WO1996012739A1

WO1996012739A1 - Immunomodulatory polypeptides

Info

Publication number: WO1996012739A1
Application number: PCT/GB1995/002487
Authority: WO
Inventors: Denis Raymond Stanworth; Janos Gergely
Original assignee: Peptide Therapeutics Limited
Priority date: 1994-10-20
Filing date: 1995-10-20
Publication date: 1996-05-02
Also published as: GB9421180D0; AU3702995A

Abstract

The present invention relates to synthetic immunomodulatory polypeptides which comprise a cyclised fragment of human IgG heavy chain polypeptide, derivative or analog thereof. A preferred immunomodulatory cyclic polypeptide of the present invention has formula (I). The invention provides synthetic immunomodulatory polypeptides capable of enhancing antibody production by acting on stimulated B cells, and such polypeptides in the form of a construct with an antigen or fragment thereof capable of down regulating undesirable immune responses.

Description

Immunomodulatory Polypeptides

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.

Many primary immunodeficiency syndromes involve abnormalities of T cells which would not be affected by a polypeptide designed to upgrade B cell responses. However, there are a number of B cell deficiencies characterised by an increased incidence of infections with pyrogenic bacteria, which would benefit from immunomodulatory therapy. These include:

X-linked hypogammaglobulinaemia

late onset hypogammaglobulinaemia

selective immunoglobulin deficiency

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.

The development of a 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.

The use of synthetic linear polypeptides which act on stimulated B cells to enhance antibody production has been proposed by Sarmay, G., et al, Eur. J. Immunol. 18, 289-294, (1988), wherein the following amino acid sequences in exposed regions of the Fc region of human IgG 1. are disclosed:

*This linear polypeptide is hereafter called "F21".

Work carried out by Stanworth and Gergely on short linear polypeptides having immunomodulatory activity indicated that linear polypeptides comprising amino acid residue sequences within the human IgG Fc region could enhance IgM production by B lymphocytes. Figure 1 shows a schematic of the Fc region of human IgG and the domains Y48, Y65, Y51 are labelled.

IgM synthesis by in vi tro cultures of human mononuclear cells, stimulated with suboptimal doses of pokeweed mitogen (PWM), was determined by measurement of [³H] thymidine incorporation, at a concentration of 10⁶ cells/ml. Synthetic linear polypeptides comprising both C_H2 domain sequences (Y48, Y51, Y91) of the Fc region and C_H3 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. In contrast, when 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. The same linear polypeptides cultured alone with peripheral blood mononuclear cells, in the absence of PWM, failed to stimulate cellular IgM production.

A linear polypeptide derived from Y75 comprising amino acid residue sequences within the human IgG C_H3 domain, which was shown to enhance IgM production by B. lymphocytes in vi tro, was shown to exert a marked immunostimulatory effect in vivo on antibody responses in mice. For instance, administration (intravenously) of 10 mMoles of the polypeptide brought about an increase (threefold) of the number of plaque forming cells (PFCs) in mice immunised with sheep erythrocytes. The same linear polypeptide was shown to give rise to an increase in the primary and secondary antibody responses of mice of the T cell-dependent contact sensitising hapten oxazalone . 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. In other 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 μg enveloped 1:1 in CFA), followed 12 days later by immunisation with sheep erythrocytes (0.1 μl, 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.

Preferably, 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.

Preferably, 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.

Preferably, the immunomodulatory cyclic polypeptide of the present invention has any one of the formulae (1) to (5) below:

wherein n and m are independently 0, 1, 2 or 3 ; p is 0 or 1; R is hydrogen or C₁-C₃ alkyl; and R₁ is OH or NR₃R₄ in which R₃ and R₄ are independently hydrogen or C₁-C₃ alkyl.

More preferably the immunomodulatory cyclic polypeptide of formula (1) above 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_H2 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.

Surprisingly, immunopotentiating and immunorestorative effects can be enhanced by cyclic polypeptide compounds comprising amino acid residue sequences within the IgG heavy chain C_H2 domain (residues 289-301) to an equal or greater extent than by linear polypeptides. This is unexpected because the linear polypeptide:

Thr-Lys-Pro-Arg-Glu-Gln-Gln-Tyr-Asp-Ser-Thr-Tyr-Arg

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. Hence, 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.

Generally it is known that cyclised forms of linear compounds are more stable and less reactive than the linear form. For example, 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.

Example 1

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:

1.A Effect of Cγ2 domain peptides F21 and F31 on the in vivo response of C57B1 mice to oxazolone.

Read out:

IgG and IgM titres of sera were compared using solid phase radioimmunoassay.

Results (Figure 2)

IgG response was not affected by the treatment C57B1 mice are low responders to oxazolone).

IgM response. Peptide F31 enhanced the secondary IgM anti-oxazolone response about 30 times while the linear peptide F2l resulted in a 9 times enhancement.

In Figure 2 the results show the effect on IgM response of 100μl intraperitoneal injection of peptide. The lines show:

F21/1 = F21 at 10^-5M concentration

F21/2 = F21 at 10^-7M concentration

F21/3 = F21 at 10^-5M concentration

F31c = crude F31 at 10^-7 concentration.

1.B Effect of Cγ2 domain peptides on the proliferation of resting human B cells.

Assay: resting human B cells (Bh) were separated from blood after the depletion of T cells by sheep erythrocyte rosetting, using Percoll gradient. 10⁵ Bh cells/well were cultured in 5% FCS/RPMI 1640 medium in the presence of the peptides for 20 hours then F(ab)2 fragment of anti-human IgM was added at 40μg/ml concentration. ³H-thymidine incorporation was tested after 72 h culture. The results are expressed as stimulation indices (SI) [SI = cpm value in peptide+anti-IgM treated sample/cpm value in anti-IgM treated sample.] Fc fragment of human IgG was used as positive control.

Results;

The peptides alone had no effect. At a 5×10^-5M final concentration the peppides enhanced the anti-IgM induced Bh cell proliferation. Peptide F31 had the highest effect producing a higher SI even than the positive control Fc fragment.

F21 F31 Fc fragment

SI: 1.6±0.3 2.8±0.2 2.4±0.1

The numbers represent mean values obtained from 10 experiments. At 10 times lower concentration no effect was detected.

As shown above in Example 1, the polypeptide of the invention can show an equivalent or greater ability to stimulate, non-specifically, B cell responses than known linear polypeptides.

Furthermore, it has been found, surprisingly, that such polypeptides can also be used to down regulate undesirable immune responses as well as to stimulate desirable ones. Thus, a construct of one of the polypeptides of the invention with an antigen or fragment thereof (e.g. in peptide form) might be expected to exhibit some of the properties of an immune complex.

One property of an immune complex (comprising antibody associated with antigen) is the action of a negative feedback regulator of specific antibody synthesis. Thus, following immunisation, the production of antigen-specific antibodies (especially IgG class antibodies) in sufficient quantity will result in the formation of immune complexes which have an inhibitory effect on antibody production, particularly the production of antigen specific antibodies. This is believed to be a factor preventing the production of unnecessarily large amounts of antibody against any particular antigen.

The precise mechanism of down regulation or pre-emption of specific antibody production by immune complexes is not fully understood, although it is believed to involve the simultaneous occupancy of the surface antigen-receptors and Fc (gamma) receptors of B lymphocytes. 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. 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).

From the above realisation, it follows that 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.

Claims

1. An immunomodulatory polypeptide which comprises a cyclised fragment of human IgG heavy chain polypeptide.

2. An immunomodulatory cyclic polypeptide having any one of the formulae (1) to (5) below:

wherein n and m are independently 0, 1, 2 or 3; p is 0 or 1; R is hydrogen or C₁-C₃ alkyl; and R. is OH or NR₃R_« in which R₃ and R₄ are independently hydrogen or C₁-C₃ alkyl.

3. An immunomodulatory polypeptide having the formula:

4. A conjugate which comprises a polypeptide according to any preceding claim and an antigen or fragment thereof wherein the conjugate has a specific suppressive or pre-emptive affect on antibody response to the antigen or fragment thereof.

5. A pharmaceutical composition for enhancing antibody production which comprises a polypeptide according to any of claims 1 to 3 together with at least one pharmaceutically acceptable carrier or excipient.

6. A pharmaceutical composition for down regulating undesirable immune responses which comprises a conjugate according to claim 4 together with at least one pharmaceutically acceptable carrier or excipient.

7. Use of a polypeptide according to any of claims 1, 2 or 3 in the manufacture of a medicament for enhancing antibody production.

8. Use of a conjugate according to claim 4 in the manufacture of a medicament for specifically suppressing or preempting antibody response.

9. A process for producing a polypeptide according to any of claims 1, 2 or 3 which comprises synthesising a cyclised polypeptide from amino acid residues or fragments.

10. A process for producing a conjugate according to claim 4 which comprises synthesising a cyclised polypeptide according to claim 9 and conjugating the polypeptide to an antigen or fragment thereof.

11. A method of treatment to enhance antibody production in a patient, which comprises administering an effective amount of a polypeptide or composition according to any of claims 1, 2, 3 or 5.

12. A method of treatment to suppress or pre-empt specific antibody production m a patient, which comprises administering an effective amount of a conjugate or composition according to claim 4 or 6.