was used .
  • EBV-LCL (Priess, ECACC (Salisbury, UK) Accession No. 860521 11 ) (10 5 cells/ml) were cultured in the presence of DR- specific mAb (LB3.1, 1-1C4, CCCL20) or peptide aXA (aXAAAKTAAAAa-NH2; ref. 17) at the concentrations indicated in Fig. 1 for 16 hours.
  • DR-specific mAb [LB3.1 (a), CCCL20 (b), 1-1C4 (c)], and FITC-conjugated goat-anti mouse Ig (Southern Biotechnology Associates Inc., Birmingham, Alabama) as the primary and secondary reagents, respectively.
  • Samples were analyzed on a FACScan flow cytometer (Becton- Dickinson, San Jose, California). The results are shown in Fig. 1.
  • the X-axis represents HLA-DR expression, and the Y-axis fluorescence of propidium iodide-stained dead cells, both in arbitrary fluorescence units.
  • Background represents control cell population cultured for 16 hours in normal medium and subsequently labeled without primary reagent, respectively.
  • DR downmodulation and cytotoxicity on EBV-LCL could be induced with two more DR specific mAb, L243 and SFR-DR5, whereas four other anti-DR mAb (8D1, 9F1 , 9F2 and 10F12) were neither cytotoxic, nor reduced DR expression (data not shown).
  • EBV-LCL LG2 (Istituto Nazionale Per La Ricerca Sul Cancro (Genova, Italy) Accession No. G201 12301) (10 5 cells/ml) were cultured in the presence of DR-specific mAb L243 (Becton- Dickinson) at the concentration of 10 nM for the indicated period of time. Cells were subsequently washed and stained by the standard indirect immunofluoresence using DR-specific mAb L243 and FITC- conjugated goat-anti mouse Ig (Southern, Birmingham, Alabama) as the primary and secondary reagent, respectively. Dead cells were stained with propidium iodide, and samples were analyzed on a FACScan flow cytometer. The results are shown in Fig. 2. Histograms represent relative number of dead cells (light) and live cells expressing decreased amounts of HLA-DR (dark).
  • EBV-LCL LG2 (10 5 cells/ml) were cultured in the presence of DR-specific mAb L243 (Becton-Dickinson) at the concentration of 10 nM for the indicated period of time. Antibody was removed by 3x washing and cell culture was resumed in the same volume of the fresh medium, as indicated. Cells were subsequently washed and stained by the standard indirect immunofluoresence using DR-specific mAb L243 and FITC- conjugated goat-anti mouse Ig (Southern, Birmingham, Alabama) as the primary and secondary reagent, respectively. Dead cells were stained with propidium iodide, and samples were analyzed on a FACScan flow cytometer. The results are shown in Fig. 3. Histograms represent relative number of dead cells (light) and live cells expressing decreased amounts of HLA-DR (dark).
  • Isolation of B cells and monocytes/macrophages from fresh peripheral blood was done by removing T cells plus monocytes or B cells (where applicable) prior to culture, using magnetic beads (Dynal) precoated with mAb specific for CD3 (SK7) and CD14 (M ⁇ P9) or CD19 (4G7), respectively (Becton-Dickinson). Th and B cell blasts were generated by 3-5 days in vitro stimulation of peripheral blood mononuclear cells with phytohemagglutinin (1 ⁇ g/ml; Sigma) and pokeweed mitogen (2.5 ⁇ g/ml; Sigma), respectively. L243 was digested by pepsin and papain in order to isolate F(ab)' 2 and Fab fragments, respectively as per ref. 18.
  • APC (10 5 /ml) were cultured in the presence of equivalent concentrations of complete L243 antibody (10 nM) or its Fab fragment (20 nM) for the indicated period of time. Cells were subsequently washed and stained as in Example 6. Results are shown in Fig. 6. Presentation is as in Fig. 2.
  • Cytotoxicity of EBV-LCL depends on DR-crosslinking.
  • EBV-LCL (Priess) (10 5 cells/ml) were cultured for 16 hours in the presence of L243 Fab (20 nM) fragments and goat anti-mouse IgG precoated magnetic beads ("anti-Ig"; Dynal), in order to crosslink cell membrane bound Fab fragments, as indicated. Cells were subsequently washed and stained as in Example 6. Results are shown in Fig. 7. Presentation is as in Fig. 1.
  • cytotoxicity is the consequence of DR-crosslinking by bivalent ligands [complete mAb and F(ab) * 2, crosslinked Fab], and does not require the Fc portion of antibody.
  • HLA class II isotypes DP and DQ, HLA class I molecules, and an MHC unrelated adhesion protein CD18 was analyzed on different APC subpopulations after co-culture with F(ab)'2 or Fab fragments of L243 .
  • Modulatory mAb LB3.1, L243 and 1-1C4 are pan-DR specific, i.e. they do not distinguish between different allelic forms of HLA- DR. Therefore, they are not suitable for testing allotype specificity of DR downregulation. However, this question could be addressed using downregulatory mAb SFR3-DR5, which is exclusively specific for DRB1*110X (formerly DR5; ref. 13).
  • B cells isolated from fresh peripheral blood of a heterozygous DRB1 *0101/1101X donor as in Example 6) were cultured in the presence of DRBl*110X-specific mAb SFR3-DR5 (20% hybridoma supernatant fluid; ref 14). Cells were subsequently washed and stained by standard indirect immunofluoresence with either DRBl * 110X-specific mAb SFR3- DR5, or DRBl *1101-specific mAb CCCL20 (12), in conjunction with goat anti-mouse & rat Ig (Southern), as indicated. Results are shown in Fig. 11. Presentation is as in Fig. 4.
  • EBV-LCL TS-10 (ECACC (Salisbury, UK) Accession No. 85102911) were cultured in the presence of 1-1C4 Fab (20 nM), anti-DP mAb (10 nM) or anti-DQ mAb (10 nM) for 16 hours, as indicated. Cells were subsequently washed and stained as in Examples 6 and 10. Results are shown in Fig. 12. Histogram staining profiles of live gated cells are shown. The X-axis represents fluorescence intensity in arbitrary units, and the Y-axis relative cell number. Open histograms represent control cell populations labeled with the respective secondary reagents.
  • Fab fragments of the invention obtained from pan-class II mAbs such as 1-1C4 would be useful for treating diseases linked to HLA-DP and -DQ expression, such as multiple sclerosis, type I diabetes mellitus, myasthenia gravis, erythematosus, organ transplant rejection, and graft versus host disease. (Refs. 36-41 )
  • APC (10 5 cells/ml) were cultured in the presence of equivalent concentrations of complete L243 antibody (10 nM) or its F(ab)'2 (10 nM) and Fab (20 nM) fragments for 16 hours, as indicated.
  • TNF ⁇ concentration in culture supematants was measured using internally controlled standard ELISA kit (T cell Diagnostics, Cambridge, Massachusetts). Cytotoxic and DR- modulatory effects of monoclonal reagents were confirmed by immunofluoresence as in Example 4 (data not shown). Results are shown in Fig. 13.
  • Th cells were incubated with mitomycin C-treated APC, antigen (134 nM), and inhibitors.
  • Antigen specific Th cell proliferation was measured by the standard 3 H-thymidine incorporation assay. Results are shown in Table 3.
  • PBMC 0401 Native HA LB3.1 mAb ⁇ l 100 0.1 0.6
  • X a DR molecules that present antigen to Th clones. b Achieved at 60 nM for mAb and their fragments, and 10 ⁇ M for antagonist peptides.
  • Th cell clones KMHA25 and DSHABBIO were incubated with mitomycin C-treated Priess cells as APC, HA307-319 peptide as antigen (330 nM), and the indicated concentrations of mAb LB3.1.
  • Antigen specific Th cell proliferation (upper panel) was measured by 3 H-thymidine incorporation 3 days later.
  • Flow cytometry of Priess cells with mAb LB3.1 (lower panel) was performed as in Example 3. Results are shown in Fig. 14.
  • APC (LG-2) were fixed with glutaraldehyde (Fluka Chemie, Buchs, Switzerland). Response of Th cell clone NBHAC25 to mitomycin treated ("My") or fixed (“Fix”) LG-2 plus HA307-319 (134 nM) in the presence of mAb or Fab was measured as in Example 17. Results are shown in Fig. 15. Conclusion: Anti-class II mAbs and their Fab fragments can also inhibit Th cell response to peptide antigen presented on fixed (i.e., dead) APC, where downregulation of class II molecules is not possible. Thus, at least one additional mechanism, most likely steric hindrance of class II- Th cell antigen receptor (TCR) interaction, plays a role in Th cell inhibition.
  • TCR Th cell antigen receptor
  • Antibodies and Fab fragments are comparable in inhibitory capacity, the latter being only marginally less efficient than the former. Both were, however, several hundred fold more efficient than peptides. It is important to note that increasing the antigen concentration (30 fold higher in the lower than in the upper panel of Fig. 16) rendered the peptide antagonist less efficient, but did not affect the potency of mAb. This observation is explained by differences in the mechanism of inhibition: whereas peptides directly compete with the antigen for class II binding sites, antibodies downregulate class II expression as well as hinder MHC-TCR interaction. Example 20
  • HLA-DR was immunoprecipitated from EBV-LCL Priess using mAb L243, and the HLA-DR ⁇ and ⁇ chains were separated by SDS-PAGE.
  • a 28k electrophoretic band containing DR- ⁇ chain was cut from the gel and used to immunize a BALB/c mouse.
  • Mouse immune B cells were subsequently immortalized by fusion with myeloma line PAI- 0 [Stocker, W. et al., Research Disclosure, 217: 155-157 (1982)] in order to obtain mAb secreting hybridomas.
  • Hybridoma 1-1C4 was identified to be secreting a mAb having inhibitory capacity.

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  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Pain & Pain Management (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention comprend des fragments monovalents de liaison d'antigène (Fab) d'anticorps monoclonaux (mAb), lesquels possèdent la capacité de réguler négativement l'expression de HLA-DR (antigène du leucocyte humain de type DR) sur les cellules présentant un antigène. Ces fragments Fab peuvent ainsi réguler négativement une telle expression de HLA-DR sans la cytotoxicité du mAb parent ou de fragments bivalents (F(ab)'2) du mAb. Les fragments Fab de l'invention constituent donc de puissants composés immunosuppresseurs spécifiques du complexe majeur d'histocompatibilité (MHC) de la classe II et ne présentant pas d'effets secondaires cytotoxiques.
EP95941017A 1994-12-07 1995-11-25 Fragments d'anticorps monoclonaux possedant une activite immunodepressive Withdrawn EP0787151A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US35091594A 1994-12-07 1994-12-07
US350915 1994-12-07
PCT/EP1995/004648 WO1996017874A1 (fr) 1994-12-07 1995-11-25 Fragments d'anticorps monoclonaux possedant une activite immunodepressive

Publications (1)

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EP0787151A1 true EP0787151A1 (fr) 1997-08-06

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EP (1) EP0787151A1 (fr)
JP (1) JP2001506122A (fr)
CN (1) CN1168679A (fr)
AR (1) AR002005A1 (fr)
AU (1) AU4256096A (fr)
BR (1) BR9509902A (fr)
CA (1) CA2206471A1 (fr)
CO (1) CO4480041A1 (fr)
CZ (1) CZ172497A3 (fr)
FI (1) FI972430A0 (fr)
HU (1) HUT77342A (fr)
IL (1) IL116228A0 (fr)
MA (1) MA23739A1 (fr)
NO (1) NO972522L (fr)
PE (1) PE52996A1 (fr)
PL (1) PL320610A1 (fr)
TR (1) TR199501542A2 (fr)
WO (1) WO1996017874A1 (fr)
ZA (1) ZA9510195B (fr)

Families Citing this family (10)

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Publication number Priority date Publication date Assignee Title
EP0893507A1 (fr) * 1997-07-25 1999-01-27 Institut Gustave Roussy Utilisation de ligands de MHC classe II (CD4 et LAG-3) comme adjuvants pour la vaccination de LAG-3 dans le traitement du cancer
AU5588099A (en) 1998-08-28 2000-03-21 Dendreon Corporation Selective apoptosis of neoplastic cells by an hla-dr specific monoclonal antibody
DE60035337T2 (de) * 2000-05-12 2008-02-28 Gpc Biotech Ag Humane Peptide/Proteine, die das Töten von Zellen, einschliesslich lymphoider Tumorzellen, herbeiführen oder bewirken
EP1156062A1 (fr) * 2000-05-12 2001-11-21 GPC Biotech AG Peptides/protéines immunomodulants et liants à un antigène du CMH classe II humain
US7521047B2 (en) 2000-05-12 2009-04-21 Gpc Biotech Ag Human polypeptides causing or leading to the killing of cells including lymphoid tumor cells
AU2002340167A1 (en) 2001-10-11 2003-06-17 Protein Design Labs Inc. Anti-hla-dr antibodies and the methods of using thereof
JP3665324B2 (ja) 2001-10-15 2005-06-29 麒麟麦酒株式会社 抗ｈｌａ−ｄｒ抗体
EP1768662A2 (fr) 2004-06-24 2007-04-04 Novartis Vaccines and Diagnostics, Inc. Immuno-potentialisateurs a petites molecules et analyses visant a detecter leur presence
AU2011235328A1 (en) * 2010-04-01 2012-09-27 Immunomedics, Inc. Antibody-based depletion of antigen-presenting cells and dendritic cells
KR102647825B1 (ko) * 2021-07-22 2024-03-14 서울대학교산학협력단 항-hla-dp 단클론 항체 및 이의 용도

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Publication number Priority date Publication date Assignee Title
DE3269354D1 (en) * 1981-06-25 1986-04-03 Univ Leland Stanford Junior Allele specific immunotherapeutic method and dosage form
JPH05502586A (ja) * 1989-12-27 1993-05-13 セントカー・インコーポレーテツド Ｃｄ４受容体に対するキメラ免疫グロブリン
EP0605442B1 (fr) * 1991-07-25 2003-04-16 Idec Pharmaceuticals Corporation Anticorps de recombinaison pour therapie humaine

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Title
See references of WO9617874A1 *

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CA2206471A1 (fr) 1996-06-13
NO972522D0 (no) 1997-06-03
ZA9510195B (en) 1996-06-07
AR002005A1 (es) 1998-01-07
IL116228A0 (en) 1996-03-31
PL320610A1 (en) 1997-10-13
JP2001506122A (ja) 2001-05-15
MX9704225A (es) 1997-09-30
BR9509902A (pt) 1997-10-21
HUT77342A (hu) 1998-03-30
CO4480041A1 (es) 1997-07-09
AU4256096A (en) 1996-06-26
CN1168679A (zh) 1997-12-24
NO972522L (no) 1997-08-06
FI972430L (fi) 1997-06-06
TR199501542A2 (tr) 1996-07-21
WO1996017874A1 (fr) 1996-06-13
MA23739A1 (fr) 1996-07-01
PE52996A1 (es) 1996-12-12
CZ172497A3 (en) 1997-10-15
FI972430A7 (fi) 1997-06-06
FI972430A0 (fi) 1997-06-06

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