EP0846178A1 - Proteines de fusion entre cd32 et des allergenes - Google Patents

Proteines de fusion entre cd32 et des allergenes

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Publication number
EP0846178A1
EP0846178A1 EP96929263A EP96929263A EP0846178A1 EP 0846178 A1 EP0846178 A1 EP 0846178A1 EP 96929263 A EP96929263 A EP 96929263A EP 96929263 A EP96929263 A EP 96929263A EP 0846178 A1 EP0846178 A1 EP 0846178A1
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EP
European Patent Office
Prior art keywords
fusion protein
cells
protein according
der
acd32
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96929263A
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German (de)
English (en)
Inventor
Geert C. Mudde
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Novartis Pharma GmbH
Novartis AG
Original Assignee
Novartis Erfindungen Verwaltungs GmbH
Ciba Geigy AG
Novartis AG
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Publication of EP0846178A1 publication Critical patent/EP0846178A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43531—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from mites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • 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
    • 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
    • A61P37/08—Antiallergic agents
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/283—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 immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to complexes of human IgG and antigen/allergen (or a combination of antigens/allergens). It concerns fusion proteins between anti-CD32 molecules and antigen/allergen (or a combination of antigens/allergens). Allergens are defined herein as antigens to which atopic patients respond with allergic reactions. Antigens as used herein can be of various origins, e.g. environmental allergens (e.g. house dust mite, birch pollen, grass pollen, cat antigens, cockroach antigens), or food allergens (e.g. cow milk, peanut, shrimp, soya), or a combination of both, or non-relevant antigens such as bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • Allergy is a disease in which IgE antibodies mediate activation of effector cells (mast cells, basophils, eosinophils) but also enhance antigen presentation through low and high affinity receptors for IgE (B cells, monocytes, dendritic cells). These actions of IgE can be countered by specific IgG antibodies which interact with CD32 (Fc ⁇ RII) on both effector and inducer cells. Therefore, allergy should be regarded as a disease with a disbalance of Thl versus Th2 cells.
  • the success of the IgG antibodies in counteracting IgE in vivo depends on the relative concentration of specific IgE over specific IgG, and also on the isotype of IgG. All human isotypes have low affinity for CD32, which can be overcome by complex formation with allergen, but many allergens do not form large enough complexes with IgG molecules to allow stable binding to CD32.
  • EP 0 178 085 and 0 287 361) uses autologous IgG antibodies complexed in vitro to the relevant allergens. This approach allows far smaller amounts of allergen to be applied with less side effects. The mechanism behind both therapies is unclear. In the classical approach there seems to be a beneficial effect if the therapy induces an increase in specific IgG antibodies, although not every significant increase of specific IgG is correlated with successful immunotherapy. A possible reason for this is the relatively low affinity of IgG antibodies for CD32 on B cells, monocytes and mast cells. The Saint-Remy approach selects the specific IgG antibodies from the patient, which are subsequently mixed with relevant allergens in vitro.
  • IgG molecules are important in directing the immune response away from B cells to monocytes.
  • the Saint-Remy approach should therefore work when general allergen-specific IgG is used instead of donor-specific allergen-specific IgG.
  • a problem with the Saint-Remy approach lies in the low affinity of human IgG antibodies for their Fc ⁇ receptors on B cells, in contrast to IgE which binds readily to almost all antigen-presenting cells, including B cells, leading to spreading of allergy and induction of Th2 cells.
  • complexes should by-pass the low affinity problem, due to enhanced avidity of the complex as compared to single IgG molecules; however, again, the combination of allergen/IgG in complexes is unfavourable, because the natural number of epitopes of allergens for binding human IgG molecules is less than five, too few to enhance the avidity of the complex for the receptors.
  • IgE bound to mast cells and basophils induces histamine release when cross-linked by antigen.
  • IgE is able to enhance antigen presentation by human B cells ( Figure 1). Otherwise, in the absence of IgE B cells are poor antigen-presenting cells, except for a very small portion of B cells, which have a specific antigen receptor. Antigen uptake can occur through pinocytosis but this takes high antigen concentrations and/or long incubation times.
  • pinocytosis of allergen over 5 days is able to mimic IgE-mediated antigen presentation, but that IgG-mediated antigen presentation does not lead to stimulation of the T cells. In fact, the data indicates prevention of allergen presentation by complexing allergen to IgG.
  • IgG-allergen complexes In another series of experiments the influence of these IgG-allergen complexes on IgE-mediated antigen presentation or on allergen uptake through pinocytosis was studied.
  • Figure 3 it is shown that IgG complexes inhibit in a dose-dependent manner IgE-mediated antigen presentation, further, antigen presentation after pinocytosis is inhibited. This effect is not antigen-specific (IgG BSA complexes inhibit Der Pl - specific stimulation), monomeric (non-complexed) IgG does not inhibit antigen presentation.
  • IgG/allergen complexes Toxicity of the IgG/allergen complexes is excluded by the experiment shown in Figure 4.
  • IgG3-Der Pl complexes pre-incubated with human monocytes are able to induce a normal T cell response. This implies that IgG/allergen complexes inhibit allergen presentation by B cells and at the same time stimulate monocytes and dendritic cells.
  • aCD32 antibodies or aggregated IgG coated to the bottom of the wells in which the stimulations are performed also inhibit antigen presentation by B cells (Figure 5). The efficiency with which CD32 targeted molecules inhibit antigen presentation is most likely dependent on the amount of cross-linking of the CD32 molecule.
  • IgG/antigen complexes as well as aCD32/antigen fusion proteins direct the immune response to cells of the monocytic and dendritic cell lineage, which induce T cell responses of mainly Thl type B cells, and other APC which predominantly express CD32B will not present the antigen in the complex, therefore these complexes prevent (further) induction of Th2 cell responses by the B cells.
  • the interaction between the complex and CD32 does not depend on heavy cross-linking (more than 2 CD32 molecules), provided the binding of the complex is strong enough to allow stable interaction.
  • IgG/antigen complexes or aCD32/antigen fusion proteins also inhibit Ig production by normal human B cells.
  • FIG 14 it is shown that tonsilar B cells stimulated with aCD40 and IL-4 in the presence of aCD32 antibodies or aggregated coated human IgG, which efficiently interact with CD32, are no longer able to produce antibodies.
  • Treatment with aCD32 F(ab) fragments is as efficient as the use of complete antibodies ( Figure 15), indicating that for inhibition of Ig production after stimulation of aCD40 and IL-4 no cross-linking of the receptor is necessary.
  • B cells which are stimulated by antigen through their B cell receptor (BCR) need co-crosslinking of BCR and CD32.
  • BCR B cell receptor
  • the downregulation of Ig production is reversible and time dependent.
  • aCD32 antibodies are added to the B cells 4 days after aCD40 plus LL-4 stimulation, no inhibition of Ig production is seen.
  • Figure 16 it is shown that also antigen-specific induction of antibody production is inhibited by aCD32 treatment of B cells.
  • IgG molecules control the response of B cells through interaction with CD32.
  • IgE antibodies which in general have a positive effect on (CD23 positive) B cells, can be counteracted by IgG molecules. This prevents the organism from hyperreactive immune reactions to antigens.
  • IgG antibodies In allergy, where IgE antibodies are abundantly present, clearly the natural IgG antibodies have failed to control the immune response.
  • IgG4 In man IgE and IgG4 are regulated by the same interieukin, namely IL-4.
  • IgG4 has the lowest affinity of all human IgG subclasses for CD32.
  • the present invention concerns fusion proteins comprising a) one or more antigens and b) one or more moieties, such as from antibody molecules, interacting with human Fc ⁇ receptor ⁇ (FcyRLI) (CD32), hereinafter briefly named "the fusion proteins according to the invention”.
  • FcyRLI human Fc ⁇ receptor ⁇
  • the fusion proteins according to the invention overcome the problems due to the low affinity of human IgG molecules to CD32.
  • an aCD32 antibody having a K d ⁇ 10 "6 with antigen, both negative (B cells) and positive effects of natural IgG molecules are obtained, including selective stimulation of the immune system leading to Thl ThO memory induction in the absence of antibody production.
  • the effect is harmless and directs the immune response to antigen-presenting cells which express CD32, whereby the cells which predominantly express CD32A mediate antigen presentation leading to induction and activation of Thl cells as a result of the LL-12 produced by the CD32A-expressing antigen-presenting cells.
  • the fusion proteins according to the invention preferably are devoid of heavy cross-linking.
  • the number of binding sites for CD32 preferably is limited so as to avoid down-regulation of stimulatory co-receptors such as CD40, CD80 or CD86.
  • They comprise a) one or more antigens and b) one or more moieties, such as from antibodies, interacting with human Fc ⁇ receptor LI.
  • the parts of the fusion protein which interact with the Fc ⁇ receptors LI may e.g. be either
  • human or humanized aCD32 antibody moieties, or parts thereof e.g. Fab fragments, which still specifically recognize and bind to Fc ⁇ RII (CD32) antigen such as expressed on B cells, mast cells, monocytes and dentritic cells, e.g. manipulated human or humanized aCD32 or IgG antibody moieties, or parts thereof, which recognize FcyRLI (CD32) with higher affinity than the native aCD32 or IgG antibodies.
  • the antigens may be from complete proteins or parts thereof still having epitopes for T cells on the sequences present in the fusion protein.
  • any antigen to which allergic patients respond with IgE-mediated hypersensitivity reactions can be used, such as allergens in atopic dermatitis, allergic asthma, allergic rhinitis and allergic conjunctivitis.
  • the most common environmental allergens are: house dust mite, birch pollen, grass pollen, cat, cockroach.
  • Each of these allergens has one or more "major allergens" (e.g. for house dust mite the major allergen is Der Pl; for birch pollen the major allergen is Bet VI).
  • complete antigens are not necessary, because the fusion protein will normally only induce T cell responses, and T cells respond to small (8-12 amino acids long) peptides.
  • the fusion proteins may be produced from one or more T cell epitope - containing DNA stretches, rather than from the genes for the complete antigens. Overlapping cross-reactive epitopes between allergens are preferred.
  • the fusion protein should specifically bind to CD32 and contain one or more, e.g. two or more T cell epitopes for one or more antigens/allergens. To allow for correct antigen processing, DNA stretches slightly longer than the actual T cell epitope should preferably be included in the preparation of the fusion proteins.
  • short DNA sequences derived from cloned genes of major allergens are used, such as house dust mite major allergen I (Der Pl) or birch pollen allergen (Bet VI).
  • These short DNA sequences contain the genetic code for one or more T cell epitopes which after processing appear on the surface of antigen-presenting cells and therefore induce an immune response in the responding allergen-specific T cells.
  • the majority of the T cell epitopes can be found in the sequence at positions 101-143 in amino acid one-letter code (SEQ. LD. No.l):
  • amino acid sequence at positions 101-131 in amino acid one-letter code (SEQ. LD. No.2):
  • QSCRRPNAQRFGISNYCQIYPPNANKIREAL 101 110 120 130 contains at least three T cell epitopes, which bind to a number of HLA class ⁇ molecules.
  • the fusion between e.g. aCD32 and antigen(s) can be effected either at the protein level (chemical fusion) or at the gene level (recombinant fusion protein), and the invention also comprises a process for producing fusion proteins as defined above. It is carried out in conventional manner, and preferably comprises the use of recombinant gene techniques or chemical cross-linking.
  • Recombinant fusion proteins are preferred.
  • Preparation using recombinant gene techniques may be carried out by known methods, e.g. in the following way: a gene segment containing the antigen-binding site and parts of the Fc region down to the CH2 exon of an aCD32 monoclonal antibody (e.g. clone 1N.3) is PCR-amplified from e.g. IV.3 cD ⁇ A and cloned. Appropriate R ⁇ A is purified as a source for PCR-amplification, e.g. from house dust mites for amplification of the Der Pl gene.
  • the allergen gene is co-ligated with the isolated heavy chain segment into an appropriate mammalian expression vector such as p350.
  • the complete corresponding light chain gene of e.g. IV.3 is isolated and cloned into an appropriate mammalian expression vector such as p345, which has features similar to p350.
  • Both recombinant plasmids are used for co-transfection in e.g. COS cells to allow release of the resultant recombinant fusion protein into the culture medium.
  • Purification of the product is done preferably by immunoaffinity purification, based on anti-light chain antibody columns, which are readily available in large quantities.
  • scFv single chain antibody specific for CD32
  • an appropriate phage display library e.g. as described by De Kruif et al., Proc. ⁇ atl. Acad. Sci. USA £2 (1995) 3938-3942
  • D ⁇ A is extracted from binding phages and a D ⁇ A fragment containing a semi-synthetic VHDJH region fused to one of the VL chains via a flexible linker (scFv fragment) is isolated and cloned. Fusions with e.g.
  • the Der Pl gene are accomplished using defined synthetic oligonucleotides coding for T-cell epitopes from Der Pl and other major allergens selected by their high (T cell) crossreactive potential in order to cover as many patients as possible with one fusion protein.
  • These oligonucleotides are co-ligated with the scFv antibody fragment into an appropriate mammalian expression vector.
  • the recombinant vector e.g. a plasmid is stably transfected into appropriate cells such as COS and/or CHO cells and the resultant fusion protein is purified from the cell supernatant, e.g. by immunoaffinity chromatography as described above.
  • the resultant gene construct may be expressed e.g. in CHO cells, especially for large scale production; however, other systems for production by recombinant gene techniques are also appropriate, especially when only T cell epitopes are used, i.e. there is no need for glycosylation.
  • the aCD32 antibodies can be obtained e.g. from a human Ig phage display library which contains only F(ab) fragments of the natural antibody.
  • the source of the genetic material which codes for either antibody or antigen/allergen is not critical.
  • Preparation by chemical cross-linking may also be carried out by known methods, e.g., for a fusion protein between Mc.a-CD32 and Der Pl, by using the methods described by Caisson et al. rBiochem.J. 173 (1978) 723-737], Cumber et al. rMeth.Enzvmol. 1 12 (1985) 207-224] and Peeters et al. fJ.Immunol.Meth. 120 (1989) 133-143].
  • Der Pl and Mc.a-CD32 are separately derivatized with SPDP at molar ratios of 1/20 and 1/5 respectively for introducing 2-pyridyldisulphide residues.
  • the SPDP-derivatized Mc.a-CD32 (whose disulphide groups are reduced to thiol groups) are incubated with the SPDP-derivatized Der Pl at a molar ratio of 1/1.6.
  • the resulting Der Pl-Mc.a-CD32 product is then purified, for example by gel filtration on e.g. Superose-12 and by anion exchange chromatography on e.g. FPLC Mono-Q.
  • the starting materials are either known or may be prepared according to known procedures or analogously to known procedures or analogously as described herein, e.g. in the Example.
  • the fusion proteins according to the invention are useful for the prevention and/or treatment of allergies, particularly of food allergies. It is not uncommon for patients who suffer from anaphylactic response to a particular allergen, also to suffer from such a response to one or more other allergens. It is possible by the method of the present invention to desensitize such a patient in respect of two or more allergens simultaneously by administering a fusion protein including antigens against each of these allergens.
  • the fusion proteins according to the invention will be used for the prevention and/or treatment of allergy in newborn children at risk of food allergy e.g. to milk, or of established allergies in patients with allergy against the allergen(s) which is (are) included in the particular fusion protein used.
  • the appropriate dosages will, of course, vary depending upon, for example, the particular fusion protein used, the host, the mode of application and the intended indication. However, in general, satisfactory results are indicated to be obtained with one to three vaccinations over 1-2 years, but if necessary repeated additional vaccinations can be done. It is indicated that for these treatments the fusion proteins of the invention may be administered in dosages and with an application schedule similar as conventionally employed.
  • the invention therefore also concems the use of a fusion protein as defined above in the prevention and/or treatment of allergies, including food allergies, and a method of treating allergies which comprises administering to a subject in need of such treatment a prophylactically or therapeutically effective amount of a fusion protein as defined above together with at least one conventional pharmaceutically acceptable carrier or diluent, as well as fusion proteins as defined above for use as a pharmaceutical, especially as an anti-allergic agent.
  • the fusion proteins according to the invention may be admixed with conventional pharmaceutically acceptable diluents and carriers and, optionally, other excipients and administered parenterally, intravenously or enterally, e.g. intramuscularly, or subcutaneously.
  • concentrations of the fusion protein will, of course, vary depending i.a. on the compound employed, the treatment desired and the nature of the pharmaceutical form.
  • the invention thus also includes pharmaceutical compositions comprising a fusion protein as defined above together with at least one pharmaceutically acceptable carrier or diluent.
  • a process for the preparation of a medicament against allergies which comprises mixing a fusion protein as defined above together with a pharmaceutically acceptable carrier or diluent, and the use of a fusion protein as defined above for the manufacture of a medicament for the prevention and or treatment of allergies, including food allergies.
  • Der Pl is covalently conjugated to Mc.a-CD32 by using SPDP, a bifunctional coupling reagent.
  • Der Pl and Mc.a-CD32 are separately derivatized with SPDP at molar ratios of 1/20 and 1/5 respectively for introducing 2-pyridyldisulphide residues.
  • the SPDP-derivatized Mc.a-CD32 (whose disulphide groups are reduced to thiol groups) are incubated with the SPDP-derivatized Der Pl at a molar ratio of 1/1.6.
  • the resulting Der Pl-Mc.a-CD32 conjugate is purified by gel filtration on Superose-12 and by anion exchange chromatography on FPLC Mono-Q.
  • Monoclonal anti-CD32(IV.3) isotype: mouse IgG2b- ⁇ chain; molecular weight 150 kD; isoelectric point 6.2 (PHAST-IEF); purified from culture medium supematant on Protein-A and HPHT and dialysed against PBS.
  • a 100 ⁇ l sample is filled up to 150 ⁇ l with PBS and 50 ⁇ l of 150 mM DTT diluted in PBS is added.
  • concentration of pyridine-2-thione released after DTT addition can be determined by measuring the absorbence at 343 nm (molar extinction coefficient: 8080 M 'cm "1 ) and corresponds to the introduced 2-pyridyl-disulphide residues. For a measured OD 343 nm increase of 0.074 (corrected for dilution) the substitution degree is calculated as 1.5 Mol 2-pyridyldisulphide / Mol Der Pl.
  • Mc.a-CD32 solution (4.2 mg) is added 5.5 ⁇ l of SPDP stock solution (5 molar excess) and the mixture is stirred for 45 min at room temperature.
  • the reaction mixture is applied on a 2.5 ml Pierce GF-5 disposable desalting column (equilibrated with 10 ml of O.lM Na-acetate/HAc buffer, O.lM NaCl, pH 4.5). After application, the desalted fraction is eluted and pooled with 1.5 ml of acetate buffer (pH 4.5) and filtered with a 0.22 ⁇ m MLLLEX-GV (low protein binding) filtration unit.
  • Protein-bound 2-pyridyldisulphide groups are converted to protein-bound thiol groups by reducing with DTT.
  • 1.5 ml of desalted sample is incubated under stirring with 1 ml of 62.5mM DTT (diluted in acetate buffer) at room temperature for 30 minutes (final DTT concentration 25mM). Because 2-pyridyl disulphide increase their electrophilicity at acidic pH the reduction can still be carried out at low pH, where native protein disulphide bounds will not be affected.
  • the reaction mixture is applied on a Pharmacia PD-10 column (equilibrated with 50 ml of PBS) and after application, the desalted fraction is eluted and pooled with 3.5 ml of PBS.
  • the sample is concentrated to 0.6 ml with a CENTRIPLUS-10 (YM10 membrane) concentration unit. The concentration is about 3.1 mg/ml, corresponding to 1.86 mg.
  • the reaction mixture (3.9 ml) is filtered with a 0.22 ⁇ m MLLLEX-GV filtration unit and concentrated to 0.5 ml with a Centriplus-10 concentration unit.
  • the final concentration of the conjugate is 3.7 mg/ml, corresponding to 1.9 mg.
  • the sample is applied on a Superose-12 (HR 10/30) column (Pharmacia) equlibrated with PBS. Bed volume 24 ml; flow rate 0.2 ml/min; volume/fraction
  • the pools are sterile filtered (0.22 ⁇ m MLLLEX-GV) and stored under sterile conditions at +4°.
  • the total protein concentration is about: Pool A: 100 ⁇ g/ml;
  • 1 ml of Pool C (390 ⁇ g) is dialysed against 20 mM of ethanolamine/HCl, 0.01 % NaN 3 buffer pH 9.0 and purified by ion-exchance chromatography on FPLC-Mono Q (HR5/5) anion exchanger.
  • Starting buffer A 20 mM ethanolamine/HCl, 0.01%NaN 3 pH 9.0; limiting buffer B: A + 0.5 M NaCl pH 9.0; flow rate 1 ml/min, volume/fraction 1 ml; recorder chart speed 5 mm/min; pressure 1.2 Mpa; scanning wavelenght 280 nm.
  • a linear gradient program is started for 30 minutes (conductivity 50 - 1260 ⁇ S/cm).
  • the relevant peak is eluted in fraction #8 (at 340 ⁇ S/cm).
  • the protein concentration is about 45 ⁇ g/ml (OD 280 nm, E 1% 14.0).
  • the sample is dialysed against PBS and sterile filtered.
  • the protein concentration of the final purified material is 20 ⁇ g/ml (OD 280 nm).
  • Solid phase ELISAs are performed in PVC microtiter plates.
  • the coating buffer 0.1 M NaHCO j /TS ⁇ COa, 0.01% NaN 3 pH 9.6, PBS with 0.05% Tween-20 is used as washing solution and 2 % fetal calf serum in washing buffer as diluent for samples, biotin- and enzyme conjugates.
  • the substrate is 1 mg/ml p-NPP diluted in 1 M diethanolamine/HCl buffer pH 9.8.
  • the stopping solution is 2 M NaOH. All incubation steps are done in a humidified chamber.
  • Equipment for sample processing and OD reading at 405 nm is Beckman Biomek-1000 laboratory workstation. Quantitative evaluation: Beckman Immunofit; curve fitting: 4-parameter logic. b)l) Determination of Der Pl (Fig. 7):
  • Mca-Der Pl(4Cl/B8/3F8)-Biotin conjugate diluted 1/500 is added and incubated for
  • Superose-12 Pool A 16.1 ⁇ g/ml
  • Superose-12 Pool B 54.9 ⁇ g/ml
  • Su ⁇ erose-12 Pool C 135 ⁇ g/ml
  • Mc.a-CD32 (mouse IgG2b1 (Fig. 8):
  • IgG2b-alkaline phosphatase conjugate (Southern) diluted 1/1000 is added and incubated for 2 hours at +37°. After washing, 100 ⁇ l/well substrate is added, incubated for 15 minutes at room temperature and stopped. Quantitative evaluation from standard curve:
  • Su ⁇ erose-12 Pool A 17.7 ⁇ g/ml
  • Superose-12 Pool B 58.8 ⁇ g/ml
  • Su ⁇ erose-12 Pool C 190 ⁇ g/ml
  • the molecular weight of the Der Pl ⁇ Mca-CD32 conjugates in the Superose-12 purified fraction pools is estimated with a native PHAST gel 4-15% gradient
  • Superose-12 Pool A band at MW 700 kD
  • Superose-12 Pool B band at MW 460 kD, 330 kD
  • Superose-12 Pool C band at MW 330 kD, 170 kD
  • the molecular weight of the final FPLC-Mono Q purified Der Pl-Mca-CD32 conjugate is estimated as 330 kD (Fig. 12).
  • the above chemically linked aCD32 (Medarex clone IV.3) with purified Der Pl is used to stimulate Der Pl - specific T cell clone CFTS4:3.1 in different concentrations using a standard LST.
  • pool A and pool B from the chemically fused preparation are used, pool A containing a single band of 700 kD (2 aCD32 molecules fused with 10 Der Pl molecules), and pool B consisting of two bands, at 460 kD and 330 kD (1 aCD32 with 10 Der Pl, and 1 aCD32 with 5 Der Pl molecules, respectively).
  • the purified protein from pool C is used, consisting of a single band of approximately 330 kD (1 aCD32 molecule fused with 5 molecules of Der Pl).
  • Monocytes and B cells are preincubated for 1 hour at room temperature with the various fractions as indicated in Figures 13a and 13b.
  • B cells are able to stimulate CFTS4:3.1 with DPT but not with the pool A, pool B, or pool C
  • monocytes are able to stimulate CFTS4:3.1 with DPT and with pool A, pool B, and pool C.
  • aCD32 antibodies are stimulated with aCD40 and LL-4 in the presence and absence of commercially available aCD32 antibodies, added in the indicated concentrations (Fig. 14). After 9 days the supernatants of the cultures are tested for IgE and IgGl content. All aCD32 antibodies inhibit IgE as well as IgGl production in a dose-dependent way. In Fig. 15 it is shown that even Fab fragments from the Medarex clone IV.3 are able to inhibit IgE synthesis. This indicates that B cells which are not activated via their BCR can be blocked in their antibody production by monomeric interaction with CD32. For B cells which are stimulated in an antigen-specific way, co-cross-linking between CD32 and BCR is necessary.
  • B cells take up antigen (e.g. Der Pl) through IgE and CD23 and subsequently become stimulated Der Pl - specific Th2 cells leading to IgE production by the B cells, these B cells can be shut down by interaction with CD32 on the B cell surface. Indeed, even in cognate B cell T cell interaction, aCD32 antibodies block antibody synthesis (Fig. 16).
  • antigen e.g. Der Pl
  • Monocytes (91% CD 14 positive) derived from a normal donor by elutriation are incubated with 100 U/ml LFN- ⁇ for 24 hours in the presence or absence of 5 ⁇ g/ml aggregated human IgG (coated to the wells, ovemight at 4°) and/or 10 ⁇ g/ml monoclonal mouse anti-human aCD32 (Medarex IV.3), and subsequently stained for the indicated markers for FACS analysis.
  • the LFN- ⁇ - induced up-regulation of CD80 and CD40 can be inhibited by heavy cross-linking of Fc ⁇ receptors by aggregated human IgG on the monocytes.
  • Preincubation with aCD32 counteracts the inhibition by aggregated human IgG, indicating that
  • HLA-DR expression is not influenced by the treatments.
  • Figure 1 Binding of Der-Pl-(3)NLP complexes to CD23 in relation to antigen presentation:
  • Panel A fluorescence
  • Panel B proliferation
  • EBV B cells were pulsed with pre-formed IC's, consisting of a constant concen ⁇ tration of IgE (A 7.5 ⁇ g/ml,# 5.0 ⁇ g/ml, ⁇ 2,5 ⁇ g/ml) and variable concentrations of Der-Pl-(3)NLP.
  • IgG3-Der P1-(3)NIP complexes In the absence of IgG3, a good T cell stimulation is found when IgE-Der P1-(3)NIP complexes are used. Titration of preformed IgG3-Der P1-(3)NIP complexes in the same (1:1) ratio leads to a dose-dependent inhibition of IgE-mediated antigen presentation. In order to have this effect the IgG3 complexes need to be present in the culture medium at the time of T cells stimulation. Preincubation or "pulsing" of the B cells with the IgG3 complexes is not successful due to the low affinity of IgG3 for CD32 on the B cells. A similar inhibition is seen with antigen presentation in the absence of IgE and also with IgG3-BSA-(3)NIP complexes.
  • FIG. 4 IgG3-mediated antigen presentation by fresh human monocvtes:
  • Human monocytes are pulsed with preformed IgG3-Der P1-(3)NLP complexes (ratio 1:1) for 1 hour on ice in the absence or presence of non-relevant aggregated human IgG.
  • the pulsed monocytes are subsequently washed and mixed with HLA-DP matched T cell clones specific for Der Pl, and after 5 days proliferation is measured as 3 H-thymidine inco ⁇ oration.
  • IgG3 complexes presented by human monocytes induce an allergen-specific T cell response which can be blocked by aggregated IgG, indicating specificity of the IgG3 interaction with the monocytes. Data are shown as mean + sd of triplicate wells.
  • DPT-specific T cell proliferation influence of aCD32 or aggregated human IgG on antigen presentation: Human EBV B cells are pulsed with (closed bars) or without (open bars) 250 ⁇ g/ml of DPT ovemight. After irradiation the B cells are mixed with T cells from a Der Pl - specific T cell clone and the cells are allowed to proliferate for 5 days. In the presence of different concentrations of aCD32 antibodies cross-linked by GaM a dose-dependent inhibition of proliferation is seen. The same effect is observed when aCD32 or non-specific aggregated human IgG is coated to the culture wells.
  • Figure 6 Examples of recombinant fusion proteins between aCD32 scFv (Fab) fragment(s) derived from a phage display library:
  • the Fab fragments can be obtained from a phage display library such as described by De Kruif et al., Proc Natl. Acad. Sci. USA 22 (1995) 3938-3942.
  • any molecule (or part of a molecule) efficiently interacting with human CD32 can replace the aCD32 Fab fragment.
  • Allergen may be any protein or combination of proteins that cause(s) allergy.
  • the allergen(s) may be replaced by fragments of the allergen(s) which contain T cell epitopes.
  • the recombinant protein can be produced in any expression system available, independent of glycosylation and other post-translatory modifications. Such fusion proteins may also be used for diseases which are characterized by (over)production of unwanted Ig molecules such as in rheumatoid arthritis, graft-versus-host disease or any other disease in which autoantibodies play a role. In these cases allergens can be replaced by a simple non-defined linker to combine the two aCD32 moieties, however the best results will be obtained when allergen is replaced by the "autoantigen" that causes the disease.
  • Lane 3 500 ng high molecular weight markers
  • Lane 8 250 ng high molecular weight markers
  • Lane 1 250 ng high molecular weight markers
  • Lane 2 45 ng FPLC Mono-S purified material.
  • FIG. 13a and 13b Ag-specific T cell proliferation:
  • Fig. 13a APC - Mo 250495 and CFB4:2
  • Fig. 13b Influence of monomeric purified CD32/Der Pl
  • pool A and pool B from the chemically fused preparation were used: pool A containing a single band of 700 kD (being 2 aCD32 molecules fused with 10 Der PI molecules) and pool B consisting of two bands at 460 kD and 330 kD (being 1 aCD32 fused with 10 Der Pl, and 1 aCD32 with 5 Der PI molecules, respectively).
  • a second set of experiments (Fig. 13b) the purified protein (CP230595) from pool C was used, consisting of a single band of approximately 330 kD (1 aCD32 molecule fused with 5 molecules of Der Pl).
  • Monocytes and B cells were preincubated for lhour at room temperature with the various fractions as indicated in the Figure.
  • B cells were able to stimulate CFTS4:3.1 with DPT but not with the pool A, pool B, or pool C, whereas monocytes were able to stimulate CFTS4:3.1 with DPT and with pool A, pool B, and pool C.
  • Human tonsilar B cells are pulsed with (closed bars) or without (open bars) 250 ⁇ g/ml of DPT overnight. After irradiation the B cells are mixed with T cells from a Der Pl - specific T cell clone (CFTS4:3.1) and the cells are allowed to proliferate and produce antibodies for 9 days. After 9 days the supernatants of the cultures were tested for IgE and IgGl content as described in Armerding, D. et al., supra. Results are shown as antibody production of 3 pooled fractions of 9 replicates (mean ⁇ sd). In the presence of different concentrations of aCD32 antibodies a dose-dependent inhibition of IgE production is seen. This indicates that even in cognate B cell T cell interaction, aCD32 antibodies block antibody synthesis.
  • CFTS4:3.1 Der Pl - specific T cell clone
  • ORGANISM Dermatophagoides pteronyssinus
  • ORGANISM Dermatophagoides pteronyssinus

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Abstract

Protéines de fusion comprenant au moins un antigène et au moins une fraction ayant une interaction avec le récepteur Fcη humain II (FcηRII) (CD32).
EP96929263A 1995-08-16 1996-08-16 Proteines de fusion entre cd32 et des allergenes Withdrawn EP0846178A1 (fr)

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GB9724531D0 (en) 1997-11-19 1998-01-21 Smithkline Biolog Novel compounds
WO1999028349A2 (fr) 1997-12-02 1999-06-10 Medarex, Inc. CELLULES EXPRIMANT DES COMPOSANTS DE FIXANT AU RECEPTEUR ANTI Fc
EP1221317A1 (fr) * 2000-12-28 2002-07-10 SHAN-Beteiligungsgesellschaft m.b.H. Vaccins comprenant des polypeptides hybrides constitué d'au moins deux allergènes différents
US8025873B2 (en) 2002-06-20 2011-09-27 Paladin Labs, Inc. Chimeric antigens for eliciting an immune response
US8029803B2 (en) 2002-06-20 2011-10-04 Paladin Labs, Inc. Chimeric antigens for eliciting an immune response
US8007805B2 (en) 2003-08-08 2011-08-30 Paladin Labs, Inc. Chimeric antigens for breaking host tolerance to foreign antigens
BG65715B1 (bg) * 2003-09-04 2009-08-31 Чавдар ВАСИЛЕВ Средство за селективно подтискане на патологични днк - специфични в клетки
BG65954B1 (bg) * 2005-01-05 2010-07-30 Чавдар ВАСИЛЕВ Средство за селективно подтискане активността на патологични автореактивни в-клетки
US9636415B2 (en) 2006-03-03 2017-05-02 S-Target Therapeutics Gmbh Bispecific molecule binding TLR9 and CD32 and comprising a T cell epitope for treatment of allergies
EP1829895A1 (fr) * 2006-03-03 2007-09-05 f-star Biotechnologische Forschungs- und Entwicklungsges.m.b.H. Molécule bispécifique anti-TLR9 et anti-CD32 contenant un épitope de lymphocyte T pour le traitement des allergies
HUE052481T2 (hu) 2012-06-15 2021-04-28 Immunomic Therapeutics Inc Nukleinsavak, allergiák kezelésére
WO2014009209A2 (fr) 2012-07-13 2014-01-16 S-Target Therapeutics Gmbh Vaccin immunorégulateur
DK2914285T3 (en) * 2012-11-01 2017-02-27 Veterinærinstituttet New fusion proteins for the treatment of allergic diseases
US9005630B2 (en) 2012-11-01 2015-04-14 Veterinaerinstituttet Fusion proteins for the treatment of allergic diseases
ES2665780T3 (es) 2013-05-21 2018-04-27 Tyg Oncology Ltd. Composición inmunogénica de péptido de gastrina
CN113186167B (zh) * 2021-04-28 2021-11-30 中国食品药品检定研究院 用于测定抗cd20单克隆抗体药物adcp生物学活性的方法

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