EP3830247A1 - Traitement de maladies immunes par neutralisation médiée par des anticorps de bactéries intestinales spécifiques - Google Patents

Traitement de maladies immunes par neutralisation médiée par des anticorps de bactéries intestinales spécifiques

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Publication number
EP3830247A1
EP3830247A1 EP19749717.5A EP19749717A EP3830247A1 EP 3830247 A1 EP3830247 A1 EP 3830247A1 EP 19749717 A EP19749717 A EP 19749717A EP 3830247 A1 EP3830247 A1 EP 3830247A1
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EP
European Patent Office
Prior art keywords
antibody
antigen
binding fragment
antibodies
bacterium
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Withdrawn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
EP19749717.5A
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German (de)
English (en)
Inventor
Christopher OELKRUG
Daniel Christoph WAGNER
Armin Braun
Christina HESSE
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Oelkrug Christopher
Original Assignee
Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
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Publication of EP3830247A1 publication Critical patent/EP3830247A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to the treatment of immune diseases and other diseases by the antibody-mediated neutralization of specific intestinal bacteria.
  • the invention relates to antibodies or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment binding to an antigen of the bacterium Candidatus Savagella and (i) inhibiting the adhesion of the bacterium to intestinal epithelial cells, preferably human intestinal epithelial cells, and / or (ii) the bacterium is depleted.
  • the invention further provides a pharmaceutical composition comprising the antibody according to the invention or an antigen-binding fragment thereof, or comprising an antibody which is produced by the method according to the invention.
  • the invention relates to a kit comprising an antibody according to the invention or an antigen-binding fragment thereof for reducing Th 17 cell proliferation, Th 17 cell differentiation or Th 17 cell activity, and / or inhibiting the formation of antibodies against the body's own antigens by B cells.
  • the kit according to the invention optionally contains an antibiotic.
  • the invention also provides a method for producing an antibody according to the invention, the method comprising: a) immunizing chickens with an immunogenic peptide from an antigen of the bacterium Candidatus Savagella; and b) recovering and purifying the antibodies produced in the chickens or in an egg laid by these chickens.
  • the invention relates to a method for producing a medicament according to the invention, comprising: a) producing an antibody according to the invention or an antigen-binding fragment thereof; and b) formulation of the antibody or an antigen-binding fragment thereof as a medicament.
  • dysbiosis a disturbed homeostasis between the microbiome and the immune system
  • Other causes of dysbiosis can be a change in colon colonization after birth, for example after caesarean section, and due to increased hygiene in the living environment.
  • the human microbiome however, has a significant influence on the expression of immune reactions [1]. Certain bacteria, which are non-pathological per se, can nevertheless have an immunoregulatory or immunostimulatory influence.
  • Candidatus Savagella American “segmented filamentous bacteria”, SFB
  • SFB single-chain bacterium
  • Th 17 cells which in turn can fight harmful intestinal bacteria.
  • the same immune cells are also responsible for the development and progression of immune-mediated diseases such as autoimmune diseases and atopies.
  • experimental animals without SFB colonization have significantly lower symptoms in animal models of rheumatoid arthritis, multiple sclerosis and allergic asthma [2-4]
  • Candidatus Savagella produces an ongoing physiological inflammatory reaction in the intestinal mucosa, which leads to an overall strengthening Immune defense against pathological intestinal germs.
  • This evolutionarily conserved concept can be excessively harmful (eg in the case of dysbiosis) and lead to immune-mediated diseases in the rest of the body [1]
  • this connection has increasingly been taken up by basic science and published with high priority [2; 3; 11-13]
  • WO 2011/047153 describes a modulation of the Thl7 immune response, inter alia by influencing the adherence and proliferation of SFB.
  • the invention thus relates to an antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment binding to an antigen of the bacterium Candidatus Savagella and (i) inhibiting the adhesion of the bacterium to intestinal epithelial cells, preferably human intestinal epithelial cells, and / or (ii) the bacterium is depleted.
  • the antibody or the antigen-binding fragment preferably binds to an antigen of the bacterium Candidatus Savagella and inhibits the adhesion of the bacterium to intestinal epithelial cells, preferably human intestinal epithelial cells, and depletes the bacterium.
  • An antibody according to the invention or an antigen-binding fragment thereof binds specifically to an antigen of the bacterium Candidatus Savagella (English “segmented filamentous bacteria”, SFB), preferably to a bacterial wall protein.
  • the antigen is chosen so that the binding of the antibody according to the invention or the antigen-binding fragment thereof inhibits the adhesion of the bacterium to intestinal epithelial cells or depletes the bacterium.
  • the binding of the antibody according to the invention or of the antigen-binding fragment thereof to the antigen can also inhibit the proliferation of the bacterium. Combinations of these mechanisms are also included in the sense of the invention.
  • the antigen can be selected such that the antibody according to the invention binds the antigen of the bacterium Candidatus Savagella and inhibits the adhesion of the bacterium to intestinal epithelial cells and depletes the bacterium.
  • the antigen can be chosen so that the antibody according to the invention binds the antigen of the bacterium Candidatus Savagella and inhibits the adhesion of the bacterium to intestinal epithelial cells and inhibits the proliferation of the bacterium.
  • a combination of all three mechanisms is also included, so that the antigen is determined such that the antibody or the antigen-binding fragment according to the invention binds to the antigen of the bacterium Candidatus Savagella and inhibits the adhesion of the bacterium to intestinal epithelial cells, and inhibits the proliferation of the bacterium , and the bacterium is depleted.
  • SFB-specific antibodies were obtained from the eggs of chickens immunized with SFB proteins; this method enables the fast and very cost-effective generation of strong binding antibodies, which also have special acid stability and are therefore well suited for oral treatment [5]
  • FIG. 1 Filiform Candidatus Savagella bacteria (“segmented filamentous bacteria”, SFB) colonize the intestinal wall and activate Thl7 cells via dendritic cells, which contribute to autoimmunity and atopy.
  • SFB mented filamentous bacteria
  • suitable bacterial wall proteins of the named bacterium are identified, synthesized and injected into chickens.
  • the chickens form highly specific, neutralizing anti-SFB antibodies, which can be isolated from the eggs and are available for oral antibody therapy.
  • a reduction in the number of SFB bacteria in the intestine can lead to a reduction in Thl7 effector cell activity and thus to immune tolerance.
  • a specific neutralization of SFB with orally administered antibodies has the great advantage that there is practically no transfer of the antibodies into the circulation and thus frequent side effects of antibody therapy can be avoided, or side effects such as those observed with dexamethasone for allergic respiratory diseases .
  • Examples of the successful oral use of antibodies for the reduction of intestinal pathogenic viruses [14], fungi [15] and bacteria [16; 17] have already been described.
  • Oral antibody therapies with the aim of immunomodulation have so far only been carried out with the direct target of the immune system.
  • a successful example of this is the use of antibodies against the T cell surface protein CD3 ([18]; US 7,883,703 B2).
  • a major advantage of this invention is the use of therapeutic IgY antibodies from the eggs of immunized chickens.
  • This method offers numerous advantages: In addition to a very The antibodies can be produced quickly and in large quantities at low cost. lgY are more acid-resistant than lgG and are therefore particularly suitable for oral application. A chicken can produce up to 30 g of pure antibody per year, which is also highly binding. Finally, IgY have a different Fc region than mammalian IgG; there are therefore no side effects due to activation of the recipient complement system [5]
  • IgY antibody-mediated inhibition of the bacterium Candidatus Savagella can have a direct influence on immune-mediated diseases.
  • Immune diseases such as multiple sclerosis, rheumatoid arthritis and allergic asthma are largely determined by the activity of Th 17 immune cells.
  • Th 17 activity can also be treated preventively or therapeutically by depleting SFB.
  • IgY antibodies are conceivable as a medicament for the prevention or therapy of immune-mediated diseases, in particular Th 17 dependent diseases. This is said to be a therapeutic agent for microbiome correction. This can also be used as "functional food” or “nutraceutical”, so that there may be options for a greatly simplified approval, for example as "novel food”.
  • the antibodies according to the invention are preferably used for oral administration in humans.
  • the bacterium "Candidatus Savagella” (English “segmented filamentous bacteria", SFB) is for example by Schnupf et al. (Curr Opin Microbiol. 2017 Feb; 35: 100-109. Doi: l0.l0l6 / j.mib.20l7.03.004. Epub 2017 Apr 25; Semin Immunol. 2013 Nov 30; 25 (5): 342-51. Doi : l0.l0l6 / j.smim.20l3.09.00l. Epub 2013 Oct 31) and in US 2012/276149 and WO 2011/047153.
  • the “antigen” of the bacterium Candidatus Savagella which is specifically bound by the antibody according to the invention or the antigen-binding fragment of the antibody, is involved in the attachment of the bacterium to intestinal epithelial cells, preferably human Intestinal epithelial cells, the proliferation of the bacterium, and / or mediates depletion, that is, killing the bacterium.
  • a suitable antigen from Candidatus Savagella bacteria is selected from the group of function-determining and “segmented filamentous bacteria” (SFB) -specific proteins.
  • Such a protein is characterized, for example, by the fact that it is resistant to the bacteria (bacterial wall protein) and has an essential or even unique role for adherence to the intestinal epithelium and / or the survival of Candidatus Savagella or "segmented filamentous bacteria" (SFB).
  • a suitable antigen from the bacterium mentioned is, for example, the “myosin-cross-reactive antigen” (MCRA) protein described in more detail below, the amino acid sequence of which is shown in SEQ ID No. 1.
  • MCRA myosin-cross-reactive antigen
  • Specific epitopes from the "myosin cross-reactive antigen” (MCRA) protein include e.g. the amino acid sequence SVLDEFYWLDKKDPYSL (SEQ ID No. 2), PDFKAVRFTRRNQYESMI (SEQ ID No. 3), or QATSIKILRDGKEEEIKL (SEQ ID No. 4).
  • “Specific binding” of the antibody according to the invention or a fragment thereof to an antigen from the bacterium describes the binding properties of the antibody, such as binding affinity, binding specificity and binding avidity; see e.g. David J. King, Applications and Engineering of Monoclonal Antibodies, pp. 240 (1998).
  • Detailed analyzes of antigen-antibody interactions are possible, for example, with surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • the kinetic characterization of the binding properties of antibodies and their antigens is an essential prerequisite for evaluating their applicability for different methods.
  • the rate constants for association kass
  • dissociation kdiss
  • antibody encompasses both a polyclonal and a monoclonal antibody (mAb), which can be modified, as described below.
  • the antibody specifically binds to an antigen of the bacterium Candidatus Savagella and is preferably a neutralizing antibody.
  • Neutralizing antibody means the inhibition of the adhesion or binding of the bacterium Candidatus Savagella to (preferably human) intestinal epithelial cells, the inhibition of the proliferation of the bacterium mentioned and / or its depletion or killing by the antibody.
  • Candidatus Savagella means at least 50%, 60%, 70% or 75%, preferably 80% or 85%, particularly preferably 90% or 95% inhibition of the attachment or binding of the bacterium mentioned to intestinal epithelial cells, or the inhibition of Proliferation of the bacterium, or that at least 50%, 60%, 70% or 75%, preferably 80% or 85%, particularly preferably 90% or 95% of the total number or population of the bacterium are depleted, measured in in-vitro tests.
  • altered antibody means a protein encoded by an altered immunoglobulin coding region that can be obtained by expression in a selected host cell.
  • altered antibodies include engineered antibodies (e.g., chimeric, reshaped, humanized, or vectorized antibodies) or antibody fragments that lack all or part of a constant immunoglobulin region, such as Fv, Fab or F (ab) 2, and the like.
  • Modified immunoglobulin coding region means a nucleic acid sequence that encodes a modified antibody. If the modified antibody is a CDR-grafted or humanized antibody, the sequences encoding the complementarity-determining regions (CDRs) from a non-human immunoglobulin, such as a chicken antibody, are inserted into a first immunoglobulin partner, the human variable framework sequence includes. The first immunoglobulin partner may be operatively linked to a second immunoglobulin partner, for example in order to produce a bispecific antibody.
  • CDRs complementarity-determining regions
  • First immunoglobulin partner means a nucleic acid sequence encoding a human framework or variable region of a human immunoglobulin, in which the native (or naturally occurring) CDR coding regions are replaced by the CDR coding regions of a donor antibody, e.g. of a chicken antibody.
  • the human variable region can be a heavy chain, a light chain (or both chains) of an immunoglobulin, an analog, or functional fragments thereof.
  • Such CDR regions that are within the variable region of antibodies (immunoglobulins) can be determined by methods known in the art. For example, Kabat et al. (Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)) Rules for focalizing CDRs. In addition, computer programs are known that are useful for identifying CDR regions / structures.
  • “Second immunoglobulin partner” denotes another nucleotide sequence which encodes a protein or peptide to which the first immunoglobulin partner is fused, that is to say operatively linked, in a grid or by means of an optional conventional Finker sequence. It is preferably an immunoglobulin gene.
  • the second immunoglobulin partner can include a nucleic acid sequence that spans the entire constant region for the same (ie homologous - the first and the second modified antibody is from the same source) or an additional (ie heterologous) antibody of interest. It can be a heavy chain or light chain of an immunoglobulin (or both chains as part of a single polypeptide).
  • the second immunoglobulin partner is not limited to any particular immunoglobulin class or isotype.
  • the second immunoglobulin partner can comprise part of a constant region of an immunoglobulin as found in a Fab or F (ab) 2 , ie a discrete part of a human constant region or framework region.
  • Such a second immunoglobulin partner may also include a sequence encoding an integral membrane protein exposed on the outer surface of a host cell, for example as part of a phage display library, or a sequence containing a protein for analytical or diagnostic detection coded, e.g. horseradish peroxidase, ß-galactosidase etc.
  • Fv, Fc, Fd, Fab or F (ab) 2 are used with their standard meanings (see e.g. Harlow et ab, Antibodies A Faboratory Manual, Cold Spring Harbor Faboratory (1988)).
  • a "genetically engineered antibody” describes a type of engineered antibody, i.e. a full-catch synthetic antibody (e.g., a chimeric, unshaped, or humanized antibody as opposed to an antibody fragment) in which part of the variable domains of the light and / or heavy chain of a selected acceptor antibody are replaced by analogue parts from one or more donor antibodies (e.g. Chickens antibodies) that have specificity for the selected epitope.
  • donor antibodies e.g. Chickens antibodies
  • such molecules can include antibodies characterized by a humanized heavy chain associated with an unmodified light chain or (or chimeric light chain), or vice versa.
  • Genetically engineered antibodies can also be characterized by altering the nucleic acid sequences encoding the framework regions of the light and / or heavy variable domain of the acceptor antibody to reflect the donor antibody binding specificity, e.g. of a chicken antibody. These antibodies can replace one (eg CDR-H3) or several CDRs (preferably all CDRs, ie CDR-Fl, CDR-F2, CDR-F3, CDR-HI, CDR-H2, and CDR-H3) from the acceptor. Antibodies against CDRs from a donor antibody described here, ie a chicken antibody.
  • a “chimeric antibody” refers to a type of genetically engineered antibody that has a naturally occurring variable region (light chain and heavy chains) derived from a donor antibody (e.g., a chicken antibody) associated with constant regions of the light and heavy Contains chain derived from an acceptor antibody.
  • a donor antibody e.g., a chicken antibody
  • a “humanized antibody” means a type of genetically engineered antibody whose CDRs are derived from a non-human donor immunoglobulin, such as a chicken Antibodies originate, the remaining parts of the molecule derived from immunoglobulin originating from one (or more) human immunoglobulins.
  • scaffold support residues can be altered to maintain binding affinity (see, for example, Queen et al, Proc. Natl. Acad. Sci. USA, 86: 10029-10032 (1989), Hodgson et al, Bio / Technology, 9: 421 (1991 )).
  • An agent can be bound to the antibody, for example to improve transport into the intestine.
  • the agent bound to the antibody can actively or passively influence transporter proteins in the intestine.
  • the attachment can be chemical or, alternatively, the unit can be genetically engineered into the antibody.
  • donor antibody refers to an antibody (monoclonal or recombinant) that contributes the nucleic acid sequences of its variable regions, CDRs or other functional fragments or analogues thereof for a first immunoglobulin partner to the altered immunoglobulin coding region and the resulting expressed altered antibody with the antigenic specificity and neutralizing activity property of the donor antibody, eg of the chicken antibody.
  • acceptor antibody means an antibody (monoclonal or recombinant) which is heterologous to the donor antibody and all (or a part, but preferably all) nucleic acid sequences which are its heavy and / or light chain framework regions and / or encode its heavy and / or light chain constant regions for which the first immunoglobulin partner contributes.
  • a human antibody is preferably the acceptor antibody.
  • CDRs are defined as the amino acid sequences of the complementarity-determining region of an antibody, which are the hypervariable regions of the heavy and light chains of an immunoglobulin. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain CDRs (CDR-HI, -2 and -3) and three light chain CDRs (CDR-L1, -2 and -3) (or CDR regions) in the variable part of an immunoglobulin.
  • CDRs as used herein means all three heavy chain CDRs or all three light chain CDRs (or both all heavy chain and all light chain CDRs, if appropriate).
  • the structure and protein folding of the antibody can mean that other residues are considered part of the antigen binding region and would be understood by one of skill in the art. See, for example, Chothia et al. (1989), Conformations of immunoglobulin hypervariable regions; Nature 342, pp. 877-883. CDRs provide the majority of the contact residues for binding the antibody to the antigen or epitope.
  • CDRs of interest in this invention are derived from the donor antibody variable heavy and light chain sequences, e.g., a chicken antibody, and include analogs of naturally occurring CDRs, the analogs having the same antigen binding specificity and / or neutralizing ability as that Share or retain donor antibodies from which they are derived.
  • a “functional fragment” is a partial variable heavy or light chain sequence (e.g., minor deletions at the amino or carboxy terminus of the immunoglobulin variable region) that maintains the same antigen binding specificity and / or neutralizing ability as the antibody from which the fragment was derived.
  • an “analog” is an amino acid sequence modified with at least one amino acid, wherein the modification is chemical or a substitution or rearrangement of a few amino acids (ie preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 Amino acid residue (s)), the modification of the amino acid sequence allowing the biological properties, for example antigen specificity and high affinity, of the unmodified sequence to be retained.
  • (silent) mutations can be constructed via substitutions if certain endonuclease restriction sites are created within or around CDR coding regions.
  • the present invention contemplates the use of analogs of the antibody of the invention.
  • analogs of the antibody of the invention include those in which the CDRs in the hypervariable region of the heavy and light chains are at least 80% homologous, preferably at least 85%, at least 90% homologous and particularly preferably at least 95%, 96%, 97%, 98% or 99% are homologous to the CDRs as defined above as CDR-HI, CDR-H2, CDR-H3, CDR-Ll, CDR-L2 and CDR-L3 and the specific binding to an antigen of the bacterium Candidatus Savagella and neutralizing Maintain activity.
  • Amino acid sequences are at least 80% homologous if they have 80% identical amino acid residues in a similar position when the sequences are optimally matched, with gaps or insertions counted as non-identical residues.
  • Algorithms for determining sequence identity and programs for sequence comparison are well known in the prior art.
  • Analogs can also occur as allelic variations.
  • An "allelic variation or modification” is a change in the nucleic acid sequence. Such variations or modifications may be based on the degeneracy of the genetic code or may be deliberately engineered or recombinantly made to the desired ones Deliver properties. These variations and modifications may or may not result in changes in an encoded amino acid sequence.
  • effector agent refers to non-protein carrier molecules to which the modified antibodies and / or natural or synthetic light or heavy chains of the donor antibody, such as a chicken antibody, or other fragments of the donor antibody can be associated by conventional means .
  • non-protein carriers can include conventional carriers used in the diagnostic field, for example polystyrene or other plastic beads, polysaccharides, e.g. as used in the BIAcore system [Pharmacia], or other non-protein substances that are useful in the medical field and safe for administration to humans and animals.
  • Other effector agents can include a macrocycle to complex a heavy metal atom or radioisotopes. Such effector agents can also be useful for increasing the half-life of the modified antibodies, e.g. Polyethylene glycol (PEG).
  • PEG Polyethylene glycol
  • a neutralizing antibody specific for the bacterium Candidatus Savagella has not yet been described in the prior art and is made available for the first time by the present invention.
  • Another aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody of the invention together with a pharmaceutically acceptable diluent or carrier.
  • antibodies, altered antibodies and fragments can be constructed by immunizing a non-human species (e.g. cattle, sheep, monkeys, chickens, rodents (e.g. mice, hamsters and rats) etc.) to contribute to a desirable immunoglobulin
  • a non-human species e.g. cattle, sheep, monkeys, chickens, rodents (e.g. mice, hamsters and rats) etc.
  • rodents e.g. mice, hamsters and rats
  • Candidatus Savagella of any kind against which antibodies which are cross-reactive to the native or recombinant antigen from the bacterium
  • Candidatus Savagella can be generated, for example humans or chickens.
  • hybridoma techniques are used to provide a hybridoma cell line that secretes a non-human monoclonal antibody, such as a chicken antibody, against the native antigen from the Candidatus Savagella bacterium.
  • Such hybridomas are then bound to using Candidatus Savagella bacterial or recombinant antigen coated on 384 or 96-well plates, with biotinylated native or recombinant Savagella bacterial antigen bound to a streptavidin-coated plate, or in a screened homogeneous Europium-APC-linked immunoassay using biotinylated native or recombinant antigen from the Candidatus Savagella bacterium.
  • a native human antibody can be generated, for example, in a human antibody mouse such as the "Xenomouse” (Abgenix), in which the mouse immunoglobulin genes are removed and genes encoding the human immunoglobulins have been inserted into the mouse chromosome. The mice are immunized normally and develop an antibody response derived from the human genes. Thus, the mouse produces human antibodies bypassing the need for humanization after selection of positive hybridomas (see LL Green, J. Immunol. Methods, Dec. 10, 1999; 231 (1-2): 11-23).
  • a Fab fragment contains the entire light chain and the amino-terminal part of the heavy chain; and an F (ab ') 2 fragment is the fragment formed by two Fab fragments linked by disulfide bonds.
  • Fab fragments and F (ab ') 2 fragments can be obtained by conventional means, e.g. Cleavage of monoclonal antibodies (mAb) with the appropriate proteolytic enzymes, papain and / or pepsin, or can be obtained by recombinant methods.
  • the Fab and F (ab ’) 2 fragments themselves are useful as a therapeutic or prophylactic and as donors for sequences that include the variable regions and CDR sequences that are useful in the generation of recombinant or humanized antibodies as described herein.
  • the Fab and F (ab ') 2 fragments can also be constructed via a combinatorial phage library (see, for example, Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994)), or via immunoglobulin Chain exchange ("chain shuffling") (see, for example, Marks et al., Bio Technology, 10: 779-783 (1992)).
  • human antibody fragments that are specific for a native or recombinant antigen from the bacterium Candidatus Savagella can be isolated using human antibody fragment phage display libraries.
  • a library of bacteriophage particles representing human antibody fragment proteins is tested against the native or recombinant antigen from the Candidatus Savagella bacterium.
  • Those phages that represent antibody fragments that bind the native or recombinant antigen from the bacterium Candidatus Savagella are retained from the library and amplified by cloning.
  • human antibody genes are then excised from the specific bacteriophage and inserted into human IgG expression constructs containing the constant regions of human IgG to contain the intact human IgG molecule with the variable regions from that for native or recombinant antigen from the bacterium Candidatus Savagella to form specific, isolated bacteriophages.
  • the donor antibodies can contribute sequences such as variable heavy and / or light chain peptide sequences, framework sequences, CDR sequences, functional fragments and analogues thereof, and the nucleic acid sequences encoding them useful in the Construction and maintenance of various modified antibodies, which are characterized by the antigen binding specificity of the donor antibody, for example a chicken antibody.
  • various coding sequences can be constructed that encode the amino acid sequences of the variable heavy and light chain, and CDR sequences as well as functional fragments and analogues thereof that share the antigen specificity of the donor antibody, such as a chicken antibody .
  • Isolated nucleic acid sequences or fragments thereof that encode the variable chain peptide sequences or CDRs can be used to generate modified antibodies, for example chimeric or humanized antibodies, or other genetically engineered antibodies when operatively combined with a second immunoglobulin partner.
  • Modified immunoglobulin molecules can encode modified antibodies, including genetically modified antibodies such as chimeric antibodies and humanized antibodies.
  • a desired altered immunoglobulin coding region contains CDR coding regions that encode peptides with the antigen specificity of an anti-Candidatus Savagella antigen antibody, preferably a high affinity antibody, inserted into a first immunoglobulin partner (a human framework region or a variable region of a human immunoglobulin).
  • the first immunoglobulin partner is preferably operatively connected to a second immunoglobulin partner.
  • the second immunoglobulin partner is defined above and can include a sequence encoding a second antibody region of interest, e.g. an Fc region.
  • Second immunoglobulin partners can also include sequences encoding another immunoglobulin to which the constant region of the light or heavy chain is fused in a raster or by means of a linker sequence.
  • Genetically engineered antibodies directed against functional fragments or analogs of native or recombinant antigen from the Candidatus Savagella bacterium can be engineered to elicit increased binding.
  • the second immunoglobulin partner can also be associated with effector agents as defined above, including non-protein carrier molecules to which the second immunoglobulin partner can be operably linked by conventional means.
  • the fusion or binding between the second immunoglobulin partner, for example antibody sequences, and the effector agent can be carried out by any suitable means, for example by conventional covalent or ionic bonds, protein fusions or heterobifunctional crosslinking agents, for example carbodiimide, glutaraldehyde and the like.
  • suitable means for example by conventional covalent or ionic bonds, protein fusions or heterobifunctional crosslinking agents, for example carbodiimide, glutaraldehyde and the like.
  • linker sequences that simply provide a desired amount of space between the second immunoglobulin partner and the effector agent can also be constructed into the modified immunoglobulin coding region. The construction of such linkers is well known to those skilled in the art.
  • the antibody can have an additional agent attached to it.
  • the recombinant DNA technique can be used to generate a genetically engineered antibody in which the Fc fragment or the CH2-CH3 domain of a complete antibody molecule is generated by an enzyme or other detectable molecule (ie, a polypeptide Effector or a reporter molecule) was replaced.
  • the second immunoglobulin partner can also be operatively linked to a non-immunoglobulin peptide, protein or fragment thereof which is heterologous to the CDR-containing sequence with the antigen specificity of anti-Candidatus Savagella antigen antibodies.
  • the resulting protein can have both anti-Candidatus Savagella antigen specificity and properties of the non-immunoglobulin when expressed.
  • the property of the fusion partner can e.g. a functional property such as another binding or receptor domain or a therapeutic property if the fusion partner is itself a therapeutic protein, or additional antigenic properties.
  • Another desirable protein of this invention can be a complete, full length, heavy and light chain antibody molecule, or any discrete fragment thereof, such as the Fab or F (ab ') 2 fragments, a heavy chain dimer, or any minimal recombinant fragments thereof, such as an Fv or a single chain antibody (SCA) or any other molecule with the same specificity as the selected donor antibody, such as a chicken antibody.
  • a protein can be used in the form of an altered antibody or can be used in its unfused form.
  • Antibodies produced by genetic engineering or recombinant means can comprise constant regions of the immunoglobulin (Ig) and variable framework regions from a source, for example the acceptor antibody, and one or more (preferably all) CDRs from the donor antibody, for example a chicken antibody.
  • Ig immunoglobulin
  • variable framework regions from a source, for example the acceptor antibody, and one or more (preferably all) CDRs from the donor antibody, for example a chicken antibody.
  • deletions, substitutions or additions, of the framework region of the light and / or heavy variable domain of the acceptor monoclonal antibody (mAb) at the nucleic acid or amino acid level or of the donor CDR regions can be carried out to the antigen binding specificity of the donor antibody, such as a chicken antibody.
  • Such genetically engineered or recombinantly produced antibodies are designed to use one (or both) of the variable heavy and / or light chains of the anti-Candidatus Savagella antigen antibody or one or more of the CDRs of the heavy or light chain.
  • the genetically engineered or recombinantly produced antibodies can be neutralizing as defined above.
  • Such genetically engineered or recombinantly produced antibodies may include a humanized antibody containing the framework regions of a selected human immunoglobulin or subtype, or a chimeric antibody containing the constant regions of the human heavy and light chain fused to the functional fragments of the anti-Candidatus Savagella Antigen antibody.
  • a suitable human (or other animal) acceptor antibody can be obtained from a conventional database, e.g. the KABAT® database, the Los Alamos database and the Swiss Protein database, by homology with the nucleotide and amino acid sequences of the donor antibody, for example a chicken antibody.
  • a human antibody characterized by homology with the donor antibody framework regions may be suitable to provide a heavy chain constant region and / or a heavy chain variable framework region for insertion of the donor CDRs .
  • a suitable acceptor antibody that can donate constant or variable framework regions of the light chain can be selected in a similar manner. It should be noted that the heavy and light chains of the acceptor antibody do not have to come from the same acceptor antibody.
  • the heterologous scaffold regions and the constant regions are desirably selected from human immunoglobulin classes and isotypes such as IgG (subtypes 1 to 4), IgM, IgA and IgE.
  • the acceptor antibody need not only include human immunoglobulin protein sequences.
  • a gene can be constructed in which a DNA sequence encoding part of a human immunoglobulin chain is fused to a DNA sequence encoding a non-immunoglobulin amino acid sequence such as a polypeptide effector or a reporter molecule.
  • variable domains in both the human heavy and light chains were preferably genetically engineered by one or more CDR exchanges. It is possible to use all six CDRs or different combinations of less than the six CDRs. All six CDRs are preferably replaced. It is possible to exchange the CDRs only in the human heavy chain, the unmodified light chain from the human acceptor antibody being used as the light chain. Alternatively, a compatible light chain from another human antibody can be selected by using the conventional antibody databases. The rest of the engineered antibody can be from any suitable human acceptor immunoglobulin.
  • the genetically engineered or recombinantly produced humanized antibody thus has the structure of a natural human antibody or a fragment thereof and has the combination of properties which are required for effective therapeutic use.
  • variable domain amino acids can be further modified by altering the variable domain amino acids without necessarily affecting the specificity and high affinity of the donor antibody, such as a chicken antibody (i.e. an anologon). It is anticipated that heavy and light chain amino acids can be substituted with other amino acids in either the variable domain frameworks or CDRs or both.
  • the constant region can be changed to increase or decrease selective properties of the molecules of the present invention, such as dimerization, binding to Fc receptors, or the ability to bind and activate complement (see, e.g., Angal et al., Mol Immunol. 30: 105-108 (1993), Xu et al, J. Biol. Chem. 239: 3469-3474 (1994), Winter et al., EP 307,434-B).
  • a modified antibody which is a chimeric antibody, differs from the humanized antibodies described above in that it connects the entire variable regions of the heavy chain and light chain of the non-human donor antibody, such as a chicken antibody, including the framework regions with the constant regions of human immunoglobulin for both chains.
  • variable heavy and light regions which contain at least the CDR coding regions and those parts of the framework regions of the light and / or heavy variable domain of the acceptor mAb which are required to determine the binding specificity of the donor mAb, such as a chicken monoclonal antibody and the remaining immunoglobulin-derived portions of the antibody chain derived from a human immunoglobulin are obtained using polynucleotide primers and reverse transcriptase.
  • the CDR coding regions are identified using a known database and by comparison with other antibodies.
  • a chicken / human chimeric antibody can then be made and tested for binding ability.
  • Such a chimeric antibody contains the entire VH and VL regions of the non-human chicken donor antibody in conjunction with the human lg constant regions for both chains.
  • Homologous framework regions of a variable region of the heavy chain from a human antibody can be identified using computerized databases, eg KABAT®, and a human antibody with homology to the chicken donor antibody will be selected as the acceptor antibody.
  • a suitable variable framework region of the light chain can be created in a similar manner.
  • a humanized antibody can be derived from the chimeric antibody or, preferably, synthetically produced by inserting the CDR coding regions of the chicken donor mAb from the heavy and light chains appropriately within the selected heavy and light chain framework.
  • a humanized antibody can be produced using standard mutagenesis techniques.
  • the resulting humanized antibody contains human framework regions and CDR-coding regions of the chicken donor mAb. Subsequent manipulation of scaffold residues can occur.
  • the resulting humanized antibody can be found in recombinant host cells, e.g. COS, CHO or myeloma cells can be expressed.
  • a conventional expression vector or a recombinant plasmid is produced by placing these coding sequences for the antibody in operative association with conventional regulatory control sequences which can control the multiplication and expression in and / or secretion from a host cell. Regulatory sequences include promoter sequences, for example a CMV promoter, and signal sequences that can be derived from other known antibodies.
  • a second expression vector can be made with a DNA sequence encoding a complementary light or heavy chain of an antibody. This second expression vector is preferably identical to the first, except as far as the coding sequences and selectable markers are concerned, in order to ensure as far as possible that each polypeptide chain is functionally expressed.
  • the heavy and light chain coding sequences for the altered antibody can rest on a single vector.
  • a selected host cell is co-transfected (or simply transfected with a single vector) by conventional techniques with both the first and second vectors to produce the transfected host cell that includes both the recombinant or synthetic light and heavy chains.
  • the transfected cell is then cultured by conventional techniques to generate the genetically engineered or recombinantly produced antibody of the invention.
  • the humanized antibody which includes the association of the recombinant heavy chain and / or light chain, is screened from the culture by a suitable assay, for example ELISA or RIA. Similar conventional techniques can be used to construct other engineered antibody molecules.
  • Suitable vectors for the cloning and subcloning steps used in the methods and construction of the compositions provided herein can be selected by one of skill in the art.
  • the conventional pUC series of cloning vectors can be used.
  • a vector, pUCl9 is commercially available from suppliers such as Amersham (Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden).
  • any vector that can be easily replicated has a variety of cloning sites and selectable genes (eg, antibiotic resistance), and is easily manipulated can be used for cloning.
  • the vectors used for the expression of the antibodies can be selected by a person skilled in the art from any conventional vector.
  • the vectors also contain selected regulatory sequences (such as CMV promoters) that target the replication and expression of heterologous DNA sequences in selected host cells.
  • These vectors contain the DNA sequences described above which code for the antibody or the modified immunoglobulin coding region.
  • the vectors can take up the selected immunoglobulin sequences, which are modified by the insertion of desired restriction sites for easy manipulation.
  • the expression vectors can also be characterized by genes which are suitable for amplifying the expression of the heterologous DNA sequences, for example the dihydrofolate reductase gene (DHFR) from mammals.
  • Other preferred vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH); and the beta globin promoter sequence (beta glopro).
  • BGH bovine growth hormone
  • beta globin promoter sequence beta glopro
  • the components of such vectors e.g. Replicons, selection genes, enhancers, promoters, signal sequences and the like can be obtained from commercial or natural sources, or obtained by known methods for use in directing the expression and / or secretion of the product of the recombinant DNA in a selected host.
  • Other suitable expression vectors of which numerous types are known in the art for mammalian, bacterial, insect, yeast and fungal expression, can also be selected for this purpose.
  • the vectors can be used to generate a cell line that is transfected with a recombinant plasmid that contains the coding sequences of the antibodies or modified immunoglobulin molecules thereof.
  • Host cells useful for cloning and other manipulations of these cloning vectors are also conventional.
  • bacterial cells such as cells from different strains of E. coli, yeast cells, insect cells, or mammalian cells, can be used to multiply the cloning vectors and for other steps in the construction of modified antibodies of this invention.
  • Suitable host cells or cell lines for expressing the antibody of the invention are preferably mammalian cells such as NS0, Sp2 / 0, CHO, COS, a fibroblast cell (e.g. 3T3) and myeloid cells and particularly preferably a CHO or myeloid cell.
  • Human cells can be used, making it possible to modify the molecule with human glycosylation patterns.
  • other eukaryotic cell lines can be used.
  • the selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product manufacture and purification are known in the art; see e.g. Sambrook et al., Cited above.
  • Bacterial cells can prove useful as host cells suitable for expression of the recombinant Fabs of the present invention (see, e.g., A. Plückthun, Immunol. Rev., 130: 151-188 (1992)).
  • any recombinant Fab produced in a bacterial cell would have to be screened for retention of antigen binding ability. If the molecule expressed by the bacterial cell were generated in an appropriately folded form, the bacterial cell would be a desirable host.
  • various strains of E. coli used for expression are commonly known as host cells in the field of biotechnology.
  • strains of yeast cells known to those skilled in the art are also available as host cells, as are insect cells, e.g. Drosophila and Fepidoptera, and viral expression systems. See e.g. Miller et al., Genetic Engineering, 8: 277-298, Plenum Press (1986) and references cited therein.
  • the transfection methods required to generate the host cells, and culture methods required to generate the modified antibody of the invention from such host cells are all conventional techniques.
  • the antibodies of the invention can be purified from the cell culture contents according to standard procedures in the art, which include ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Such techniques are within the skill of those skilled in the art.
  • the antibody is then assayed for in vitro activity using an appropriate assay.
  • Conventional ELISA test formats are currently used to assess the qualitative and quantitative binding of the antibody to MAG.
  • other in vitro assays for verifying specific binding to the antigen and neutralizing efficacy as described herein can be used prior to subsequent human clinical trials performed to evaluate antibody persistence in the body despite the usual clearance mechanisms.
  • the antibody or the antigen-binding fragment is selected from the group consisting of: immunoglobulin molecule, polyclonal antibody, monoclonal antibody, chimeric antibody, antibody generated by CDR grafting, humanized antibody, Fab, Fab ', F (ab ') 2, Fv, disulfide-linked Fv, scFv, single domain antibody, diabody, multispecific antibody, dualspecific antibody, and bispecific antibody.
  • the antibody is a chicken immunoglobulin molecule or antibody.
  • the antibody is preferably generated against an antigen or a suitable peptide of the bacterium Candidatus Savagella and is preferably a polyclonal or monoclonal chicken antibody.
  • the chicken antibody can be humanized or altered as defined elsewhere.
  • the chicken immunoglobulin molecule is particularly preferably an IgY immunoglobulin molecule or polyclonal IgY antibody.
  • Immunoglobulin Y is a class of immunoglobulin molecules which are contained in the serum of chickens and, in high concentrations, in particular in the egg yolk of chicken eggs. As with the other immunoglobulins, IgY is a protein that is produced by the immune system in response to certain foreign structures and recognizes them specifically.
  • Immunoglobulin Y is the functional equivalent of IgG in chickens and, like this, is made up of two light and two heavy chains. Structurally, the two immunoglobulin classes differ primarily in the heavy chains, which have a molecular mass of around 65.1 kilodaltons in IgY and are therefore larger than those in IgG.
  • the light chains of IgY with a molar mass of about 18.7 kilodaltons are somewhat smaller compared to IgG.
  • the molar mass of IgY is thus about 167 kilodaltons.
  • the steric flexibility of the IgY molecule is less than that of IgG.
  • IgY is partially comparable to both IgE and IgG. In contrast to IgG, however, IgY does not bind to protein A or protein G and also not to cellular ones Fc receptors. In addition, IgY does not activate the complement system.
  • the name immunoglobulin Y was proposed by GA Leslie and LW Clem in 1969 after showing differences between the immunoglobulins and immunoglobulin G found in chicken eggs.
  • IgY offers various advantages over the use of mammalian antibodies. Since the antibodies are obtained from the egg yolk of laid eggs, it is a non-invasive method of antibody production. No blood has to be taken from the chickens to obtain blood serum. Repeated egg laying from the same chicken increases the available amount of a certain antibody considerably. Cross-reactivity with mammalian proteins is also significantly lower than that of IgG. In addition, the immune response to certain antigens is more pronounced in chickens than in rabbits or other mammals. Since only IgY of the immunoglobulins produced during the immune response can be found in chicken eggs, no contaminations with IgA or IgM are contained in corresponding preparations.
  • the yield of IgY from a chicken egg is high and comparable to that of IgG from rabbit serum.
  • IgY is used as a component of food, particularly in Asian countries such as Japan.
  • yoghurt products that contain specific IgY are sold there. This prevents bacteria from the Helicobacter pylori species from attaching to the stomach.
  • the IgY used for this is obtained from the eggs of immunized chickens.
  • Antibodies are also produced against Salmonella and other bacteria, but also against viruses and used as part of the food to protect against these pathogens.
  • the IgY according to the invention can thus be used, for example, in the form of “novel food” or “nutraceuticals”.
  • the antibody or the antigen-binding fragment (indirectly) leads to a reduction in Th 17 cell proliferation, Th 17 cell differentiation or Th 17 cell activity.
  • the antibody or the antigen-binding fragment inhibits the formation of antibodies against the body's own antigens by B cells.
  • Th 17 cells develop from so-called naive T helper cells, i.e. T helper cells that have not yet come into contact with their specific antigen, only after they have been activated Antigen contact.
  • IL-6 and TGF-ß are necessary for the differentiation of Thl7 cells.
  • Thl7 cells are named after the interleukin IL-17 they produce and have an important role in the activation of neutrophil granulocytes, but are also associated with the development of chronic inflammation and autoimmune diseases.
  • Other secretion products from Thl7 cells are TNF-a and IL-6.
  • Receptors for IL-17 are found on various cell types of the immune system, for example on myeloid cells, which include the neutrophils granulocytes, and on lymphocytes.
  • the messenger substances released by the Thl7 cells cause inflammatory processes in which neutrophil granulocytes play a dominant role.
  • IL-17 causes G-CSF, IL-6 and IL-8 to be released in the target cells mentioned, which induce and activate neutrophil granulocytes.
  • inflammatory proteins such as IL-1, IL-6, PGE-2, cyclooxygenase 2 and matrix metalloproteases are increasingly expressed.
  • the antibody of the invention contributes to an increased immune tolerance.
  • Immune diseases such as multiple sclerosis, rheumatoid arthritis, type 1 diabetes and allergic asthma are largely determined by the activity of Th 17 immune cells.
  • the gut bacterium, Candidatus Savagella induces precisely these Thl7 immune cells (see e.g. US 2012/276149; WO 2011/047153).
  • the present invention comprises the neutralization of this bacterium with orally administered chicken protein antibodies, in order to treat Thl7 immune-cell-mediated diseases.
  • Tests for reducing Th 17 cell proliferation, reducing Th 17 cell differentiation, reducing Th 17 cell activity or inhibiting the formation of antibodies against endogenous antigens by B cells are known to the person skilled in the art and are described in the prior art; see for example US 2012/276149 or WO 2011/047153.
  • the antigen of the Candidatus Savagella bacterium is a bacterial wall protein, particularly preferably the "myosin cross-reactive antigen" (SEQ ID No. 1)
  • an antigen in the sense of the invention is a cell wall protein of the bacterium Candidatus Savagella, such as the "myosin cross-reactive antigen", the amino acid sequence of which is shown in SEQ ID No. 1.
  • the antibody or the antigen-binding fragment binds to an epitope from the myosin cross-reactive antigen, comprising or consisting of the amino acid sequence S VLDEF YWLDKKDP Y SL (SEQ ID No. 2),
  • the invention also relates to a method for producing an antibody according to the invention, comprising:
  • a suitable protein is selected from the group of function-determining and “segmented filamentous bacteria” (SFB) -specific proteins, which is characterized in that it is a bacterial wall protein and a non-redundant role for adherence to the intestinal epithelium and / or survival of Candidatus Savagella or "segmented filamentous bacteria” (SFB) plays, such as the "myosin-cross-reactive antigen” (MCRA) protein.
  • MCRA myosin-cross-reactive antigen
  • Suitable epitopes are then determined within the protein sequence of the selected target antigen, for example using “epitope prediction programs” and / or using databases. The target sequence with the mathematically best antigenicity / immunogenicity is then selected for the antibody production.
  • the corresponding peptide is synthesized in sufficient quantity with the amino acid sequence.
  • small molecules such as peptides have to be coupled to carrier proteins.
  • ovalbumin hen's egg albumin
  • bovine or human serum albumin the snail protein KLH is a carrier protein widely used in biotechnology for the immunization of animals.
  • the peptide can then e.g. can be coupled to the slotted screw hemocyanin (keyhole limpet hemocyanin, KLH) protein.
  • KLH keyhole limpet hemocyanin
  • a chicken produces up to 3 grams of IgY per month.
  • the antibodies are then isolated from the egg yolk and tested for their neutralizing effect, i.e. whether the antibody is able to inhibit the adhesion or binding of the bacterium Candidatus Savagella to intestinal epithelial cells, to inhibit the proliferation of the said bacterium and / or to deplete or kill the bacterium.
  • the process is the myosin-cross-reactive antigen as the antigen of the bacterium Candidatus Savagella.
  • the epitope sequences from the myosin cross-reactive antigen with the amino acid sequence SEQ ID No. 1, ie peptides with the amino acid sequence S VLDEF YWLDKKDP Y SL (SEQ ID No. 2), PDFKAVRFTRRNQYESMI (SEQ ID No. 3), or QATSIKILRDGKEEEIKL (SEQ ID No. 4).
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or an antigen-binding fragment of the invention, or the antibody produced by the method according to the invention.
  • the antibody or an antigen-binding fragment of the invention is suspended in a pharmaceutically acceptable carrier.
  • the patient to be treated is preferably a human.
  • the present invention further provides a medicament comprising the antibody or an antigen-binding fragment of the invention, or the antibody produced by the process according to the invention, and preferably suitable additives and / or auxiliaries.
  • suitable additives and / or auxiliary substances are, for example, a physiological saline solution, suitable stabilizers, proteinase inhibitors, etc.
  • Suitable stabilizers are, for example, Tween 80 (0.02%), sugar solutions, such as. B. sucrose solution (20-30%) or amino acid solutions, such as. B. glycine or cysteine solutions.
  • the medicament according to the invention is usually produced by adding suitable additives and / or auxiliary substances to the antibody or an antigen-binding fragment of the invention.
  • the therapeutic agents of this invention can be administered as a prophylactic or otherwise as needed.
  • the dose and duration of treatment are related to the relative duration of the molecules of the present invention in human circulation and can be adjusted by one of skill in the art depending on the condition being treated and the general health of the patient.
  • the mode of administration of the therapeutic agent of the invention can be any suitable route that delivers the agent to the host.
  • the antibodies and pharmaceutical compositions / drugs of the invention are particularly useful for oral administration.
  • the medicament according to the invention is for use in the prevention or treatment of an oncological disease, allergic disease or an immune disease, preferably an autoimmune disease, the diseases mentioned being mediated by the activity of Th 17 cells.
  • an allergy sufferer or patient with autoimmune disease takes the specific SFB antibodies of the invention orally over a defined period of time in a defined treatment regimen.
  • the reduction in the allergic reaction is then determined, for example, using a prick test.
  • the effect of the specific SFB antibodies on a patient with autoimmune disease can be demonstrated, for example, by reducing the formation of autoantibodies.
  • the reference corresponds to the strength of the reaction without SFB antibody treatment.
  • the allergic disease, immune disease or autoimmune disease is preferably selected from the group consisting of: multiple sclerosis, type 1 diabetes, rheumatoid arthritis, allergic respiratory disease and allergic asthma.
  • the antibody or an antigen-binding fragment thereof is administered orally.
  • Chicken antibodies are particularly acid-stable and are therefore particularly suitable for oral use in patients.
  • all previous routes that are used for the transfer of immunoglobulins are also included, e.g. intramuscular, intravenous, intraperitoneal, or intratecal administration.
  • the antibody or an antigen-binding fragment thereof is used in combination with an antibiotic, preferably a beta-factam and / or glycopeptide antibiotic.
  • the present invention relates to a method for producing a medicament according to the invention, comprising:
  • the present invention relates to a kit comprising an antibody according to the invention or an antigen-binding fragment thereof for reducing Th 17 cell proliferation, Thl7 cell differentiation or Thl7 cell activity, and / or inhibiting the formation of antibodies against the body's own antigens by B cells.
  • the kit can also comprise an antibiotic such as beta-factam and / or a glycopeptide antibiotic, as well as instructions for using the components of the kit.
  • an antibiotic such as beta-factam and / or a glycopeptide antibiotic
  • determining encompass qualitative, semi-quantitative and / or quantitative determination, for example the quantitative determination of the antibody of the invention in the serum or plasma of a patient to whom this antibody is used for therapeutic purposes was administered.
  • the content of all references cited here is hereby incorporated by reference to the respective specific disclosure content and in their entirety.
  • FIG. 1 Graphical summary of the concept on which this invention is based: Special filiform Candagatus Savagella bacteria ("segmented filamentous bacteria", SFB) colonize the intestinal wall and activate Thl7 cells via dendritic cells (DC), which contribute to autoimmunity and atopy.
  • SFB mented filamentous bacteria
  • DC dendritic cells
  • suitable bacterial wall proteins of the named bacterium are identified (1), synthesized and injected into chickens (2).
  • the chickens form highly specific anti-SFB antibodies, which can be isolated from the eggs (3) and are available for oral antibody therapy in humans (4).
  • a reduction in the number of SBF bacteria in the intestine can lead to a reduction in Th 17 effector cell activity and thus to immune tolerance.
  • Example 1 Production and production of neutralizing antibodies against the "myosin-cross-reactive antigen” (MCRA) protein
  • the "myosin-cross-reactive antigen” (MCRA) protein was selected from the group of function-determining and "segmented filamentous bacteria" (SFB) -specific proteins.
  • the protein is located in the bacterial wall and the non-redundant role for adherence to the intestinal epithelium in humans and the survival of Candidatus Savagella or "segmented filamentous bacteria” (SFB) is a suitable target for a neutralizing antibody.
  • Suitable epitopes were then determined within the known protein sequence of the MCRA protein (SEQ ID No. 1). The target sequence with the best calculated antigenicity was selected for antibody production. With the known amino acid sequence, the corresponding peptide with the SEQ ID Nm.
  • the inventors' underlying working hypothesis states that a reduced colonization density with SFB in the intestine of patients leads to a reduced TH17 response. In general, this would have great therapeutic relevance in TH 17-mediated inflammatory diseases.
  • the inventors assume that the SFB also exists in the intestines of human patients and that oral therapy with a neutralizing antibody could be suitable for the treatment of a whole range of allergic diseases, immune diseases, autoimmune diseases and oncological diseases.
  • chicken antibodies is a basic concept of the present invention.
  • the use of chicken antibodies (IgY) has numerous advantages over classic approaches: IgY, for example, does not react with the human complement system and has a particularly high acid stability, which makes it attractive for the development of an orally administered therapeutic agent.
  • the oral application can look like this:
  • An allergy sufferer takes the specific SFB antibodies orally over a defined period in a defined treatment regimen.
  • the reduction in the allergic reaction is then determined, for example, using a prick test.
  • the reference corresponds to the strength of the reaction without SFB antibody treatment.
  • the appropriate SFB bacterial wall proteins were identified, synthesized and injected into chickens in this project.
  • the chickens produced highly specific, neutralizing anti-SFB antibodies that were isolated from the eggs.
  • This product with pharmaceutical potential isolated from the eggs can then be used for oral antibody therapy in the patient for the treatment of a whole range of allergic diseases, Immune diseases, autoimmune diseases and oncological diseases can be used.

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Abstract

La présente invention concerne un anticorps ou un fragment de liaison à l'antigène correspondant, l'anticorps ou le fragment de liaison à l'antigène se liant à un antigène de la bactérie Candidatus savagella et (i) inhibant l'adhérence de la bactérie aux cellules épithéliales intestinales, de préférence aux cellules épithéliales intestinales humaines, et/ou (ii) provoquant une déplétion de la bactérie. L'invention concerne en outre un médicament, comprenant l'anticorps ou un fragment de liaison à l'antigène correspondant selon l'invention ou comprenant un anticorps qui a été préparé par le procédé selon l'invention. En outre, l'invention concerne un kit comprenant un anticorps ou un fragment de liaison à l'antigène correspondant selon l'invention pour la réduction de la prolifération des cellules Th17, de la différenciation des cellules Th17 ou de l'activité des cellules Th17 et/ou pour l'inhibition de la formation d'anticorps contre les antigènes propres au corps par les lymphocytes B. Le kit selon l'invention comprend éventuellement un antibiotique. En outre, l'invention concerne un procédé pour la préparation d'un anticorps selon l'invention, le procédé consistant à : a) immuniser des poules à l'aide d'un peptide immunogène constitué par un antigène de la bactérie Candidatus savagella ; et b) extraire et purifier les anticorps formés dans les poules ou dans un œuf pondu par ces poules. Enfin, l'invention concerne un procédé pour la préparation d'un médicament selon l'invention, consistant à : a) préparer un anticorps ou un fragment de liaison à l'antigène correspondant selon l'invention ; et b) formuler l'anticorps ou un fragment de liaison à l'antigène correspondant sous forme de médicament.
EP19749717.5A 2018-08-03 2019-08-02 Traitement de maladies immunes par neutralisation médiée par des anticorps de bactéries intestinales spécifiques Withdrawn EP3830247A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102018213030.2A DE102018213030A1 (de) 2018-08-03 2018-08-03 Behandlung von Immunerkrankungen durch die antikörpervermittelte Neutralisierung spezifischer Darmbakterien
PCT/EP2019/070910 WO2020025801A1 (fr) 2018-08-03 2019-08-02 Traitement de maladies immunes par neutralisation médiée par des anticorps de bactéries intestinales spécifiques

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JPH08214900A (ja) * 1995-02-17 1996-08-27 Yakult Honsha Co Ltd Ｒｎａ配列、新規セグメント細菌、検出方法、およびその用途
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ES2393674T3 (es) 2003-11-14 2012-12-27 Brigham And Women's Hospital, Inc. Métodos para modular la inmunidad
WO2011046616A2 (fr) 2009-10-15 2011-04-21 New York University Procédés pour la modulation d'infection bactérienne
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EP3978598B1 (fr) * 2011-12-01 2024-08-28 The University of Tokyo Bactéries d'origine humaine qui induisent la prolifération ou l'accumulation de lymphocytes t régulateurs
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US20210198346A1 (en) 2021-07-01
KR20210040998A (ko) 2021-04-14
CN112867785A (zh) 2021-05-28
WO2020025801A1 (fr) 2020-02-06
DE102018213030A1 (de) 2020-02-06
JP2021534229A (ja) 2021-12-09
DE102018213030A8 (de) 2020-04-02

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