SELF-ASSEMBLING SYNTHETIC PROTEINS

Abstract

The present disclosure provides for a synthetic immunogenic protein for use as an immuno-modulatory agent to enhance mammalian immune reactions towards conjugated protein or peptide containing antigens that are otherwise poorly immunogenic, including but not limited to self-antigens. The chimeric immunogenic proteins of the present disclosure can be used in the treatment of many illnesses, including but not limited to cancers, infectious disease, autoimmune disease, allergies and any clinical indication involving or affected by the immune response of a mammalian host.

Claims

1. A recombinant synthetic protein, comprising: a monomeric sequence that is able to assemble into stable pentamers, including a Cholera Toxin β (CTB) subunit having missense mutations T1F, P2T, Q3D, N4I, M37I, A38I, I39L, I40V, T41N, T78S, E79N, A80S, A95S, A102V, and N103R; a peptide spacer; and a polypeptide including a full-length growth factor, or part thereof, selected from the group consisting of IGF-1, IGF-2, FGF1, FGF2, TGF-α, TGF-β, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PDGF, NGF, EGF, HGF, BMP's, PDL1 and IL-1, IL-2, IL-3, IL-4, IL-5, and IL-6, wherein the part thereof includes a neutralizing domain of the growth factor, wherein the polypeptide is separated from the CTB subunit by the peptide spacer, which prevents the polypeptide from sterically inhibiting assembly of the pentamers by the CTB subunit.

2. The recombinant protein according to claim 1, wherein the monomeric sequence further comprises one or more additional polypeptide sequences.

3. (canceled)

4. The recombinant protein according to claim 2, wherein the one or more additional polypeptide sequences include a full-length growth factor, or part thereof, selected from the group consisting of IGF-1, IGF-2, FGF1, FGF2, TGF-α, TGF-β, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PDGF, NGF, EGF, HGF, BMP's, PDL1 and IL-1, IL-2, IL-3, IL-4, IL-5, and IL-6.

5. The recombinant protein according to claim 1, wherein the growth factor is TGF-α, TGF-β, or EGF.

6. The recombinant protein according to claim 4, wherein the part thereof includes a neutralizing domain of the growth factor, optionally wherein the part thereof includes a full length or neutralizing domain of one or more growth factors in the protein as a single domain or as two or more multiple repeats.

7-10. (canceled)

11. The recombinant protein according claim 1, wherein the spacer comprises, in part, a growth factor or neutralizing domain thereof, or is selected from the group consisting of SSG, SSGGG, SGG, GGSGG, GGGGS, SSGGGSGGSSG, GGSGGTSGGGSG, SGGTSGGGGSGG, GGSGGTSGGGGSGG, SSGGGSGGSSG, SSGGGGSGGGSSG, SSGGGSGGSSGGG, and SSGGGGSGGGSSGGG, or includes one or more host T-cell epitopes.

12. (canceled)

13. A process of preparing a stable homo-pentamer complex comprising assembling monomeric sub-units of claim 1 to form one or more stable homo-pentamer complexes.

14. A process of preparing a multivalent vaccine formulation comprising mixing one or more single monomeric sub-units of claim 1 to form a multivalent vaccine.

15. A process for treating a patient comprising administering an immunogenic dose of the multivalent vaccine formulation of claim 14 to the patient during a treatment period.

16-29. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0042] The embodiments described in the present disclosure are illustrated in the figures of the accompanying drawings which are meant to be exemplary and not limiting, in which like references are intended to refer to like or corresponding parts, and in which:

[0043] FIG. 1. Illustrates the ELISA signals produced by a panel of soluble mutant clones binding to an immobilized galactose-containing molecule (GM1), following 3 rounds of selection;

[0044] FIG. 2. Illustrates a Western blot of soluble mutant proteins from one of the mutant libraries electrophoresed on an SDS gel, some of which retain the ability to form pentamers, and others that do not;

[0045] FIGS. 3a and 3b. Illustrate a selection of mutations identified after selecting and screening several mutant libraries FIG. 3(a) Sequences of a selection of mutants, each isolated from one of 7 separate mutant libraries and illustrating the distribution of mutated residues, and FIG. 3(b) sequences of synthetic proteins derived from compiling mutations derived from several different mutant clones;

[0046] FIG. 4. Illustrates GM1-binding ELISA data from four compilation mutants, showing that two clones have retained galactose binding activity, and two have not;

[0047] FIG. 5. Illustrates the structure of the complete cholera holotoxin molecule;

[0048] FIG. 6. Illustrates sequences of the synthetic protein according to the disclosure (Sequence ID: 1); and a further sequence of synthetic protein according to the disclosure comprising sequences encoding human TGF Beta1 (Bold), a flexible linker (Italicised), and synthetic protein carrier (Sequence ID: 2);

[0049] FIG. 7. Illustrates the distribution of targeted residues in several mutant libraries (a), and sequences of synthetic proteins (templates) successively obtained (b);

[0050] FIGS. 8A and 8B Illustrate GM1-binding ELISA (a) and Western blot (b) of synthetic carrier (SEQUENCE-3: 5B5−/+G33D) and its immediate precursor (1B6). Specific detection was via cMyc-tag;

[0051] FIGS. 9A and 9B. Illustrate impaired detection of synthetic carrier by anti-CTB monoclonal antibody (a) and polyclonal anti-CTB serum (b);

[0052] FIG. 10. Illustrates immunogenicity of synthetic carrier (precursor) fused to hFGF2. Depicted are IgG responses to hFGf2, in mice sera 28 days after immunisation;

[0053] FIGS. 11A and 11B. Illustrate GM1-binding ELISA (a) and culture medium and crude periplasm preps signals on Western blot (b) of carrier 5B5_I74D, potentially deficient in assembling with CTA2. Including 5B5, 5B5_G33D and 5B5_T78D;

[0054] FIG. 12. Illustrates sequences of the synthetic protein according to the disclosure comprising amino acid sequence (Sequence ID: 3) of synthetic carrier protein indicated are glycine-33 and isoleucine-74 (bold and underlined), resulting in absence of GM1-binding and assembly with CTA, respectively, when changed to an aspartate (G33D and I74D in sequence-4); and

[0055] FIG. 13. Illustrates a further sequence of synthetic protein according to the disclosure (Sequence ID:4) comprising amino acid sequence of synthetic protein comprising sequences encoding synthetic carrier, a flexible linker (in italics), and human growth factor FGF2 (underlined).

DETAILED DESCRIPTION

[0056] Detailed embodiments of the present recombinant proteins or vaccines are disclosed herein, however, it is to be understood that the disclosed embodiments are merely exemplary, which may be embodied in various forms. Therefore, specific functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the recombinant protein disclosed herein.

[0057] The present disclosure provides a synthetic recombinant protein for improving the presentation of the maximum number of growth factor epitopes, tumor antigen epitopes, and/or receptor binding sites as elements of an immunogenic recombinant protein.

[0058] In one illustrative embodiment, a synthetic recombinant protein as shown in Sequence ID: 2 containing at least one growth factor including but not limited to human transforming growth factor (TGF), a tumor antigen, and/or a receptor is described. In alternative illustrative embodiments, the protein may express other immunogenic recombinant proteins that are modeled based upon known immunogenic proteins. It is contemplated within the scope of the disclosure that such recombinant proteins will be expressions of polypeptides that are highly immunogenic to the human immune system. Preferably, the recombinant proteins confer additional properties to the chimeric protein, for example, high expression yield and ease of manufacture, oral stability and the ability to cross from gut to blood stream, and/or previous safe use in humans.

[0059] Certain illustrative embodiments as provided herein include recombinant proteins according to the disclosure within vaccine compositions and immunological adjuvant compositions, including pharmaceutical compositions, that contain, in addition to recombinant proteins at least one adjuvant, which refers to a component of such compositions that has adjuvant activity.

[0060] An adjuvant having such adjuvant activity includes a composition that, when administered to a subject such as a human (e.g., a human patient), a non-human primate, a mammal or another higher eukaryotic organism having a recognized immune system, is capable of altering (i.e., increasing or decreasing in a statistically significant manner, and in certain preferred embodiments, enhancing or increasing) the potency and/or longevity of an immune response. In certain illustrative embodiments disclosed herein a desired antigen and or antigens contain within a protein carrier, and optionally one or more adjuvants, may so alter, e.g., elicit or enhance, an immune response that is directed against the desired antigen and or antigens which may be administered at the same time or may be separated in time and/or space (e.g., at a different anatomic site) in its administration, but certain illustrative embodiments are not intended to be so limited and thus also contemplate administration of recombinant protein in a composition that does not include a specified antigen but which may also include but is not limited to one or more co-adjuvant, an imidazoquinline immune response modifier.

[0061] Accordingly and as noted above, adjuvants include compositions that have adjuvant effects, such as saponins and saponin mimetics, including QS21 and QS21 mimetics (see, e.g., U.S. Pat. No. 5,057,540; EP 0 362 279 B1; WO95/17210), alum, plant alkaloids such as tomatine, detergents such as (but not limited to) saponin, polysorbate 80, Span 85 and stearyl tyrosine, one or more cytokines (e.g., GM-CSF, IL-2, IL-7, IL-12, TNF-alpha, IFN-gamma), an imidazoquinoline immune response modifier, and a double stem loop immune modifier (dSLIM, e.g., Weeratna et al., 2005 Vaccine 23:5263).

[0062] Detergents including saponins are taught in, e.g., U.S. Pat. No. 6,544,518; Lacaille-Dubois, M and Wagner H. (1996 Phytomedicine 2:363-386), U.S. Pat. No. 5,057,540, Kensil, Crit. Rev Ther Drug Carrier Syst, 1996, 12 (1-2):1-55, and EP 0 362 279 B1. Particulate structures, termed Immune Stimulating Complexes (ISCOMS), comprising fractions of Quil A (saponin) are haemolytic and have been used in the manufacture of vaccines (Morein, B., EP 0 109 942 B1). These structures have been reported to have adjuvant activity (EP 0 109 942 B1; WO 96/11711). The haemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A) have been described as potent systemic adjuvants, and the method of their production is disclosed in U.S. Pat. No. 5,057,540 and EP 0 362 279 B1. Also described in these references is the use of QS7 (a non-haemolytic fraction of Quil-A) which acts as a potent adjuvant for systemic vaccines. Use of QS21 is further described in Kensil et al. (1991. J. Immunology 146:431-437).

[0063] Combinations of Q S21 and polysorbate or cyclodextrin are also known (WO 99/10008). Particulate adjuvant systems comprising fractions of QuilA, such as QS21 and QS7 are described in WO 96/33739 and WO 96/11711. Other saponins which have been used in systemic vaccination studies include those derived from other plant species such as Gypsophila and Saponaria (Bomford et al., Vaccine, 10(9):572-577, 1992). [0203] Escin is another detergent related to the saponins for use in the adjuvant compositions of the embodiments herein disclosed. Escin is described in the Merck Index (12.sup.th Ed.: entry 3737) as a mixture of saponin occurring in the seed of the horse chestnut tree, Aesculus hippocastanum. Its isolation is described by chromatography and purification (Fiedler, Arzneimittel-Forsch. 4, 213 (1953)), and by ion-exchange resins (Erbring et al., U.S. Pat. No. 3,238,190). Fractions of escin (also known as aescin) have been purified and shown to be biologically active (Yoshikawa M, et al. (Chem Pharm Bull (Tokyo) 1996 August; 44(8):1454-1464)). Digitonin is another detergent, also being described in the Merck index (12th Ed., entry 3204) as a saponin, being derived from the seeds of Digitalis purpurea and purified according to the procedure described by Gisvold et al., J. Am. Pharm. Assoc., 1934, 23, 664; and Rubenstroth-Bauer, Physiol. Chem., 1955, 301, 621.

[0064] Other adjuvants or co-adjuvants for use according to certain herein disclosed embodiments include a block co-polymer or biodegradable polymer, which refers to a class of polymeric compounds with which those in the relevant art will be familiar. Examples of a block co-polymer or biodegradable polymer that may be included in a vaccine composition or an immunological adjuvant include Pluronic® L121 (BASF Corp., Mount Olive, N.J.; see, e.g., Yeh et al., 1996 Pharm. Res. 13:1693),

[0065] Certain further illustrative embodiments contemplate immunological adjuvants that include but are not limited to an oil, which in some such embodiments may contribute co-adjuvant activity and in other such embodiments may additionally or alternatively provide a pharmaceutically acceptable carrier or excipient. Any number of suitable oils are known and may be selected for inclusion in vaccine compositions and immunological adjuvant compositions based on the present disclosure. Examples of such oils, by way of illustration and not limitation, include squalene, squalane, mineral oil, olive oil, cholesterol, and a mannide mono-oleate.

[0066] Immune response modifiers such as imidazoquinoline are also known in the art and may also be included as adjuvants or coadjuvants in certain presently disclosed embodiments. As also noted above, one type of adjuvant or co-adjuvant for use in a vaccine composition according to the disclosure as described herein may be the aluminium co-adjuvants, which are generally referred to as “alum.” Alum co-adjuvants are based on the following: aluminium oxy-hydroxide; aluminium hydroxyphosphoate; or various proprietary salts. Alum co-adjuvants are be advantageous because they have a good safety record, augment antibody responses, stabilize antigens, and are relatively simple for large-scale production. (Edelman 2002 Mol. Biotechnol. 21:129-148; Edelman, R. 1980 Rev. Infect. Dis. 2:370-383.)

Pharmaceutical Compositions

[0067] In certain illustrative embodiments, the pharmaceutical composition is a vaccine composition that comprises both the recombinant protein according to the disclosure and may further comprise one or more components, as provided herein, that are selected from TLR agonist, co-adjuvant (including, e.g., a cytokine, an imidazoquinoline immune response modifier and/or a dSLIM) and the like and/or a recombinant expression construct, in combination with a pharmaceutically acceptable carrier, excipient or diluent.

[0068] Illustrative carriers will be nontoxic to recipients at the dosages and concentrations employed. For vaccines comprising recombinant protein, about 0.01 mu.g/kg to about 100 mg/kg body weight will be administered, typically by the intradermal, subcutaneous, intramuscular or intravenous route, or by other routes. It will be evident to those skilled in the art that the number and frequency of administration will be dependent upon the response of the host. “Pharmaceutically acceptable carriers” for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, sterile saline and phosphate-buffered saline at physiological pH may be used. Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. In addition, antioxidants and suspending agents may be used.

[0069] The pharmaceutical compositions may be in any form which allows for the composition to be administered to a patient. For example, the composition may be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral (e.g., sublingually or buccally), sublingual, rectal, vaginal, and intranasal (e.g., as a spray). The term parenteral as used herein includes iontophoretic, sonophoretic, passive transdermal, micro-needle administration and also subcutaneous injections, intravenous, intramuscular, intrasternal, intracavernous, intrathecal, intrameatal, intraurethral injection or infusion techniques. In a particular embodiment, a composition as described herein (including vaccine and pharmaceutical compositions) is administered intradermally by a technique selected from iontophoresis, micro-cavitation, sonophoresis or micro-needles.

[0070] The pharmaceutical composition is formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of one or more compounds of the invention in aerosol form may hold a plurality of dosage units.

[0071] For oral administration, an excipient and/or binder may be present. Examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose and ethyl cellulose. Coloring and/or flavoring agents may be present. A coating shell may be employed.

[0072] The composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain one or more of a sweetening agent, preservatives, dye/colorant and flavour enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

[0073] A liquid pharmaceutical composition as used herein, whether in the form of a solution, suspension or other like form, may include one or more of the following carriers or excipients: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as squalene, squalane, mineral oil, a mannide monooleate, cholesterol, and/or synthetic mono or digylcerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[0074] In a particular embodiment, a pharmaceutical or vaccine composition of the invention comprises a stable aqueous suspension of less than 0.2 um and further comprises at least one component selected from the group consisting of phospholipids, fatty acids, surfactants, detergents, saponins, fluorodated lipids, and the like.

[0075] It may also be desirable to include other components in a vaccine or pharmaceutical composition, such as delivery vehicles including but not limited to aluminium salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules, and liposomes. Examples of additional immunostimulatory substances (co-adjuvants) for use in such vehicles are also described above and may include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), glucan, IL-12, GM-CSF, gamma interferon and IL-12.

[0076] While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration and whether a sustained release is desired. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention.

[0077] Pharmaceutical compositions may also contain diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates, including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents. Preferably, product may be formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents.

[0078] In an illustrative embodiment, the epitope or receptor supporting domain of the recombinant protein, whether derived from a natural or synthetic polypeptide sequence, should have the capacity to self-assemble into oligomeric multimers under appropriate chemical/environmental conditions, or to be reduced to monomers under alternative conditions. Ideally, multimerisation domains will assemble into stable multimers with a discreet number of sub-units, for example dimers, trimers, tetramers, pentamers, etc., such that a product of homogeneous size is generated. Without being bound to any particular theory, it is thought that the recombinant synthetic protein, as set forth in Sequence ID: 1, will allow assembly into stable multimers with an insignificant number of sub-units. Examples of natural polypeptides include, but are not limited to, leucine zippers, lac repressor protein, streptavidin/avidin, cholera toxin B subunit, Pseudomonas trimerization domain, and viral capsid proteins.

[0079] According to the disclosure, the recombinant proteins, whether growth factors or parts thereof, cellular receptors or parts thereof, tumor antigens or parts thereof, are related to broad range of either cellular pathways involved in chronic disease or cancers for growth factors and receptors and to broadest possible range of solid tumors for use of tumor antigens within the said synthetic proteins. The proteins are in the form of a recombinant protein and may be useful in treating chronic diseases, for example, breast, lung, bladder, ovarian, vulva, colonic, pulmonary, brain, colorectal, intestinal, head and neck, and esophageal cancers. As different tumor antigens can be expressed and multiple cellular receptors and growth factors over expressed in the said diseases, the proteins described hereunder can contain one or more different tumor antigens, one or more different receptors or growth factors of one or multiple cellular pathways associated with the disease. These proteins are called “multivalent.”

[0080] In the context of the present disclosure, “neutralizing domain” is defined as a region or regions of either or both member(s) of a specific binding pair, e.g. a growth factor and its cognate receptor, wherein the binding of a third molecule that is not a member of the specific binding pair to the aforementioned region(s) will prevent the subsequent binding of the two members of the specific binding pair. The third molecule can be another protein molecule including but not limited to an antibody, or can be a small non-protein molecule, and can be either natural or synthetic in origin. The neutralizing domain will normally include those regions of the members of the specific binding pair that are in direct contact during binding, and will also include regions out-with said regions where upon binding of a third molecule introduces sufficient stearic hindrance to prevent the members of the specific binding pair from binding directly.

[0081] It is well established in the field that specific recognition of a ligand by its cognate receptor is defined by an interaction between the binding site of the receptor and a particular molecular signature (epitope) of the ligand. Thus an antibody that either binds to or otherwise blocks the receptor binding site, or binds to or otherwise blocks the recognition epitope of the ligand, will prevent ligand receptor interactions. Such antibodies are described as being “neutralizing.” In the context of the present disclosure it is desirable that neutralizing antibodies are generated by the host upon administration of the recombinant protein, and thus the protein sequence may express or include one or more of all of, or a suitable sequence derived from, a growth factor or tumor antigen such that epitopes required for receptor binding are presented in a functional (native) conformation.

[0082] In addition to expressing multiple copies of a single tumor antigen, receptor, and/or growth factor, presented as a single tumor antigen, receptor, and/or growth factor or part thereof per physical site, and/or as chains of repetitive tumor antigen, receptor, and/or growth factor sequences (for example, n=1 or more); the protein according to the disclosure may also include expressions of one or more epitopes or binding sites from two or more different tumor antigens, receptors, and/or growth factors present as single or as chains at different positions within the sequence of the recombinant protein.

[0083] In an illustrative embodiment, a protein comprised of a homogeneous recombinant protein expressing one or more epidermal growth factor (EGF) neutralizing domains is disclosed. The protein is in the form of a recombinant protein and may be useful in treating chronic diseases, for example, breast, lung, bladder, ovarian, vulva, colonic, pulmonary, brain, colorectal, head and neck, and esophagus cancers. In an illustrative embodiment, the protein is a recombinant protein expressing or including EGF sequences and synthetic polypeptide sequences according to the disclosure. In one illustrative embodiment the synthetic polypeptide sequence is substantially similar to Sequence ID: 1.

[0084] In another illustrative embodiment, a protein comprised of a homogeneous recombinant protein expressing one fibroblast growth factor (FGF) is disclosed.

[0085] In a further illustrative embodiment, the protein is a recombinant protein expressing or including FGF sequences and synthetic polypeptide sequences according to the disclosure.

[0086] In yet a further illustrative embodiment, a protein comprised of a homogeneous recombinant protein expressing one transforming growth factor Beta 1 (TGF-β1) is disclosed. In an illustrative embodiment, the protein is a recombinant protein expressing or including TGF-β1 sequences and synthetic polypeptide sequences according to the disclosure.

[0087] In yet another illustrative embodiment, a protein comprised of a homogeneous recombinant protein expressing one transforming growth factor-Beta 1 (TGF-β1) is disclosed. In an illustrative embodiment, the protein is a recombinant protein expressing or including TGF-β1 sequences and synthetic polypeptide sequences substantially similar to Sequence ID: 2 according to the disclosure.

[0088] In another illustrative embodiment, expressing one insulin-like growth factor-1 (IGF-1) is disclosed. In an illustrative embodiment, the protein is a recombinant protein expressing or including IGF-1 sequences and synthetic polypeptide sequences a protein comprised of a homogeneous recombinant protein expressing one hepatocyte growth factor (HGF) is disclosed.

[0089] In a further illustrative embodiment, the protein is a recombinant protein expressing or including HGF sequences and synthetic polypeptide sequences according to the disclosure.

[0090] In a further illustrative embodiment, a protein comprised of a homogeneous recombinant protein expressing one Insulin-like growth factor-1 (IGF-1) and one insulin-like growth factor-2 is disclosed. In an illustrative embodiment, the protein is a recombinant protein expressing or including IGF-1 sequences, IGF-2 sequences and synthetic polypeptide sequences according to the disclosure.

[0091] In yet another illustrative embodiment, a protein comprised of a homogeneous recombinant protein expressing one vascular endothelial growth factor-A (VEGF-A) and one vascular endothelial growth factor-C (VEGF-C) is disclosed. In an illustrative embodiment, the protein is a recombinant protein expressing or including VEGF-A neutralizing domain sequences, VEGF-C sequences and synthetic polypeptide sequences according to the disclosure.

EXAMPLES

[0092] Aspects of the disclosure are further described in detail as in the following examples. However, the following examples are not intended to limit the scope of the disclosure to the precise details of methodology or construction set forth below. Practical and illustrative embodiments are illustrated and described in the following examples. However, it will be appreciated that those skilled in the art may make modifications and improvements within the spirit and scope of the present disclosure.

Example 1: Construction of a Selectable Carbohydrate-Binding Display System

[0093] A system whereby libraries of mutants could be generated and screened for mutants with desirable characteristics was needed that included a physical linkage between the selectable phenotype and the encoding genotype of a diverse population of clones. Such a system was developed using established technologies employed to great effect in the field of antibody engineering amongst others.

[0094] A gene encoding the chosen carbohydrate-binding domain was cloned in-frame and upstream of the minor coat protein gene (gene III) of M13 bacteriophage, in both a complete independently functional M13 ‘phage’ vector with a suitable selectable marker (M13-KO7 ‘phage’), and a ‘phagemid’ vector that includes only the F1 packaging region and minor coat protein-encoding gene from the virus. The former vector when introduced into a suitable bacterial host such as E. coli and propagated appropriately, will generate virus particles that display five copies of the chosen carbohydrate-binding domain at one end of the filamentous virus particle, as N-terminal fusions of the minor coat protein P3 (encoded by gene III).

[0095] The latter, due to the nature of virus propagation when using a phagemid/helper phage system, and familiar to those practiced in the art, will generate virus particles in which only a small minority of the population will display typically one or less copies of the same carbohydrate-binding domain-P3 fusion protein.

[0096] Clones derived from each vector were propagated under appropriate conditions, well known to those familiar with the technology, and the culture supernatants containing virus particles were screened by ELISA for binding activity to the major carbohydrate recognised by the carbohydrate-binding domain. In this example a complex molecule that includes a distal galactose group, and which is amenable to immobilisation by adsorption onto a typical 96 well immunoassay plate was used. In order to bind to the carbohydrate group, it is necessary for two or more of the carbohydrate-binding domains to associate in a complex as a carbohydrate binding pocket is formed between adjacent domains.

[0097] In the former ‘phage’ example, this could potentially be formed between adjacent P3 fusion proteins on a single virion, subject to acceptable orientation constraints of the fusion protein, or between separate virus particles. Using the phagemid system, such associations would most likely form between two or more separate virus particles as the proportion of viruses including two or more fusion proteins on a single virion is anticipated to be very small.

[0098] Upon screening the aforementioned clones, a significantly stronger ELISA signal was generated from the ‘mono-valent’ phagemid-derived clone than from the penta-valent phage clone, the latter also producing a much lower titre of infective virus particles. The poor performance of the phage system in this example does not limit the utility of the system in the process described, but more likely is believed to be a function of either steric/orientation constraints imposed on the system by the particular genetic linkage employed here, and/or infectivity restrictions imposed by the fusion protein (the P3 minor coat protein is the mediator of virus infection). It was therefore the phagemid based system that was chosen to generate and select variant mutant proteins.

Example 2: Generation of Mutant Clone Libraries

[0099] Libraries of mutant polypeptide-encoding clones were generated from a genetic template derived from a carbohydrate-binding self-assembling protein domain. In the present example, the protein domain was selected from the A1B5 group of bacterial holotoxins. Structural and functional information obtained from published data bases such as the Protein Data Base, and the scientific literature, were used to identify regions and/or specific residues thought potentially or proven to be involved in the ability of the template protein domain to form stable homo-pentamers, or to interact with specific carbohydrate-containing moieties found on the surface of many mammalian cell types. These regions/residues were excluded from subsequent mutagenesis and mutant screening rounds.

[0100] Regions or residues defined as above as being potentially amenable to mutation and not involved in desired characteristics were selected and targeted for rational or random mutagenesis, as deemed appropriate. In order to maximise the chances of generating and selecting carbohydrate-binding variants that were divergent from the template domain, mutations were restricted to a limited number of residues in close proximity for each sequential round of library construction screening.

[0101] Depending on the region or residue being targeted, mutation was either random (i.e. including potentially all 20 amino acids), or functionally restricted as far as reasonably possible to those residues that display similar bio-physical and/or chemical properties to those found in the template domain.

[0102] Characteristics that were considered relevant in such cases included, but were not limited to, side chain size, charge, polarity, hydrophobicity/hydrophilicity, and the ability to participate in formation of specific secondary structures such as α-helices. Where possible/feasible, the residue encoded by the template in any given position was omitted from the mutant libraries to avoid selection of non-mutants.

[0103] Libraries were generated by designing oligonucleotide primers that included both regions of sequence that were homologous to the template gene such that the primer (and specifically its 3′-end) would anneal to the template DNA under appropriate conditions, and further regions in which appropriate degeneracy was included such as to encode a diversity of amino acids in one or more positions.

[0104] In the instant example, such oligonucleotides were constructed using defined degeneracy familiar to those instructed in the art whereby the DNA base in any diversified position comprised equal quantities of 2-4 bases (G, A, T or C) as required. Alternatively, more controlled diversity is envisaged whereby oligonucleotides generated from positional mixes of tri-nucleotide phosphoramidites are used such that the specific residues (amino acids) and their relative proportions are precisely controlled at each diversified position.

[0105] Where mutations were to be introduced at either terminus, a single PCR reaction was performed (and repeated when necessary) using one or two degenerate primers in order to introduce the required genetic diversity. The resulting product, which included (primer-derived) flanking restriction sites, was cloned into the phagemid vector outlined in example 1 above such that a library of variant proteins was encoded as in-frame genetic fusions to the minor P3 protein-encoding gene (gene III) of the M13 bacteriophage.

[0106] Where is was desired to introduce diversity into region(s) distal to the termini of the template gene, two pairs of primers were used such that each pair included one terminal (5′ or 3′) primer that exactly matched the template sequence, and one degenerate primer that encoded some, all or none of the required diversity. The 3′-end of the degenerate primers were designed such that i) each exactly matched the template sequence, and ii) each was exactly complementary to the degenerate primer from the second primer pair.

[0107] In this way, two PCR products were generated, and then annealed to and linked to each other via the complementary overlapping sequence derived from the degenerate primers. The resulting product was subsequently cloned into the phagemid vector as an in-frame genetic fusion with the gene III minor coat protein gene. Successive libraries were either built upon the outputs from screening and selecting one or more clones from the previous library, or were built and screened in parallel from the template or one or more selected mutant clones.

[0108] All genetic construct libraries were introduced into host E. coli TG1 cells by electroporation using standard methodologies, and transformants plated onto selective media. Following appropriate incubation, colonies of bacteria were scraped off plates and stored and/or screened as required.

Example 3: Screening of Mutant Libraries

[0109] In order to carry out selections of mutants from the libraries, cultures (of libraries) were inoculated into liquid media (2×TY, 100 μg/ml ampicillin, 1% glucose) such that sufficient cells to include 100-1000 fold representation of the anticipated diversity were included, and sufficient volume to ensure that the OD.sub.600 was in the region of 0.1. Cultures were then incubated (with shaking) at about 37° C. until the OD.sub.600 reached 0.4-0.6 (i.e. log phase growth). The cultures were then infected by the addition of M13 KO7 helper phage at the ratio of ˜20 helper phage per bacterial cell (OD.sub.600 of 1.0 taken to be ˜8.0×10.sup.8 cells/ml). After approximately 30 min static incubation, cells were incubated for about 30 additional min with shaking. Kanamycin was added to ˜50 μg/ml and the cultured incubated overnight at ˜30° C. with shaking.

[0110] The following morning, cultures were centrifuged for about 25-30 min at ˜8,000 g to pellet cells, and the supernatant removed and retained. Cell pellets were discarded. A 20% volume of 200 mM NaCl, 20% PEG 6,000 was added to the culture supernatant, and incubated on ice for about 1 h to precipitate phage particles. Phage were pelleted at ˜8,000 g for about 25-30 min., and re-suspended in 20 percent of the original volume PBS (phosphate-buffered saline). Again, 20% new volume of 200 mM NaCl, 20% PEG 6,000 was added and the phage incubated on ice for about 25-30 min. The phage were pelleted again at ˜8000 g for 25-30 min, and the pellet re-suspended in ˜2 ml phosphate buffered saline (PBS). The resulting suspension was transferred to Eppendorf tube(s) and pelleted at maximum speed for 5 min to pellet any remaining bacterial cells/debris. The phage suspension was then used for selections.

[0111] In order to carry out selections, an immunotube was coated with a suitable antigen such as an immobilizable derivative of galactose, or a natural ligand of the B-subunit, at 1-10 μg/ml (typically 5 ml) overnight at 4-8° C. or at room temperature for 1 h. After washing 3-5 times with PBS (by simply pouring into the immunotube and pouring out again), the tube was blocked by the addition of MPBS (PBS containing 2% milk powder) for 1-2 h at 37° C. The MPBS was washed out as described above, and approximately 1×10.sup.11 to 1×10.sup.12 or more phage particles added.

[0112] The volume was made up to ˜5 ml, and the immunotube sealed e.g. with para-film. It was then ‘tumbled’ (end-over-end) for about 30 min, then incubated standing for about 90 min to allow those phage that display a protein from the mutant library that is able to recognise the immobilised ligand, to bind to it. Phage that had not bound, or were only weakly bound, were removed by washing. The stringency of the selection was varied as required by the number of washes used, the use of PBST (PBS containing 0.1% Tween 20) to wash, or adjusting the coating concentration of ligand.

[0113] Bound phage particles were eluted from the immunotube by adding 1 ml 100 mM triethylamine (TEA) and tumbling for a maximum of 10 min (longer incubation adversely affects phage viability), then pouring immediately into 0.5 ml 1 M Tris-HCl (pH 7.4) to neutralise. About 0.75 ml eluted phage was added to ˜10 ml log phase culture of a suitable E. coli strain such as TG1 (Agilent). The culture was incubated at about 37° C. without shaking to allow infection. Serial dilutions of a sample of the infected cells were spread onto small TYE plates containing 100 μg/ml ampicillin and 1% glucose. The remaining cells were plated onto larger bioassay plates with the same media. All were incubated overnight at about 30° C. Of the other 0.75 ml phage, ˜75 μl were infected as above into ˜1 ml log phase E. coli HB2151 cells and serial dilutions plated out as and incubated as above. The remaining phage were stored at about −80° C. as a glycerol stock (˜15% glycerol).

[0114] The following day large bio-assay plates were scraped to remove the cells, which were then stored as glycerol stocks or grown and ‘rescued’ with M13 KO7 helper phage as described earlier, to prepare an enriched population of phage for the next round of selection. Typically two to three sequential rounds of selection were carried out. Upon completion of selections, individual colonies from the small serial dilution plates of HB2151 cells were picked into 96 well culture plates containing 100 ˜μl/well 2×TY with 100 μg/ml ampicillin, and incubated with shaking overnight at 37° C. The following day, approximately 5 μl culture from each well was inoculated into a fresh plate with ˜150 μl media/well, and incubated at about 37° C. with shaking for about 2 hours. Additional media containing 1 M IPTG was added to give a final concentration of 1 mM IPTG, and the plate incubated with shaking overnight at 30° C. In HB2151 cells, induction with IPTG results in the expression of soluble mutant proteins including a detectable c-myc peptide tag, rather than proteins fused to a component of a virus particle as is the case with E. coli TG1 cells. The initial 96 well culture plate was stored at about −80° C.

[0115] The next day, culture supernatants from the induced 96 well plate were assayed for binding to immobilised carbohydrate using typical ELISA protocols, and binding detected with a readily available HRP-labelled anti-c-Myc antibody (FIG. 1). Clones that were strongly positive for binding to GM1 are shown in shaded cells. Positive and negative controls are boxed.

[0116] Culture supernatants from clones that were strongly positive by binding ELISA were further analysed by SDS PAGE and Western blot to assess the presence of pentamers and other multimeric states as illustrated in FIG. 2. Samples were not reduced or boiled prior to electrophoresis, and protein was detected with HRP-labelled anti-c-Myc antibody. In the example shown, clones C1 and C3 are positive controls that readily assemble into pentamers of ˜60 kDa. Clone C2 is a negative control that runs as a 12 kDa monomer under the conditions used. Clones A1 and A3 can be seen to run primarily as pentamers, whereas a significant proportion of clone B12 forms monomers. The remaining clones form either only monomer (B1, B7) or form many multimeric forms.

Example 4: Compilation of Mutations

[0117] In order to assess the capacity of the maximum number of residues to accommodate mutations, several different libraries each targeting different residues or regions of the template, were constructed and screened. As a result, a large number of clones were identified that exhibited the desired selection criteria of binding to immobilised carbohydrate derivatives and of forming predominantly pentamers, however each of which included a number of unique mutations/differences as illustrated in FIG. 3A. In order to generate clones with potentially enhanced characteristics it was necessary to combine these varied sequences into a smaller number of clones.

[0118] As the mutant generation and selection process had identified several positions where three or more different residues could be accommodated, a total of four combination mutants (A-D) were designed as illustrated in FIG. 3B. The four clones containing combined mutations were expressed, and the synthetic proteins evaluated for their binding compared to appropriate controls by ELISA as illustrated in FIG. 4. It was found that two of the clones (A and C) had only weak binding, however the other two (B and D) were able to bind to GM1 very strongly. This indicates that mutations derived from separate clones can be combined successfully, however the enhancement or even retention or of selected binding characteristics is not guaranteed.

Example 5: Stabilization of Homo-Pentamers

[0119] In their native fully functional form, the bacterial holotoxins described by the group name ‘A1B5’ comprise five B-subunits and a single A-subunit. The B subunits assemble into a pentameric ring with a central ‘pore’. A single A-subunit sits atop this ring and projects down into the void as illustrated in FIG. 5. The A-subunit itself comprises 2 distinct domains. The A1 domain, which is released when the holotoxin is translocated into a cell, contains the enzymatic activity associated with the toxicity of the protein complex. It is held as a part of the holotoxin by the A2 domain. This has an alpha-helical structure in which the A1 domain sits at the N-terminus, and the C-terminus projects into the pore formed by the 5 B-subunits. Some residues of the C-terminus of A2 form interactions with residues of the B-subunits. As there are confirmed interactions between specific residues of the B-subunits and the A2 domain (Zhang R. G., et al (1995)), it is reasonable to suggest that the presence of the A-subunit, and specifically the A2 domain, contributes to stabilizing the pentameric B-subunit ring. Due to the structural and functional distinctions between the two A-subunit domains, without being bound to any particular theory, it is thought that the A2 domain alone would confer this stabilizing effect on a B-subunit pentamer. Similarly, as it is known that B-subunits are able to form pentameric rings in the complete absence of the A-subunit, it is probable that the A2 domain could associate with, and therefore exert a stabilizing effect on B-subunit pentamers by either being co-expressed with the B-subunits, or by being added exogenously.

[0120] It is therefore proposed that a synthetic or non-naturally occurring polypeptide, that shares a sufficiently similar conformation to holotoxin B-subunits, that is able to assemble into multimers including pentameric rings, and in which the residues corresponding to those that interact with A2 in holotoxin B-subunits are the same or share the same bio-physical properties, then such a synthetic protein pentamer would experience a similar stabilizing benefit. Moreover, it is suggested that in the absence of a natural A2 domain, a synthetic polypeptide could be isolated from a suitable polypeptide library using technologies familiar to those practiced in the art such as ‘phage display’, that would bind at least in part to the exposed surfaces of two or more natural or synthetic monomers when assembled into a pentamer, and so stabilize the structure.

[0121] As naturally occurring A2 domains support a structurally independent A1 domain that does not in itself interact with any other component of the holotoxin, then it is probable that an A2 domain or a functional synthetic equivalent could provide a means of anchoring another polypeptide domain. Without being bound to any particular theory, it is thought that non-naturally occurring polypeptide, that shares a sufficiently similar conformation to polypeptide described in Sequence ID: 1 contributes by itself to form pentameric rings exhibiting a stabilized pentameric ring structure allowing for the attachment of epitopes as described within this disclosure.

Example 6

[0122] Reference is to FIG. 7 for subsequent mutation events. A major binder from the screening that targeted mutations (library 6) in a confined region (the β3 strand) of CTB was selected. Starting off with this template (L6e5) one mutagenesis intermezzo without library construction and screening was carried out. It involved the same DNA methodology as forth in EXAMPLE 2, i.e. generation of PCR products and subsequent annealing/linking via complementary overlapping sequence. The β5-α2 loop (positions 55-64), a ‘permissive’ site proposed by Backström et al. (Gene, 1994), was addressed for inserting peptides. Some non-human epitope peptides were tested and substitution with FVSTLQAA passed criteria (GM1 binding and pentamer formation). The DNA sequence of this carrier was the template for generating the next library, targeting the flexible ends of the protein (positions 1-4 and 102-3). Screening did deliver mutants meeting criteria and the DNA sequence of one of them was used as template in a library targeting two untouched middle regions (approximate β1-β2 and α2-β5 loops). Once more did screening release GM1-binding pentameric proteins. One of them is notable (5B5, 20% changed v CTB), (FIGS. 8A and 8B, SEQUENCE-3).

[0123] Synthetic carrier 5B5 was assessed in ELISAs with anti-CTB antibodies. Detection by a monoclonal was significantly impaired (FIG. 9A). Polyclonal antibodies (three suppliers tested) exhibited a trend towards reduced detection of 5B5. This is illustrated for one of them (a sheep serum source) with normalised amounts of carrier (FIG. 9B).

[0124] In addition, we propose the practicality of the glycine (G) to aspartate (D) mutation at position 33 of the carrier/subunit sequence (Jobling & Holmes, Mol. Microbiol. 1991). The G33D mutation disables ganglioside GM1 binding but subunits still form a pentamer. This was confirmed for binders selected for next mutation round (FIG. 7), but its G33D DNA version was obviously not used as next-round template, and the final 5B5 (FIGS. 7 and 8: the introduced negatively charged aspartate gives it a slower electrophoretic mobility). GM1 is present on all cells. A synthetic carrier that is not able to bind GM1 will be available/presented to immune cells in a higher dosage. Moreover, GM1-binding inability, 20% sequence difference, impaired detection with anti-CTB antibodies, and (pending, see below) absence of assembly with CTA makes it distinct from CTB.

[0125] During processing of the last two libraries, precursor carrier d8_G33D (12% substituted) was fused to a growth factor, this time hFGF2, and His.sub.6-tag purified recombinant conjugate was used to immunise mice, via i.m. administration. Significant IgG antibody responses against FGF2 were obtained (FIG. 10). Immunisations of 5B5 and follow-ups, with and without G33D, fused to hFGF2 (SEQUENCE-4) are planned (pending purification and characterisation).

Example 7

[0126] The cholera holotoxin is an AB5 hexamer, i.e. CTA non-covalently interacts with the CTB pentamer, entering cells via ganglioside GM1 cell surface receptor. The proposed synthetic carrier pentamer is distinct form CTB, besides at least 20% sequence difference and its inability to bind GM1, if it does not bind (the CTA2 component) of CTA. Tinker et al. (Infect. Immun. 2003) have shown that substituting a single residue in CTB (I74D or T78D, in the region interacting with CTA2) disables holotoxin assembly. Synthetic carrier 5B5 with these single mutations still formed pentamers, as shown in FIGS. 11A and 11B for I74D, but absence of interaction with CTA2 in vitro has not been established yet.

Example 8

[0127] It is anticipated that a monomeric carrier will be useful to fuse to and purify complicated antigens, like dimeric TGFβ1 growth factor. With the latter it will still be exposed to the immune cells/system as a relatively large (four entities) complex. Modelling of CTB has revealed exposed hydrophobic residues potentially involved in subunit interaction/multimerisation. By changing them to hydrophilic/polar amino acids (e.g. F25R, L31E, A32Q, L77N) the current pentameric synthetic carrier (5B5), may well be converted to a monomeric version.

[0128] Although the devices, systems, and methods have been described and illustrated in connection with certain embodiments, many variations and modifications will be evident to those skilled in the art and may be made without departing from the spirit and scope of the disclosure. The discourse is thus not to be limited to the precise details of methodology or construction set forth above as such variations and modification are intended to be included within the scope of the disclosure.

[0129] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.