Title:  Delivery of biologically active polypeptides

United States Patent:  6,605,286

Issued:  August 12, 2003

Inventors:  Steidler; Lothar (Ghent, BE); Remaut; Erik (Ghent, BE); Wells; Jeremy Mark (Cambridge, GB); Le Page; Richard William Falla (Cambridge, GB)

Assignee:  Vlaams Interuniversitair Instituut voor Biotechnologie (Zwijnaarde, BE); Microbial Technics Limited (Cambridge, GB)

Appl. No.:  060878

Filed:  April 16, 1998

Abstract

Biologically active polypeptides and/or antigens are delivered by administering to a subject a non-invasive or non-pathogenic bacterium which expresses one or more antigens or polypeptides. The non-invasive or non-pathogenic bacterium can be included in delivery systems or pharmaceutical formulations.

SUMMARY OF THE INVENTION

Thus, the present invention provides:

(i) a method of delivering one or more biologically active polypeptides which comprises administering to a subject a non-invasive or non-pathogenic bacterium which expresses said one or more polypeptides;

(ii) A method of delivering one or more antigens which comprises administering to a subject a non-invasive or non-pathogenic bacterium which expresses said one or more antigens; and

(iii) A method of delivering one or more antigens and/or one or more biologically active polypeptides which comprises administering to a subject a non-invasive or non-pathogenic bacterium which expresses both said one or more antigens and said one or more heterologous biologically active polypeptides.

The biologically active polypeptides can be either homologous to the bacterium or heterologous, derived from either eukaryotic sources or prokayotic sources, or their viruses.

According to another aspect of the present invention, there is provided a non-invasive or non-pathogenic bacterium expressing (i) one or more heterologous biologically active polypeptides and (ii) one ore more antigens.

DETAILED DESCRIPTION OF THE INVENTION

"Biological activity" refers to ability to perform a biological function and with reference to a polypeptide implies that the polypeptide adopts a stable conformation ("folded form") which is the same or closely analogous to its native configuration. When folded correctly or substantially correctly, for example with formation of proper folded units, .alpha.-helices, .beta.-sheets, domains, disulphide bridges etc., a polypeptide should have the ability to perform its natural function. Generally, the unit of function in a polypeptide is a domain.

Mere ability to be bound by an antibody or other receptor, either with or without elicitation of an immune response, is passive and does not constitute "biological activity". Any antigen has the ability to be bound by an antibody but is not necessarily biologically active.

A "heterologous" polypeptide is one not native to the bacterium, i.e. not expressed by the bacterium in nature or prior to introduction into the bacterium, or an ancestor thereof, of encoding nucleic acid for the polypeptide.

A bacterium according to the present invention will in general be Gram-positive, and may in principle be any innocuous bacterium, for example Listeria innocua, Staphylococcus xylosus or a Lactococcus. Lactococci, in particular Lactococcus lactis, represent a preferred embodiment of the present invention. Such bacteria are non-colonising.

The skilled person will appreciate that the methods of the present invention could be used to deliver a range of biologically active polypeptides. Examples of suitable polypeptides include ones which are capable of functioning locally or systemically, eg is a polypeptide capable of exerting endocrine activities affecting local or whole-body metabolism and/or the biologically active polypeptide(s) is/are one(s) which is/are capable of the regulation of the activities of cells belonging to the immunohaemopoeitic system and/or the one or more biologically active polypeptides is/are one(s) which is/are capable of affecting the viability, growth and differentiation of a variety of normal or neoplastic cells in the body or affecting the immune regulation or induction of acute phase inflammatory responses to injury and infection and/or the one or more biologically active polypeptides is/are one(s) which is/are capable of enhancing or inducing resistance to infection of cells and tissues mediated by chemokines acting on their target cell receptors, or the proliferation of epithelial cells or the promotion of wound healing and/or the one or more biologically active polypeptides modulates the expression or production of substances by cells in the body.

Specific examples of such polypeptides include insulin, growth hormone, prolactin, calcitonin, luteinising hormone, parathyroid hormone, somatostatin, thyroid stimulating hormone, vasoactive intestinal polypeptide, a structural group 1 cytokine adopting an antiparallel 4.alpha. helical bundle structure such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, GM-CSF, M-CSF, SCF, IFN-.gamma., EPO, G-CSF, LIF, OSM, CNTF, GH, PRL or IFN.alpha./.beta., a structural group 2 cytokine which are often cell-surface asociated, form symetric homotrimers and the subunits take up the conformation of .beta.-jelly roll described for certain viral coat proteins such as the TNF family of cytokines, eg TNF.alpha., TNF.beta., CD40, CD27 or FAS ligands, the IL-1 family of cytokines, the fibroblast growth factor family, the platelet derived growth factors, transforming growth factor .beta. and nerve growth factors, a structural group 3 cytokine comprising short chain .alpha./.beta. molecules, which are produced as large transmembrane pre-cursor molecules which each contain at least one EGF domain in the extracellular region, eg the epidermal growth factor family of cytokines, the chemokines characterised by their possession of amino acid sequences grouped around conserved cysteine residues (the C--C or C--X--C chemokine subgroups) or the insulin related cytokines, a structural group 4 cytokine which exhibit mosaic structures such as the heregulins or neuregulins composed of different domains, eg EGF, immunoglobulin-like and kringle domains.

Alternatively, the biologically active polypeptide can be a receptor or antagonist for biologically active polypeptides as defined above.

The bacterium expresses the biologically active polypeptide and the antigen from nucleic acid contained within it. The nucleic acid may comprise one or more nucleic acid constructs in which nucleic acid encoding the biologically active polypeptide and nucleic acid encoding the antigen are under control of appropriate regulatory sequences for expression in the bacterium.

Suitable vectors comprising nucleic acid for introduction into bacteria can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. `phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.

In a preferred embodiment, the coding sequences for the biologically active polypeptide and the antigen are contained in an operon, i.e. a nucleic acid construct for multi-cistronic expression. In an operon, transcription from the promoter results in a mRNA which comprises more than one coding sequence, each with its own suitably positioned ribosome binding site upstream. Thus, more than one polypeptide can be translated from a single mRNA. Use of an operon enables expression of the biologically active polypeptide and the antigen to be coordinated.

In an alternative embodiment, the coding sequences for the biologically active polypeptide and the antigen are part of the same nucleic acid vector, or separate vectors, and are individually under the regulatory control of separate promoters. The promoters may be the same or different.

A nucleic acid construct or vector comprising a coding sequence for a biologically active polypeptide and a coding sequence for an antigen wherein each coding sequence is under the control of a promoter for expression in a non-invasive bacterium (as disclosed--especially a non-commensal and/or non-colonising bacterium e.g. a Lactococcus), whether as an operon or not, is provided by a further aspect of the present invention.

A promoter employed in accordance with the present invention is preferably expressed constitutively in the bacterium. Use of a constitutive promoter avoids the need to supply an inducer or other regulatory signal for expression to take place. Preferably, the promoter directs expression at a level at which the bacterial host cell remains viable, i.e. retains some metabolic activity, even if growth is not maintained. Advantageously then, such expression may be at a low level. For example, where the expression product accumulates intracellularly, the level of expression may lead to accumulation of the expression product at less than about 10% of cellular protein, preferably about or less than about 5%, for example about 1-3%. The promoter may be homologous to the bacterium employed, i.e. one found in that bacterium in nature. For example, a Lactococcal promoter may be used in a Lactococcus. A preferred promoter for use in Lactococcus lactis (or other Lactococcus) is "P1" derived from the chromosome of Lactococcus lactis (Waterfield N. R.; Le Page, R. W. F.; Wilson P. W. and Wells J. M., gene, 165:9-15, 1995), the sequence of which is shown in the following (SEQ ID NO. 1):

GATTAAGTCA TCTTACCTCT TTTATTAGTT TTTTCTTATA ATCTAATGAT AACATTTTA TAATTAATCT ATAAACCATA TCCCTCTTG GAATCAAAT TTATTATCTA CTCCTTTGTA GATATGTTAT AATACAAGTA TC.

The nucleic acid construct or constructs may comprise a secretory signal sequence. Thus, in a preferred embodiment the nucleic acid encoding the biologically active polypeptide may provide for secretion of the biologically active polypeptide (by appropriately coupling a nucleic acid sequence encoding a single sequence to the nucleic acid sequence encoding the polypeptide). Ability of a bacterium harbouring the nucleic acid to secrete the polypeptide may be tested in vitro in culture conditions which maintain viability of the organism.

Suitable secretory signal sequences include any of those with activity in Gram positive organisms such as Bacillus, Clostridium and Lactobacillus. Such sequences may include the .alpha.-amylase secretion leader of Bacillus amyloliquefaciens or the secretion leader of the Staphylokinase enzyme secreted by some strains of Staphylococcus, which is known to function in both Gram-positive and Gram-negative hosts (see "Gene Expression Using Bacillus", Rapoport (1990) Current Opinion in Biotechnology 1:21-27), or leader sequences from numerous other Bacillus enzymes or S-layer proteins (see pp341-344 of Harwood and Cutting, "Molecular Biological Methods for Bacillus", John Wiley & Co. 1990). For Lactococcus the leader sequence of the protein designated Usp45 may be preferred (SEQ ID NO. 2):

ATG AAA AAA AAG ATT ATC TCA GCT ATT TTA ATG TCT ACA

met lys lys lys ile ile ser ala ile leu met ser thr

GTG

val

ATA CTT TCT GCT GCA GCC CCG TTG TCA GGT GTT TAC GCT

ile leu ser ala ala ala pro ley ser gly val tyr ala

However, it may be preferable that the antigen accumulates intracellularly. As discussed, preferably the level of accumulation should allow the bacterium to remain viable, i.e. retain some metabolic activity, and may be less than about 10 of cellular protein, preferably about or less than about 5% of cellular protein.

The antigen may in principle be any peptide or polypeptide to which a receptor of the immune system, such as an antibody, can bind. In a preferred embodiment, the antigen is a bacterial toxoid form of a toxin or an antigenic fragment thereof. For good compatibility of expression in Lactococcus, which has a bias towards A/T usage over G/C in its coding sequences (60% A/T), the antigen may be one whose coding sequence is A/T rich (has a higher A/T content than G/C). For instance, the antigen may be a toxoid (or an antigenic fragment thereof), or another immunogenic component from Clostridium or Pneumococcus or other Streptococcus species. Clostridial coding sequences, for example, often have >70% A/T base pair content, as do genes from the important human malarial parasites belonging to the genus Plasmodium.

For use in enhancing an immune response to the antigen, i.e. antigenic peptide or polypeptide, as discussed herein, the biologically active polypeptide preferably has cytokine activity. Cytokines are discussed in "The Cytokine Facts Book", Callard and Gearing (1994), Academic Press. Preferred polypeptides with cytokine activity are interleukins, including Interleukin-2 (IL-2) and Interleukin 6 (IL-6). Many cytokines contain a disulphide bridge and all are secreted from the cells which naturally produce them. The reducing nature of the cytoplasm of bacterial cells would be expected to prevent formation of disulphide bridges. It would not be obvious that a polypeptide which is naturally secreted, especially on which naturally contains a disulphide bridge, would be biologically active when retained in a bacterial cell.

Thus, in one embodiment, the biologically active polypeptide is one which is secreted from cells which naturally produce it.

The use of a cytokine to enhance an immune response to the antigen in accordance with the present invention is particularly apposite for antigens of low immunogenicity. Furthermore, application of an immunogen to a mucosal membrane generally elicits an IgA response. The ability of a vaccine to elicit a good (protective level) mucosal immune response is a highly desirable feature, since it is now known that sIgA antibodies play a vital role in protecting mucosal surfaces against infection. For example, sIgA which binds to the surface of the cholera bacillus has been shown to be capable of preventing experimental cholera in mice. sIgA which effectively neutralised HIV-1 may play an important role in protecting against infection with this virus, since once the virus has gained access to the body a lifelong infection is established. Methods for the reliable and long-lasting induction of mucosal sIgA responses are therefore much sought after, since the great majority of human viruses and bacterial pathogens initiate infections by colonising mucosal surfaces.

Thus, antigens of low immunogenicity from a parasite against which an enhanced IgA response is beneficial may be employed particularly advantageously in the present invention, for instance the P28 immunogen (glutathione-S-transferase) of Schistosoma mansoni.

To generate a bacterium according to the present invention, nucleic acid is introduced into a bacterial host cell. Thus, a further aspect of the present invention provides a method comprising introducing nucleic acid as disclosed into a non-invasive bacterium, preferably a Gram-positive bacterium and most preferably a non-commensal, non-colonising bacterium (such as Lactococcus). The introduction may employ any available technique. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene. Growing the cells in culture under conditions for expression of the biologically active polypeptide and the antigen may be employed to verify that the bacteria contain the encoding nucleic acid and are able to produce the encoded material.

In a further aspect, the present invention provides a method of delivering a biologically active dose of a polypeptide in vivo, the method comprising administering to an individual a non-invasive bacterium containing nucleic acid for expression of a biologically active polypeptide heterologous to the bacterium. As discussed supra, preferred bacteria include Lactococci such as Lactococcus lactis and a preferred route of administration may be by application to mucosa.

Although, it has previously been shown possible to express in such bacteria a heterologous polypeptide in a biologically active form, this has only ever been done in vitro in culture conditions which are optimised for bacterial viability and growth. In vivo, for instance on the mucosal membrane, the bacteria are in an environment which would not be expected to support their growth or viability. It is thus surprising that such bacteria are able to deliver a polypeptide in a dose (amount) which is sufficient for the biological activity of the polypeptide to result in a detectable biological effect.

In a preferred embodiment, the biologically active polypeptide has cytokine activity and the bacterium may also express an antigen. Interleukins such as IL-2 and IL-6 may advantageously be delivered.

It will be appreciated that the methods of the present invention and the use of a non-invasive or non-pathogenic bacterium as described herein provide a wide range of therapeutic methods which would enable the skilled person manipulate, for instance, the immume response of a subject. Thus, the present invention provides, in various other aspects:

(i) a method of regulating the survival, growth, differentiation, effector functions or susceptibility to infection of cells or tissues which comprises administering to a subject a non-invasive or non-pathogenic bacterium as defined herein;

(ii) a method of boosting an immune response against tumour cells or an infection colonising a mucosal surface or adjacent or distant tissue which comprises administering to a subject a non-invasive or non-pathogenic bacterium as defined herein;

(iii) a method of modulating the type of immune response (antibody versus cell-mediated) against a pathogenic infectious agent which comprises administering to a subject a non-invasive or non-pathogenic bacterium as defined herein;

(iv) a method of modulating the infiltration of normal tissues with inflammatory or tumour cells which comprises administering to a subject a non-invasive or non-pathogenic bacterium as defined herein;

(v) a method of controlling the rate of growth, rate of invasion or survival of tumour cells which comprises administering to a subject a non-invasive or non-pathogenic bacterium as defined herein;

(vi) a method of inducing apoptosis in tumour cells which comprises administering to a subject a non-invasive or non-pathogenic bacterium as defined herein;

(vii) a method of downregulating an immune response which comprises administering to a subject a non-invasive or non-pathogenic bacterium which expresses a biologically active polypeptide; and

(viii) a method of treating an allergic autoimmune or other immune dysregulative disease state, which comprises administering to a subject a non-invasive or non-pathogenic bacterium which expresses a biologically active polypeptide.

Alternatively stated, when a cytokine and an antigen are both expressed by a bacterium, an aspect of the present invention provides a method of enhancing an immune response to an antigen, the method comprising administering to an individual a non-invasive bacterium containing nucleic acid for expression of a polypeptide with cytokine activity and an antigen.

Enhancement of an immune response, such as an antibody response, preferably provides a level of immune response which is protective of the individual against subsequent challenge with the antigen in a pathogenic context. For example, if the antigen is a bacterial toxoid or a toxin fragment, the level of an antibody response to administration of a bacterium in accordance with the present invention may subsequently protect the individual against pathogenic consequences of challenge with the bacterial toxin, e.g. upon infection with bacteria which produce the toxin.

Administration of the bacterium by application to a mucosal surface may be advantageous in certain contexts by virtue of generating an enhanced immune response at the mucosal membrane (e.g. IgA response) in addition to a systemic response.

The bacterium may be applied in a nutrient medium, i.e. medium containing a substance or substances which sustain (at least in vitro) metabolic activity in the bacterium. Such substances may sustain viability if not growth of the bacterium. Such substances may include an energy source such as glucose, amino acids and so on.

The individual to which the bacterium is administered may be human or animal, i.e. a non-human mammal. Administration may conveniently be nasal, and may be oral, vaginal or anal. In contexts where mucosal administration is not preferred, the bacterium may be administered by any other suitable means within the capacity of those skilled in the art, e.g. by parental routes (i/v, i/p, s/c, i/m).

In a therapeutic context, i.e. where the biological effect of delivery of the polypeptide to an individual is beneficial to that individual, administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. In a prophylactic context, the amount may be sufficient to reduce the deleterious effect on the individual of a subsequent pathogenic challenge, for instance by enhancing the immune response. The actual amount administered, and rate and time-course of administration, will depend on the aim of the administration, e.g. the biological effect sought in view of the nature and severity of the challenge, and is the subject of routine optimisation. Prescription of treatment, including prophylactic vaccination, for example decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.

A composition comprising bacteria may be administered in accordance with the present invention alone or in combination with other treatments, either simultaneously or sequentially.

The present invention also provides a pharmaceutical composition comprising a bacterium as disclosed. Such a pharmaceutical composition is in one embodiment preferably suitable for application to a mucosal membrane.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the bacterium, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration. For intravenous, cutaneous or subcutaneous injection, or injection at the site of an affliction, a parenterally acceptable aqueous solution may be employed which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required. As discussed, a pharmaceutical comprising a bacterium S for administration in accordance with the present invention may comprise one or more nutrient substances, e.g. an energy source such as glucose, amino acids and so on.

In another aspect, the present invention provides a method of manufacture of a pharmaceutical comprising formulating bacteria as disclosed with a suitable carrier medium for administration to an individual. In one embodiment, the pharmaceutical is suitable for application to a mucosal membrane of an individual.

The present invention also provides a non-invasive bacterium expressing a heterologous biologically active polypeptide, and possibly also an antigen, for pharmaceutical use, i.e. use in a method of treatment of the human or animal body by surgery or therapy, including prophylaxis ("vaccination"). As disclosed, the bacterium may be Gram-positive, is preferably non-commensal and/or non-colonising and suitable examples include Lactococcus. The method preferably comprises administration to a mucosal membrane of an individual, e.g. to enhance an immune response in the individual.

A further aspect of the invention provides the use of any bacterium as disclosed in the manufacture of a composition, i.e. a pharmaceutical composition or medicament, for administration to an individual. Such administration is preferably to a mucosal membrane of the individual and may be to enhance an immune response in the individual, e.g. to an antigen expressed by the bacterium.

Claim 1 of 30 Claims

What is claimed is:

1. A method of delivering at least one heterologous biologically active polypeptide to a subject in need of same which comprises administering to the subject a non-invasive or non-pathogenic bacterium which constitutively expresses at least one heterologous biologically active polypeptide to illicit a biological response from the subject.

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