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WO1996012739A1 - Polypeptides immunomodulateurs - Google Patents

Polypeptides immunomodulateurs Download PDF

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Publication number
WO1996012739A1
WO1996012739A1 PCT/GB1995/002487 GB9502487W WO9612739A1 WO 1996012739 A1 WO1996012739 A1 WO 1996012739A1 GB 9502487 W GB9502487 W GB 9502487W WO 9612739 A1 WO9612739 A1 WO 9612739A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
fragment
immunomodulatory
antigen
conjugate
Prior art date
Application number
PCT/GB1995/002487
Other languages
English (en)
Inventor
Denis Raymond Stanworth
Janos Gergely
Original Assignee
Peptide Therapeutics Limited
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Peptide Therapeutics Limited filed Critical Peptide Therapeutics Limited
Priority to AU37029/95A priority Critical patent/AU3702995A/en
Publication of WO1996012739A1 publication Critical patent/WO1996012739A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to synthetic immunomodulatory polypeptides capable of enhancing antibody production by acting on stimulated B cells, and to such polypeptides in the form of a construct with an antigen or fragment thereof capable of down regulating undesirable immune responses.
  • Amino acids and amino acid residues are represented herein by their standard codes as identified by IUPAC-IUB Biochemical Nomenclature Commission and represent D and L amino acids, their analogues or derivatives.
  • Antibodies are produced by B cells and form one of the body's major defences against invasion by pathogenic organisms. They also contribute to the elimination of cells rendered foreign to the body by using antibody dependent cell mediated cytotoxicity.
  • Immune suppression can vary in severity from almost total - as would be the case in patients treated with cytotoxic drugs, through moderate to mild - as might well be the situation induced by inadvertent exposure to immunotoxicants. Moreover, it is known that immunological competence decreases with advancing age, making it difficult for patients with debilitating disease to combat infectious episodes. Table 1 gives examples of the clinical spectrum of secondary human deficiency conditions conceivably amenable to interaction with an immunomodulatory peptide.
  • polypeptide capable of stimulating B cell function is desirable; it could be used as an adjunct to primary therapy when a depressed immune response contributes to pathogenesis of a disease.
  • Linear polypeptides synthesised to resemble secjuences within the IgG Fc region have been shown to be immunomodulatory in model systems.
  • Figure 1 shows a schematic of the Fc region of human IgG and the domains Y48, Y65, Y51 are labelled.
  • Synthetic linear polypeptides comprising both C H 2 domain sequences (Y48, Y51, Y91) of the Fc region and C H 3 domain sequences (Y75) of the Fc region - in contrast to a linear polypeptide comprising a partial sequence of Y75 - brought about a significant enhancement (by two-threefold) in IgM production.
  • these linear polypeptides were added on the third day to PWM-containing cultures of human B. lymphocytes they had hardly any effect; indicating that their, influence on B cell stimulation occurs in the early phases.
  • Inj ection of the polypeptide brought about a doubling of serum IgG-2b anti-oxazalone and a trebling of the IgG3 antibody levels (in both the primary and secondary responses).
  • the immunorestorative capacity of the Y75 polypeptide was evaluated in three different animal models. In one of these, mice were injected (intraparenterally 200 mg/kg) and two days later primed with oxazalone. Injection of linear polypeptide on the day of hapten priming brought about a threefold enhancement of the secondary antibody response (at day 24), greater than that effected by human Fc region fragments.
  • mice suppressed by injection (intramuscularly) with 6 mg/kg Adreson/cortisone suspension in saline, injection of linear polypeptide one day later (i.e. on the day of priming with oxazalone) brought about a considerable (between 2-3 fold) enhancement in the secondary antibody response (i.e. at day 28).
  • Other mice were experimentally immunosuppressed by antigenic competition by injection (intraparenterally and sub-cutaneously) of NP hapten-BSA conjugate (20 ⁇ g enveloped 1:1 in CFA), followed 12 days later by immunisation with sheep erythrocytes (0.1 ⁇ l, 4% suspension). Injection of the Y75 linear polypeptide (intravenously or intraparenterally) brought about a threefold increase in the number of plaque forming cells in the suppressed mice.
  • the present invention can provide immunomodulatory cyclic polypeptides having an equivalent or greater ability to stimulate, non-specifically, B cell responses than known polypeptides.
  • the present invention can provide an immunomodulatory polypeptide which comprises a cyclised fragment of human IgG heavy chain polypeptide, derivative or analog thereof.
  • the immunomodulatory cyclic polypeptide of the present invention can comprise an amino acid residue sequence of the IgG heavy chain polypeptide cyclised directly (for example by way of a disulphide or amide bond) or indirectly (for example by way of a spacer such as a polypeptide or a hydrocarbon chain) to another amino acid residue of the IgG heavy chain polypeptide.
  • an amino acid residue near to or at the end of the IgG heavy chain polypeptide fragment is bound to an amino acid residue near to or at the other end of the polypeptide fragment.
  • a cysteine residue is near to or at each end of the IgG heavy chain polypeptide, the cysteine residues being bound together by a disulphide bond to form a cystine residue.
  • the present invention can also provide an immunomodulatory polypeptide cyclised via an amide linkage.
  • the immunomodulatory cyclic polypeptide of the present invention has any one of the formulae (1) to (5) below:
  • n and m are independently 0, 1, 2 or 3 ; p is 0 or 1; R is hydrogen or C 1 -C 3 alkyl; and R 1 is OH or NR 3 R 4 in which R 3 and R 4 are independently hydrogen or C 1 -C 3 alkyl.
  • immunomodulatory cyclic polypeptide of formula (1) is the compound named "F31" shown below:
  • the immunomodulatory cyclic polypeptide of the present invention can be used in the manufacture of a medicament for the treatment of immunodeficiency.
  • the present invention can provide a medicament comprising an effective amount of the immunomodulatory cyclic polypeptide preferably together with a pharmaceutically acceptable carrier.
  • the present invention can provide a method of treatment of immunodeficiency comprising administering an effective dose of the immunomodulatory cyclic polypeptide of the present invention.
  • the immunomodulatory cyclic polypeptide of the present invention can be derived from a linear polypeptide within the C H 2 or 0,3 domain of human IgG or analogues or derivatives thereof wherein one or more amino acid residues is added, deleted or substituted by residues having similar physico-chemical properties (vide peptides 1 and 2 above wherein a Tyr residue present in peptide 1 is deleted in peptide 2).
  • the cyclic polypeptide according to the present invention can be synthesised by the solid-phase procedure of Merryfield, R.B., J. Amer. Chem. Soc, 1963, 85:2419, using T-Boc amino acid derivatives described by Stanworth, D.R., et al, Mol. Immunol. 1984, 21:243, or preferably using F-moc chemistry.
  • SEQ ID NO: 3 forms part of the Y48 domain of the IgG Fc region which, as can be seen from figure 1, is a linear ß sheet with a loop at the Thr-Lys-Pro-Arg end and a bend at the other.
  • a cyclised version of this polypeptide would be constrained to adopt a non-natural configuration vis a vis the natural configuration adopted in the linear ß sheet environment. Consequently, it is surprising that the cyclised polypeptide shows any activity at all.
  • cyclised forms of linear compounds are more stable and less reactive than the linear form.
  • cyclic peptides have been shown to be less active than linear peptides by G.P. Zecchini, et al, Archiv der Pharmazie 326. 955-958 (1993).
  • Side chain-side chain cyclisation was used to cyclise For-Cys-Leu-Phe-Cys-OMe to form a conformationally constrained analog of the prototypical chemotactive tripeptide For-Met-Leu-Phe-OMe.
  • the cyclotetrapeptide was shown to have a reduced biological activity compared to the linear peptide.
  • the following example is a cyclic polypeptide according to the invention.
  • This cyclic polypeptide F31 can have an increased ability to enhance antibody production compared to the linear polypeptide F21. Data supporting this is shown below:
  • IgG and IgM titres of sera were compared using solid phase radioimmunoassay.
  • Peptide F31 enhanced the secondary IgM anti-oxazolone response about 30 times while the linear peptide F2l resulted in a 9 times enhancement.
  • F21/1 F21 at 10 -5 M concentration
  • F31c crude F31 at 10 -7 concentration.
  • the numbers represent mean values obtained from 10 experiments. At 10 times lower concentration no effect was detected.
  • polypeptide of the invention can show an equivalent or greater ability to stimulate, non-specifically, B cell responses than known linear polypeptides.
  • polypeptides of the invention can also be used to down regulate undesirable immune responses as well as to stimulate desirable ones.
  • a construct of one of the polypeptides of the invention with an antigen or fragment thereof e.g. in peptide form
  • an immune complex comprising antibody associated with antigen
  • an immune complex comprising antibody associated with antigen
  • a negative feedback regulator of specific antibody synthesis is the action of a negative feedback regulator of specific antibody synthesis.
  • B lymphocytes have low affinity receptors for IgG, i.e. Fc (gamma) receptor-II, which are only engaged efficiently by polyvalent IgG, or IgG held in the proximity of the membrane of the cell in the form of an immune complex. It is believed that the co-clustering of the antigen-receptors and Fc receptors of a B lymphocyte results in the transduction of a negative signal to the cell.
  • the present invention lead to the realisation that the polypeptides of the invention and certain other fragments of IgG (such as the Fc fragment) can be used to represent the antibody moiety of an immune complex.
  • IgG immunomodulatory polypeptides
  • By coupling our immunomodulatory polypeptides (or IgG Fc fragment) to antigen it is possible to produce an artificial immune complex capable of antigen specific suppression of the immune responses of B-lymphocytes (i.e. capable of suppression or pre-emption of the production of antibodies recognising the antigen moity of the artificial complex).
  • the antigen moiety of the complex need not be a whole antigen but could be a fragment, e.g. a peptide fragment. Such a fragment could be used to switch off immune response in an epitope-specific manner, i.e. affecting one epitope of an antigen yet sparing others. This could be utilised in the design of vaccines against certain infectious agents where there is a risk of eliciting antibodies with undesirable properties.
  • Such epitopes could comprise molecular mimics, whereby a whole antigen or organism could carry a risk of autoimmunity resulting from antigenic cross-reactivity of a single epitope of the infectious agent or antigen with a host protein.
  • Another example is the avoidance of generating injection-enhancing antibodies such as have been described in Dengue haemmhoragic fever, and in HIV infected individuals.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des polypeptides immunomodulateurs comprenant un fragment cyclisé de polypeptide à chaîne lourde d'IgG humaine, ou d'un dérivé ou d'un analogue de celui-ci. Un polypeptide cyclique immunomodulateur préféré correspond à la formule (I). L'invention concerne des polypeptides immunomodulateurs capables de renforcer la production d'anticorps en agissant sur des cellules B stimulées, ainsi que de tels polypeptides se présentant sous forme de produit d'assemblage comprenant un antigène ou un fragment d'antigène, capable de réguler à la baisse les réponses immunitaires indésirables.
PCT/GB1995/002487 1994-10-20 1995-10-20 Polypeptides immunomodulateurs WO1996012739A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU37029/95A AU3702995A (en) 1994-10-20 1995-10-20 Immunomodulatory polypeptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9421180.2 1994-10-20
GB9421180A GB9421180D0 (en) 1994-10-20 1994-10-20 Immunomodulatory Polypeptides

Publications (1)

Publication Number Publication Date
WO1996012739A1 true WO1996012739A1 (fr) 1996-05-02

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1995/002487 WO1996012739A1 (fr) 1994-10-20 1995-10-20 Polypeptides immunomodulateurs

Country Status (3)

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AU (1) AU3702995A (fr)
GB (1) GB9421180D0 (fr)
WO (1) WO1996012739A1 (fr)

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* Cited by examiner, † Cited by third party
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CN112724239B (zh) * 2021-01-22 2022-04-08 浙江辉肽生命健康科技有限公司 具有氨基酸结构nkeldpvqklfvdkireyk的生物活性肽及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992009628A1 (fr) * 1990-11-23 1992-06-11 Immunodynamics, Inc. Peptides immunoactifs synthetiques presentant une activite immunomodulante et therapeutique
WO1993008815A1 (fr) * 1991-10-28 1993-05-13 Cytoven Compositions pharmaceutiques dipeptidiques et leurs procedes d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992009628A1 (fr) * 1990-11-23 1992-06-11 Immunodynamics, Inc. Peptides immunoactifs synthetiques presentant une activite immunomodulante et therapeutique
WO1993008815A1 (fr) * 1991-10-28 1993-05-13 Cytoven Compositions pharmaceutiques dipeptidiques et leurs procedes d'utilisation

Also Published As

Publication number Publication date
AU3702995A (en) 1996-05-15
GB9421180D0 (en) 1994-12-07

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