RU2801230C2 - Self-assembly synthetic proteins - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to new recombinant fusion proteins, and can be used in medicine as a therapeutic agent in the treatment of various malignant neoplasms (cancer). The invention discloses a synthetic protein based on a mutant B-subunit of the A1B5 group of bacterial holotoxins, obtained from the B-subunit of cholera toxin (CBT), in which the specified subunit is connected through a peptide linker with a polypeptide comprising a tumor antigen or growth factor, the increased expression of which is observed in cells tumors.

EFFECT: such a fusion protein has immunogenic properties and may be useful as an immunomodulatory agent to enhance immune responses in mammals.

15 cl, 5 ex, 6 dwg

Description

FIELD OF THE INVENTION

The present invention relates to the creation of a synthetic protein scaffold that exhibits certain functional and biophysical characteristics that are useful in using the synthetic protein to deliver and present antigens to the host's immune system, even when said antigens are weakly immunogenic or non-immunogenic in the host, thereby inducing the host to produce specific humoral immune response to antigens. Describes a synthetic protein that is immunogenic to mammalian immune systems and that can assemble into stable defined multimers. In addition, a method is described for using said protein to provide immunogenicity and induce the formation of specific antibodies against weakly immunogenic or non-immunogenic peptides.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority to US Patent Application No. 61/791,268, filed March 15, 2013, entitled "Synthetic Self-Assembling Immunogenic Proteins", which is incorporated herein by reference in its entirety.

PRIOR ART

The introduction of a foreign (non-self) substance, i.e. an antigen, into the immune system of a vertebrate animal usually leads to the induction of the host's immune response directed against this antigen. This usually causes stimulation of B- and/or T-lymphocytes and the production of immunoglobulin molecules (antibodies) that recognize and bind this antigen. There are many factors that affect the extent to which a substance induces an immune response in the host. The degree of foreignness is important because the immune system evolved and evolved to be insensitive to self. Size is also an important factor, since larger molecules are usually more immunogenic than smaller molecules. Molecules that have a molecular weight of less than ~1000 Da (belong to the category of haptens) are too small to be seen by the immune system in isolation and are therefore non-immunogenic, although they may still be antigenic.

Larger molecules will be more complex and therefore more likely to contain numerous immunogenic epitopes and will also be more readily taken up and processed by antigen presenting cells (APCs). The composition of the substance is also important, with proteins being by far the most immunogenic. Polysaccharides are much less immunogenic (in isolated form), while nucleic acids and lipids are mostly non-immunogenic. Similarly, particulate or denatured antigens are more immunogenic than soluble and natural molecules. The route of entry and biological activities of foreign substances can also significantly influence the nature and extent of any host immune response. For example, a parenteral injection of a substance that interacts with components or cells of the immune system will result in a much stronger response to a relatively inert or inactive substance than a mucosal (oral/inhaled) route.

T cells and B cells recognize foreign antigens and respond differently to them. Specialized antigen presenting cells or APCs (macrophages, dendritic cells, and B cells) continuously explore their environment by taking molecules from the extracellular space, including macromolecules and whole microorganisms, and processing their protein composition. Exogenous proteins are digested by a group of proteolytic enzymes in endosomes, and the resulting peptides are presented on the cell surface in the groove of major histocompatibility complex (MHC) class II molecules. These, in turn, are recognized by specialized T cell surface receptors (TCRs). The T cell development process ensures that T cells bearing receptors that respond to MHC II containing autologous peptides are eliminated and only those cells that recognize foreign sequences mature successfully. The peptides that are recognized by T cells (T cell epitopes) are without exception linear, but they are not always exposed or accessible on the native fold from which they were derived.

In contrast, B cell surface receptors or immunoglobulins (BCRs) recognize and interact primarily with soluble proteins (including conformational and denatured epitopes), haptens, polysaccharides, and to a lesser extent some lipids and nucleic acids. The specificity of BCR is identical to that of an antibody that a B cell can secrete. After binding to the cognate antigen, the BCR is internalized and the associated antigen is processed. Only when the antigen is a protein, or when it is associated with a protein component, will the antigen be presented on the cell surface as part of the MHC II complex. Under these conditions, a B cell can be made available for activation by a T helper cell that contains a TCR that recognizes the presented peptide. In the case of a large or complex protein, the B cell may be activated by many different T cells, none of which will necessarily recognize the same antigenic epitope as the BCR, but all of which will recognize the peptide component of the same protein. It is this ability of the immune systems of vertebrates that allows them to produce antibodies against antigenic determinants that are not immunogenic in themselves.

To develop effective vaccines, it is necessary to present antigenic epitopes to the host's immune system in such a way as to stimulate a strong immune response involving T and B lymphocytes. Immune responses that do not involve the activation of effector (helper) T cells and their subsequent stimulation of B cells are usually short-lived and do not lead to antigenic memory, i. do not result in a more aggressive and faster humoral immune response when the host is exposed to that immunogen a second time.

Vaccines are also often required to activate antibodies capable of inhibiting, blocking, or otherwise neutralizing the functional activity of the target, and thereby provide protection to the host. This can be a difficult task for many reasons. Often, those epitopes that should be selected as a target for the humoral immune response have not been identified due to the lack of structural and functional data regarding this target. Even when detailed information about the target and its interactions is established, the identified epitope(s) may not be immunodominant(s) and therefore may not elicit the responses sought for by most patients. In other cases, key protective antigenic determinants may not be a protein, but, for example, polysaccharides in pathogenic glycoproteins, which are usually non-immunogenic (T-dependent) in isolation.

The vast majority of vaccines are administered by parenteral routes; however, there are many advantages to mucosal delivery such as patient compliance, self-administration, reduced risk of infection, and the potential to stimulate both mucosal and systemic immunity. There are also many hurdles to overcome such as dilution of the vaccine, the presence of microflora, the need to withstand low oral pH, membrane permeability and the need for strong adjuvants (Vajdy et al. 2004). In addition, mucosal administration may result in B-cell tolerance rather than an immune response. Dosage can also have a large effect on immune responses. If the immunogen is practically not accepted by the immune system, or if the system is loaded with too high a dose, then tolerance can be induced. Conversely, too low a dose may also lead to tolerance or may simply not be able to stimulate immune cells sufficiently.

Numerous approaches have been developed to help overcome these difficulties. In most cases, vaccines are administered along with some form of adjuvant. Essentially, adjuvants are any formulation that, when co-administered with an immunogen, causes one or more of the following: persistence of the immunogen at the injection site, enhancement of co-stimulatory signals, non-specific stimulation of lymphocyte proliferation, or granuloma formation. There are many forms of adjuvants, for example Freund's complete adjuvant contains inactivated Mycobacterium, while others contain an emulsion of oil (eg squalene) in water. Such adjuvants are commonly used in animals because they can cause adverse reactions at the injection site. Some organic adjuvants are used in human vaccines, such as Montanide (based on mineral oil with added plant ingredients), although in most cases they are inorganic, such as aluminum salts.

Among the most common and widely used methods to overcome low immunogenicity may be the association of a known or desired antigen or antigenic determinant with a highly immunogenic carrier. This is a protein or polypeptide derived from various organisms, such as bovine serum albumin (BSA) and fissure limpet hemocyanin (KLH), which are often used as carriers for chemically conjugated haptens and small peptides to induce antibodies in animals (Berzofsky and Berzofsky , 1993). The carrier presents the haptens on a molecule large enough to be recognized and processed by the host's immune system, and also stimulates the host's immune response, being inherently immunogenic.

In general, the farther phylogenetically distant sources of carrier proteins are from the recipient, the better. It is then likely that the carrier will be very different from the host proteins, and therefore more foreign. Another important consideration when choosing a carrier protein is the possibility that if the protein is a homologue of the host protein and therefore has significant homology, then the elicited immune response may also react against the host proteins and lead to adverse side effects. Non-protein antigens can only be linked chemically, which can limit control over where on the carrier they are attached and how they are presented. Small peptides can be linked chemically or genetically. In other branches of scientific research, recent developments in bioinformatics have led to the development of rational design of immunogens, and in particular peptides.

The concept of peptide vaccines is based on the identification and chemical synthesis of B-cell and T-cell epitopes that are immunodominant and can induce specific immune responses, such as binding the B-cell epitope of the target molecule to a well-known T-cell epitope to ensure its immunogenicity. (Naz R.K. and Dabir P. 2007). Peptides are considered to be relatively easy to manufacture compared to larger and more complex protein antigens. They may also have the necessary chemical stability and lack of oncogenic or infectious potential, making them attractive vaccine candidates. However, several barriers limit the widespread use of peptide vaccines, including their often poor intrinsic immunogenicity and the need for strong adjuvants and carriers. Other studies have suggested that more immunogenic recombinant chimeric proteins can be produced if T helper epitopes are linked in tandem repeats (Kjerrulf M., et al. 1997).

Another popular class of carrier protein are bacterial toxoids. In the case of vaccines against bacterial infection, when the symptoms of the infection are caused by the action of toxins, then these toxins can be used as vaccines. Of course, it is necessary to make them inactive, either chemically or with a non-toxic component. Such attenuated toxins, such as the diphtheria and tetanus vaccines that were developed in the 20th century, are called toxoids. Polysaccharide-protein conjugate vaccines, which are being used or are in the final stages of development by companies such as Wyeth (Pfizer), Aventis Pasteur, GSK, Merck and others, use tetanus, diphtheria or other toxoids.

The cholera toxin subunit B or heat-resistant enterotoxin (LT) of E. coli has been proposed by many as useful protein carriers for various vaccine applications (Nemchinov, L. G et al. 2000, George-Chandy, A. et al. 2001, US Pat. No. 6,153,203). They are highly immunogenic and, in the absence of CT-A, the subunits are non-toxic. While forming the basis for the widespread use of cholera vaccine, it has a proven safety profile when administered systematically. Relatively small (~12 kDa), it can assemble into stable pentamers which give it a much higher molecular weight.

Of particular interest to many researchers is the use of the affinity of CTB and enterotoxin pentamers for G _MI -ganglioside, a branched penta-saccharide that has been found on the surface of nucleated cells. During cholera infection, it is this binding that facilitates the transport of the holotoxin through the intestinal epithelium. There have been many reports in the scientific literature that vaccines based on CTB compounds, either chemically or genetically, can be effective in stimulating mucosal immune properties (George-Chandy, A. et al. 2001, Flouhui Song et al. 2003, Shenghua Li et al. 2009, Harakuni, A. et al. 2005) if administered orally or intranasally. To retain the ability to interact with G _MI -gangliosides that attach in the cavity formed between adjacent CTB subunits, it is important that the target antigen does not block access to the G _MI binding site and does not prevent the assembly of CTB multimers.

It has been demonstrated that fusions at the genetic level can be carried out with CTB, which successfully retains the G _MI binding, although there are also limitations. Liljeqvist, S. et al. (1997) showed that the binding domain of the serum albumin of the streptococcal G protein can be genetically fused to either the N-terminus or the C-terminus of CTB, or to both ends simultaneously while maintaining the G _MI binding. However, it was noted that the N-terminal fusion protein and the double fusion protein were significantly less efficient in forming stable pentamers and less efficient in binding to G _MI . Similarly, it has been demonstrated that large fusions at the genetic level are unable to form pentamers unless a heterogeneous mixture of both chimeric and wild-type CTB is present (Harakuni, A. et al. 2005).

BRIEF DESCRIPTION OF THE INVENTION

According to the present disclosure, the highly immunogenic nature of the synthetic carrier is capable of increasing the host's immune response to incorporated variable sequences due to its intrinsic immunostimulatory and adjuvant-like properties. Additionally, a synthetic carrier is disclosed which, according to the invention, will elicit a humoral immune response in the host against "self" antigenic determinants that are encoded, at least in part, by variable sequences.

In an exemplary embodiment, the recombinant synthetic protein may assemble into stable homopentamers, where each monomer includes one or more antigenic determinants derived from the target proteins.

In another illustrative embodiment, the recombinant synthetic protein can be assembled into stable heteropentamers, where monomers that express different antigenic determinants are assembled together.

In a further illustrative embodiment of the invention, the recombinant synthetic protein is substantially similar to the following sequence:

FTDHTDICGEYHNTQIHTLNDKILSYTESLVGKREIILVNFKGGATFQVEVP GSQFIIDSQKKAIERMKDTLRIAYLSNSKIEKLCVWNNKTPFISIAAISMVR (SEQUENCE ID: 1).

And in yet another further exemplary embodiment, the recombinant synthetic protein includes linker or spacer sequences whereby the variable sequences located at one or both ends of the synthetic carrier are separated from the synthetic carrier in such a manner as to allow the synthetic carrier elements of multiple recombinant proteins to be contacted. In one illustrative embodiment, a recombinant synthetic protein containing a linker sequence that attaches to a growth factor is substantially similar to the following sequence:

ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCL GPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRK PKVEQLSNMIVRSCKCSGGSGGTSGGGGGSGFTDIITDICGEYHNTQIHTL NDKILSYTESLYGKREIILVNFKGGATFQVEVPQHIDSQ KKAIERMKDT LRIAYLSNSKIEKLCVWNNKTPHSIAAISMVR (SEQUENCE ID: 2).

In another exemplary embodiment, the recombinant synthetic protein will assemble into stable ring-shaped pentamers. Other multimeric assemblies, such as but not limited to dimers, trimers, tetramers, and large multimers, are also contemplated within the scope of this disclosure. The linker sequences may vary, but at a minimum they should be of sufficient length to prevent steric inhibition of multimer assembly of different antigenic determinant sequences upon linking the synthetic carrier domains. It is contemplated within the scope of this disclosure that the linkers or spacers may be flexible, allowing the formation of stable pentamers.

In another embodiment, the pentameric structure of the multimer is stabilized by the introduction of at least one stabilizing molecule. The stabilizing molecule may be co-expressed in the same cells as the synthetic molecule, or alternatively may be added exogenously.

In one embodiment, the stabilizing molecule comprises sequences substantially similar to the cholera toxin CT-A2 domain.

In another embodiment of the invention, the stabilizing molecule contains sequences that interact with the synthetic protein in order to stabilize the multimers. It is further contemplated that this stabilization may result in a greater proportion of pentameric forms using conventional manufacturing techniques known in the art.

In a further exemplary embodiment, the stabilizing molecule may serve to attach the antigenic domain to the multimeric form of the synthetic protein.

In yet another further embodiment, the interrupted repeats of the stabilizing molecule may serve to link two or more multimeric forms of the synthetic protein.

In an exemplary embodiment, the linker or spacer sequences will be generally hydrophilic and will be flexible, thus they will not include a defined secondary structure. In another illustrative embodiment of the invention, the linker sequences will be those that do not directly or indirectly affect the folding of the synthetic carrier domain or the various antigenic determinant domains.

In a further illustrative embodiment of the invention, the linker sequences will be proteolysis-resistant extracellular proteases.

In another illustrative embodiment of the invention, the linker or spacer sequences include, without limitation, the following:

SSG, SSGGG, SGG, GGSGG, GGGGS, SSGGGSGGSSG, GGSGGTSGGGSG, SGGTSGGGGSGG, GGSGGTSGGGGSGG, SSGGGSGGSSG, SSGGGGSGGGSSG, SSGGGSGGSSGGG and SSGGGGSGGGSSGGG.

In one illustrative embodiment, the linker sequences will include sequences that, either alone or together with flanking sequences from various antigenic determinants or a synthetic carrier, will form T-cell epitopes. Preferably, the T cell epitopes will be T cell epitopes that are recognized by human T helpers when they are associated with MHC (major histocompatibility complex) class II proteins. Preferably, the T cell epitopes will be associated with MHC II HLA (human leukocyte antigen) subclass DR molecules.

In another illustrative embodiment of the invention, the synthetic recombinant protein is structurally homologous to the B subunit of the A1B5 group of bacterial holotoxins.

In a further exemplary embodiment, the recombinant synthetic protein multimers are capable of binding to a G _MI -ganglioside.

In an exemplary embodiment of the invention, the synthetic recombinant protein is an immunogenic protein that induces an immune response in mammalian hosts.

In another exemplary embodiment of the invention, the synthetic recombinant protein is an immunogenic protein comprising one or more variable sequences that present antigenic determinants to which it is desired to elicit an immune response in patients. The variable sequences may be located at the N- and/or C-terminus of the synthetic carrier coding sequence, or may be included in the synthetic carrier coding sequence such that they are properly presented to cells of the immune system. In a preferred embodiment of the invention, the variable sequences are presented in such a way as to reproduce the conformation that these sequences exhibit in the natural molecules from which they are derived and be accessible to immunoglobulin surface receptors on B cells.

In another illustrative embodiment of the invention, various antigenic determinants are derived from signaling molecules, including, without limitation, growth factors such as adrenomedullin (AM), angiopoietin (Ang), bone morphogenetic proteins, epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), nerve growth factor (NGF) and other neurotrophins, platelet growth factor (PDGF), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β) , tumor necrosis factor alpha (TNF-α) and vascular endothelial growth factor (VEGF).

In another embodiment, the variable antigenic determinants are derived from other ligand molecules that are involved in the development or progression of the disease, such as, but not limited to, PDL1.

In another embodiment of the invention, various antigenic determinants are derived from receptors found on the surface of cells and involved in signaling events that regulate cell growth, such as natural growth factor receptors.

In a further embodiment, the variable antigenic determinants are derived from tumor antigens.

In yet another embodiment, the various antigenic determinants are derived from bacterial, viral, fungal, or other pathogens.

BRIEF DESCRIPTION OF GRAPHICS

The embodiments described in this disclosure are illustrated in the accompanying drawings, which are for illustrative purposes and not limitation, in which like references are intended to refer to like or corresponding parts, in which:

Figure 1 shows ELISA signals produced by a group of soluble mutant clones binding to an immobilized galactose containing molecule (GM1) after 3 rounds of selection;

Figure 2 shows a Western blot of soluble mutant proteins from one of the mutant libraries that were subjected to SDS (sodium dodecyl sulfate) gel electrophoresis, some of which retain the ability to form pentamers and others that do not;

figure 3 shows the selection of mutations that were identified after selection and screening of several mutant sequence libraries in the selection of mutants in figure 3(a), each of which is isolated from one of 7 separate mutant libraries and illustrates the distribution of mutant residues, and synthetic protein sequences on figure 3(b), which were obtained from selected mutations obtained from several different mutant clones;

Figure 4 shows GM1 binding ELISA results obtained from four selected mutants showing that two clones retained galactose binding activity and two did not;

figure 5 shows the structure of the whole molecule of cholera holotoxin and

Figure 6 shows the synthetic protein sequences of the present disclosure (Sequence ID: 1) and additional synthetic protein sequences of the present disclosure, including sequences encoding human TGF-beta 1 (bold), flexible linker (in italics), and synthetic carrier protein (Sequence ID: 2).

DETAILED DESCRIPTION OF THE INVENTION

Detailed embodiments of these recombinant proteins or vaccines are disclosed herein, however, it should be understood that the disclosed embodiments of the invention are illustrative only and may be embodied in various forms. Therefore, the specific functional details disclosed herein should not be interpreted as limiting, but solely as the basis for formulas and as an exemplary basis for the education of those skilled in the art for the various uses of the recombinant protein disclosed herein.

The present disclosure provides a synthetic recombinant protein to improve 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.

In one exemplary embodiment, a synthetic recombinant protein shown in Sequence ID: 2 is described, containing at least one growth factor, including, without limitation, transforming human growth factor (TGF), a tumor antigen, and/or receptor. In alternative exemplary embodiments, the protein may be other immunogenic recombinant proteins that are modeled based on known immunogenic proteins. It is contemplated within the scope of this disclosure that such recombinant proteins will be polypeptides that are highly immunogenic to the human immune system. Preferably, the recombinant proteins will confer additional properties to the chimeric protein, such as high expression yield and ease of manufacture, oral stability and ability to pass from the digestive tract into the bloodstream, and/or current safe use in humans.

Certain illustrative embodiments of the invention provided herein include the recombinant proteins of the present disclosure in vaccine compositions and in immunological adjuvant compositions, including pharmaceutical formulations that contain, in addition to the recombinant proteins, at least one adjuvant that refers to a component of such compositions that have adjuvant activity.

An adjuvant having such adjuvant activity includes a composition that, when administered to a subject such as a human (e.g., a sick human), a non-human primate, a mammal, or other higher eukaryotic organisms that has a learned immune system, is capable of altering (i.e., increase or decrease in a statistically significant manner, and in certain preferred embodiments, increase or increase) in the strength and/or duration of the immune response. In certain illustrated embodiments disclosed herein, the desired antigen and/or antigens are contained in a carrier protein, and optionally one or more adjuvants can thus modify, for example, elicit or increase, an immune response that is directed against the desired antigen and/ or antigens, which may be administered simultaneously or which may be separated in time and/or space (e.g., in different parts of the body) when administered, but certain illustrated embodiments of the invention are not intended to be limited in this way, and therefore also provide for the administration of a recombinant protein in a composition that does not include said antigen, but which may also include, without limitation, one or more coadjuvants, an imidazoquinoline immunomodulator.

Accordingly, and as noted above, adjuvants include compositions that exhibit adjuvant effects, such as saponins and saponin mimetics, including QS21 and QS21 mimetics (see, for example, US Pat. No. 5,057,540; EP 0362279 Bl; WO95/17210), alum, plant alkaloids such as tomatine, detergents such as (but not limited to) saponin, polysorbate 80, Span 85, and sterile tyrosine, one or more cytokines (e.g., GM-CSF, IL-2, IL-7, IL-12, TNF-alpha, IFN-gamma), an imidazoquinoline immunomodulator, and a double-stranded stem-loop immunomodulator (dSLIM, eg, Weeratna et al. 2005 Vaccine 23:5263).

Detergents, including saponins, are described, for example, in US patent No. 6544518; Facaille-Dubois, M. and Wagner F.I. (1996 Phytomedicine 2:363-386) U.S. Patent No. 5,057,540 Kensil, Crit. Rev Ther Drug Carrier Syst, 1996, 12 (1-2): 1-55, and EP 0362279 Bl. Corpuscular structures called immunostimulatory complexes (ISCOMS), which include fractions of Quil A (saponin), are hemolytic and have previously been used in the manufacture of vaccines (Morein, B., EP 0109942 Bl). These structures have been reported to have adjuvant activity (EP 0109942 Bl; WO 96/11711). The hemolytic saponins QS21 and QS17 (HPLC-purified Quil A fractions) have been described as potent systemic adjuvants and a method for their production is described in US Pat. No. 5,057,540 and EP 0362279 Bl. These references also describe the use of QS7 (non-hemolytic fraction of Quil-A), which acts as a strong adjuvant for systemic vaccines. The use of QS21 is further described in Kensil et al., 1991. J. Immunology 146:431-437).

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

Other adjuvants or coadjuvants for use in the embodiments disclosed herein include a block copolymer or a biodegradable polymer, which are a class of polymeric compounds with which those skilled in the art will be familiar. Examples of a block copolymer or biodegradable polymer that may be included in vaccine compositions or an immunological adjuvant include Pluronic. RTM. L121 (BASF Corp., Mount Olive, NJ; see, for example, Yeh et al., 1996 Pharm. Res. 13:1693).

Some additional embodiments of the invention provide for immunological adjuvants that include, without limitation, oil, which in some such embodiments of the invention may contribute to 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 vaccines and immunological adjuvant formulations based on the present invention. Examples of such oils, by way of illustration and not limitation, include squalene, squalane, mineral oil, olive oil, cholesterol, and mannide monooleate.

Immunomodulators such as imidazoquinoline are also known in the art and may also be included as adjuvants or coadjuvants in certain embodiments disclosed herein. Also, as noted above, one type of adjuvant or coadjuvant for use in a vaccine composition in accordance with the disclosure described herein can be aluminum coadjuvants, which are commonly referred to as "alum". Alum coadjuvants are based on the following: aluminum oxyhydroxide, aluminum phosphate, or various proprietary salts. Alum coadjuvants have useful properties because they have a good safety record, increase humoral immune responses, stabilize antigens, and are relatively easy to produce on a large scale (Edelman 2002 Mol. Biotechnol. 21:129-148; Edelman, R. 1980 Rev. Infect. Dis 2:370-383).

Pharmaceutical compositions

In certain illustrative embodiments, the pharmaceutical composition is a vaccine composition that includes a recombinant protein according to this disclosure and may further include one or more of the components provided herein, which are selected from a TLR agonist, coadjuvant (including, for example, cytokine, imidazoquinoline immunomodulator and/or dSLIM) and the like, and/or a recombinant expression construct in combination with a pharmaceutically acceptable carrier, excipient or diluent.

Exemplary carriers will be non-toxic to recipients at the dosages and concentrations used. For vaccines containing the recombinant protein, administrations from about 0.01 μg/kg to about 100 mg/kg body weight will be administered, typically by intradermal, subcutaneous, intramuscular, or intravenous routes, or other routes. Those skilled in the art will appreciate that the amount and frequency of administration will depend on the response of the host organism. "Pharmaceutical acceptable carriers" for therapeutic agents 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, at physiological pH, sterile saline and phosphate buffer may be used. Preservatives, stabilizers, colorants and even flavoring agents can be present in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid, and p-hydroxybenzoic acid esters may be added as preservatives. In addition, antioxidants and suspending agents may be used.

The pharmaceutical compositions may be presented in any form that allows the composition to be administered to a patient. For example, the composition may be in solid, liquid or gaseous (aerosol) form. Typical routes of administration include, without limitation, oral, topical, parenteral (eg, sublingual or buccal), sublingual, rectal, vaginal, and intranasal (eg, as a spray). The term parenteral as used herein includes iontophoretic delivery, sonophoretic, passive transdermal, microneedle administration, as well as subcutaneous injection, intravenous, intramuscular, intrasternal, intracavernous, intrathecal, intramural, intraurethral injection or infusion techniques. In a specific embodiment of the invention, the composition described herein (including vaccine and pharmaceutical compositions) is administered intradermally using a technique selected from iontophoresis, microcavitation, sonophoresis or micro acupuncture.

The pharmaceutical composition is formulated to allow the active ingredients contained in the composition to be bioavailable upon administration of the composition to a patient. The formulations to be administered to the patient may be in the form of one or more dosage units, where, for example, a tablet may be a single dosage unit, and a container containing one or more compounds of this invention in aerosol form may contain multiple dosage units.

For oral administration, a filler and/or binder may be present. Examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose and ethylcellulose. Coloring and/or flavoring agents may be present. A covering sheath may be used. The composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion or suspension. The liquid may be used for oral administration or for delivery by injection, for example. When intended for oral administration, preferred compositions contain one or more sweeteners, preservatives, color/pigment and flavor enhancer. A composition intended for administration by injection may include one or more surfactants, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent.

The liquid pharmaceutical composition used herein, whether in the form of a solution, suspension, or other similar form, may include one or more of the following carriers or excipients: sterile diluents such as water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride solution, fixed oils such as squalene, squalane, mineral oil, mannide monooleate, cholesterol and/or synthetic mono- or di-glycerides which may serve as a solvent or suspending medium, polyethylene glycols, glycerol, propylene glycol or other solvents; bactericides such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. The parenteral preparation may be enclosed in ampoules, single-use syringes, or multi-dose vials made of glass or plastic. The injectable pharmaceutical composition is necessarily sterile.

In a certain embodiment, the pharmaceutical or vaccine composition of the present invention contains a stable aqueous suspension, less than 0.2 microns, and additionally contains at least one component selected from the group consisting of phospholipids, fatty acids, surfactants, detergents , saponins, fluorinated lipids, etc.

It is also desirable to include other components in the vaccine or pharmaceutical composition, such as delivery vehicles, which include, but are not limited to, aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules, and liposomes. Examples of additional immunostimulatory substances (coadjuvants) for use in such media are also described above and may include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), glucan, IL-12, GM-CSF, interferon gamma, and IL-12 .

Although any suitable carrier known to those skilled in the art may be used in the pharmaceutical compositions of this invention, the type of carrier may vary depending on the route of administration and whether sustained release is desired. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, fat, wax, or a buffer. For oral administration, any of the aforementioned carriers or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose and magnesium carbonate may be used. Biodegradable microspheres (eg, polylactic galactide) can also be used as carriers for pharmaceutical formulations of the present invention.

Pharmaceutical formulations 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 fillers. Neutral buffered saline or saline mixed with non-specific serum albumin are illustrative useful diluents. Preferably, the product may be formulated as a lyophilisate using suitable solutions of excipients (eg sucrose) as diluents.

In an exemplary embodiment of the present invention, the recombinant protein domain-bearing epitope or receptor derived from either a naturally occurring or synthetic polypeptide sequence should be capable of self-assembling into oligomeric multimers under appropriate chemical/environmental conditions, or reduced to monomers under alternative conditions. Ideally, multimerizable domains will assemble into stable multimers with a defined number of subunits, eg dimers, trimers, tetramers, pentamers, etc., such that a product of uniform size is formed. Without being bound to any particular theoretical explanation, it is believed that the recombinant synthetic protein listed in Sequence ID: 1 will allow assembly into stable multimers with few subunits. Examples of naturally occurring polypeptides include, but are not limited to, leucine zipper protein domains, lactose repressor protein, streptavidin/avidin, cholera toxin B subunit, Pseudomonas trimerization domain, and viral capsid proteins.

According to the present disclosure, recombinant proteins, growth factors or parts thereof, cellular receptors or parts thereof, tumor antigens or parts thereof, refer to a wide range of either cellular pathways involved in chronic disease or cancers in relation to growth factors and receptors, and to the broadest possible spectrum of solid tumors for the use of tumor antigens in the composition of the above synthetic proteins. These proteins are in the form of a recombinant protein and can be used in the treatment of chronic diseases such as cancers of the breast, lung, bladder, ovary, vulva, colon, pulmonary artery, brain, rectum, intestines, head, neck and esophagus. Since various tumor antigens may be expressed in the above diseases and many cellular receptors and growth factors may be overexpressed, the proteins described below may contain one or more different tumor antigens, one or more different receptors or growth factors of one or more cellular pathways associated with disease. These proteins are called "multivalent".

In the context of the present disclosure, a "neutralizing domain" is defined as the region or regions of one or both members of a specific binding pair, e.g., a growth factor and its cognate receptor, where the binding of a third molecule that is not a member of the specific binding pair to the aforementioned region(s) ami) will prevent subsequent binding of two members of a specific binding pair. The third molecule may be another protein molecule, including without limitation an antibody, or may be a small non-protein molecule and may be either natural or synthetic in origin. The neutralizing domain will typically include those regions of the members of the specific binding pair that are in direct contact during binding, and will also include regions outside of the regions described above where, after binding, the third molecule introduces sufficient steric hindrance to prevent the members of the specific binding pair from directly binding.

It is well known in the art that the specific recognition of a ligand by its cognate receptor is determined by the interaction between the binding site of the receptor and the specific molecular portrait (epitope) of the ligand. Thus, an antibody that either binds to a binding site or otherwise blocks the binding site of a receptor, or binds to an epitope, or otherwise blocks recognition of a ligand epitope, will prevent receptor-ligand interaction. Such antibodies are described as "neutralizing". In the context of the present disclosure, it is desirable that neutralizing antibodies be produced by the host upon administration of the recombinant protein, and thus the protein sequence may express or include one or more of the total number of suitable sequences derived from a growth factor or tumor antigen, such that the epitopes required for binding to the receptor, will be presented in a functional (natural) conformation.

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 as part of it per natural site and/or as a chain of repetitive tumor antigen, receptor and /or growth factor sequences (eg, n = 1 or more); a protein of the present disclosure may also include the expression of one or more epitopes or binding sites of two or more different tumor antigens, receptors and/or growth factors, present singly or in chains at various positions within the recombinant protein sequence.

In an illustrative embodiment, a protein is disclosed, consisting of a homogeneous recombinant protein that is one or more neutralizing domains of epidermal growth factor (EGF). This protein is presented in the form of a recombinant protein and can be used in the treatment of chronic diseases, such as cancers of the breast, lung, bladder, ovary, vulva, colon, pulmonary artery, brain, rectum, intestines, head, neck and esophagus . In an exemplary embodiment, the protein is a recombinant protein that is or includes EGF sequences and synthetic polypeptide sequences according to the present disclosure. In one illustrative embodiment, the sequence of the synthetic polypeptide is substantially similar to Sequence ID: 1.

In another illustrative embodiment, a protein is disclosed, consisting of a homogeneous recombinant protein, which is a single fibroblast growth factor (FGF).

In a further illustrative embodiment, the protein is a recombinant protein that is or includes FGF sequences and synthetic polypeptide sequences according to the present disclosure.

In yet another illustrative embodiment, a protein is disclosed, consisting of a homogeneous recombinant protein, which is one transforming growth factor beta-1 (TGF-β1). In an exemplary embodiment, the protein is a recombinant protein that is or includes TGF-β1 sequences and synthetic polypeptide sequences according to the present disclosure.

And in yet another illustrative embodiment, a protein is disclosed, comprising a homogeneous recombinant protein expressing one transforming growth factor beta-1 (TGF-βl). In an exemplary embodiment, the protein is a recombinant protein that is or includes TGF-βl sequences and synthetic polypeptide sequences substantially similar to Sequence ID: 2 of the present disclosure.

In another illustrative embodiment, a single expressed insulin-like growth factor 1 (IGF-1) is described. In an illustrative embodiment, the protein is a recombinant protein that is or includes IGF-1 sequences and synthetic polypeptide sequences, a protein that includes a homogeneous recombinant protein that is one hepatocyte growth factor (HGF).

In a further exemplary embodiment, the protein is a recombinant protein expressing or comprising HGF sequences and synthetic polypeptide sequences of this disclosure.

In a further illustrative embodiment, the present invention discloses a protein consisting of a homogeneous recombinant protein that is one insulin-like growth factor-1 (IGF-1) and one insulin-like growth factor-2. In an exemplary embodiment, the protein is a recombinant protein that is or includes IGF-1 sequences, IGF-2 sequences, and synthetic polypeptide sequences according to the present disclosure.

In yet another exemplary embodiment, a protein is disclosed consisting of a homogeneous recombinant protein that is one vascular endothelial growth factor-A (VEGF-A) and one vascular endothelial growth factor-C (VEGF-C). In an exemplary embodiment, the protein is a recombinant protein that is or includes VEGF-A neutralizing domain sequences, VEGF-C sequences, and synthetic polypeptide sequences according to the present disclosure.

EXAMPLES

Aspects of the present disclosure are further detailed in the following examples. However, the following examples are not intended to limit the scope of the present disclosure in order to clarify the details of the methodology or development set forth below. Practical and illustrative embodiments of the invention are illustrated and described in the following examples. However, those skilled in the art will appreciate that modifications and improvements can be made within the spirit and scope of the present invention.

EXAMPLE 1 Development of a selectable carbohydrate-binding display system

What was needed was a system by which mutant libraries could be created and screened for mutants with desired characteristics, which would include a physical link between a selectable phenotype and the genotype that encodes it for diverse populations of clones. Such a system was made using established techniques used with great success in antibody engineering in particular.

The gene encoding the selected carbohydrate-binding domain was cloned in-frame and upstream of the M13 bacteriophage minor coat protein gene (gene III) in both a fully independent functional M13 'phage' vector with a suitable selectable marker ('phage' M13-K07) and in a 'phagemid' vector that includes only the F1 packaging region and the gene encoding the minor viral envelope protein. The above vector, when introduced into a suitable bacterial host such as E. coli and propagated accordingly, will produce viral particles that will present five copies of the selected carbohydrate-binding domain at one end of the filamentous viral particle as N-terminal fusions of the P3 minor coat protein (encoded by the gene III).

The latter, due to the nature of virus propagation, using a helper/phagemid phage system known to one of skill in the art, will produce viral particles in which only a small fraction of the population will typically be one or fewer copies of the same P3 carbohydrate-binding domain of the fusion protein.

Clones derived from each vector were propagated under appropriate conditions, which are well known to those skilled in the art, and the culture supernatant containing viral particles was screened by ELISA to assay binding activity to a major carbohydrate that is recognized by the carbohydrate-binding domain. In this example, a complex molecule was used which included a distal galactose group and which was subjected to immobilization by adsorption on a conventional 96-well immunoassay plate. Binding to a carbohydrate group requires that two or more carbohydrate-binding domains associate into a complex, since a carbohydrate-binding pocket is formed between adjacent domains.

In the previous 'phage' example, a pocket could potentially form between adjacent P3 fusion proteins on a single virion, provided that the acceptable orientation is limited to the fusion protein or between individual viral particles. When using the phagemid system, such associations are most likely to form between two or more single virus particles, since the proportion of viruses containing two or more fusion proteins on a single virion is expected to be very small.

As a result of screening the aforementioned clones, a significantly stronger ELISA signal was obtained from the ‘monovalent’ phagemid-derived clone than from the pentavalent phage clone, the latter also producing a much lower titer of infectious viral particles. The low performance of the phage system in this example does not limit the use of this system in the process described, but is more likely to be a function of either the steric/orientational constraints imposed on the system by the specific genetic linkage used in this case and/or the infectivity constraints imposed by fusion protein (the P3 minor envelope protein is a mediator of viral infection). Therefore, a phagemid-based system was chosen to generate and select various mutant proteins.

EXAMPLE 2 Creation of Mutant Clone Libraries

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 databases such as the Protein Database and from the scientific literature was used to identify regions and/or certain residues among those potentially or proven to be involved in the ability of the matrix protein domain to form stable homopentamers or interact with specific carbohydrate-containing parts found on the surface of many types of mammalian cells. These regions/residues were excluded from mutagenesis and subsequent rounds of mutant selection.

Regions or residues identified above as being potentially mutable and not involved in the required characteristics were selected and targeted for rational or random mutagenesis as deemed appropriate. To maximize the chances of generating and selecting carbohydrate-binding variants that were different from the template domain, mutations were limited to a small number of residues in the immediate vicinity of each successive round of library development screening.

Depending on the region or residue targeted, the mutation was either random (i.e. potentially involving all 20 amino acids) or functionally limited to the extent possible by those residues that exhibit similar biophysical and/or chemical properties to those found in matrix domain.

Characteristics that were considered significant in such cases included, without limitation, side chain size, charge, polarity, hydrophobicity/hydrophilicity, and the ability to participate in the formation of a specific secondary structure, such as α-helices. Where possible/permissible, the amino acid residue encoded by the template at any given position is excluded from mutant libraries to avoid selection of non-mutants.

Libraries were created by designing oligonucleotide primers that included both sequence regions that were homologous to the template gene such that the primer (and especially its 3' end) would anneal to template DNA under appropriate conditions, and additional regions where a corresponding degeneration was provided, for example, for the purpose of encoding a variety of amino acids at one or more positions.

In this example, such oligonucleotides were produced using predetermined degeneracy known to those skilled in the art, where the DNA base at any different position includes equal amounts of 2-4 bases (G, A, T, or C) as required. Alternatively, more controlled diversity is contemplated when oligonucleotides are prepared from positional mixtures of trinucleotide-phosphoramidites used such that specific residues (amino acids) and their relative proportions are precisely controlled at each different position.

When mutations were to be introduced at one end, a single PCR reaction was performed (and repeated if necessary) using one or two degenerate primers in order to introduce the necessary genetic diversity. The resulting product, including (derived from the primer) flanking restriction sites, was cloned into the phagemid vector described in Example 1 above, such that a library of various proteins was encoded as in-frame fusions to the minor protein-coding gene P3 (gene III) of the bacteriophage M13.

Where it was desired to introduce diversity into the region(s) further to the ends 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 sequence templates, and one degenerate primer that encodes some, all, or none of the required variety. The 3' ends 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 of the second primer pair.

Thus, two PCR products were obtained and then annealed and linked to each other by a complementary overlapping sequence obtained using degenerate primers. The resulting product was then cloned into a phagemid vector as an in-frame fusion to gene III, the minor coat protein gene. Subsequent libraries were built either based on the products of screening and selection of one or more clones from the previous library, or built and screened in parallel reactions from a template or one or more selected mutant clones.

All libraries of genetic constructs were introduced into E. coli host TG1 cells by electroporation using standard methodologies, and the transformants were plated on a selective medium. After appropriate incubation, bacterial colonies were collected from the plates and stored and/or screened as required.

EXAMPLE 3 Screening for Mutant Libraries

To perform selections of mutants from libraries, cultures (libraries) were inoculated into liquid media (2xTY, 100 μg/ml ampicillin, 1% glucose) so that a sufficient number of cells included 100-1000-fold copies of the expected diversity and sufficient volume to ensure finding OD ₆₀₀ values in the region of 0.1. The cell cultures were then incubated (with stirring) at approximately 37° C. until the OD ₆₀₀ value reached 0.4-0.6 (ie, log-phase growth). The cultures were then infected with the addition of helper phage M13-K07 at a ratio of ~20 helper phages per bacterial cell (OD ₆₀₀ with a value of 1.0 was taken to be ~8.0 x 10 ⁸ cells/ml). After approximately 30 minutes of static incubation, cells were incubated for approximately 30 additional minutes with agitation. Added to ~50 μg/ml kanamycin and the culture was incubated overnight at ~30°C with stirring.

The next morning, the culture was centrifuged for approximately 25-30 minutes at ~8000 g to pellet the cells and the supernatant was removed and saved. The cell pellet was discarded. 20% volume 200 mm NaCl, 20% PEG 6000 was added to the culture supernatant and incubated on ice for approximately 1 hour to precipitate phage particles. The phage was pelleted by centrifugation at ~8000 g for approximately 25-30 minutes and resuspended in 20 percent of the original volume of PBS (sodium phosphate buffer). The resulting 20% volume of 200 mM NaCl, 20% PEG 6000 was re-added and the phage was incubated on ice for approximately 25-30 minutes. The phage was pelleted again by centrifugation at ~8000 g for 25-30 minutes and the pellet was resuspended in ~2 ml of sodium phosphate buffer (PBS). The resulting suspension was transferred to Eppendorf tube(s) and centrifuged at maximum speed for 5 minutes to pellet any remaining bacterial cells/debris. The phage suspension was then used for selection.

For selection procedures, an immunological tube is coated with a suitable antigen, such as an immobilizable galactose derivative or a natural B-subunit ligand, at a concentration of 1-10 μg/ml (typically 5 ml) and left overnight at 4-8°C or at room temperature for 1 hour. After washing 3-5 times with PBS (simply pouring into the immunological tube and pouring again), the tube was blocked by adding MPBS (PBS containing 2% milk powder) for 1-2 hours at 37°C. Washed with MPBS as described above and added from about 1x10 ¹¹ to 1x10 ¹² or more phage particles.

The volume was adjusted to ~5 ml and the immunological tube was sealed, for example, with a paraffin film. The tube was then 'tilted' (bottom to lid) for approximately 30 minutes, then incubated in an upright position for approximately 90 minutes to allow those phages presenting a protein from the mutant library capable of recognizing the immobilized ligand to bind to it. . Phage that did not bind or only weakly bound were removed by washing. The stringency of the selection was changed as required by the number of washes applied, by using PBST (PBS containing 0.1% Tween 20) for washes, or by changing the ligand concentration in the coating.

Bound phage particles were eluted from the immunoassay tube with 1 ml of 100 mM triethylamine (TEA) and mixed by inversion for a maximum of 10 minutes (longer incubation has a negative effect on phage viability), and then immediately poured into 0.5 ml of 1 M Tris -HCl (pH 7.4) for neutralization. Approximately 0.75 ml of the eluted phage was added to ~10 ml of a log-phase cell culture of a suitable E. coli strain such as TG1 (Agilent). The culture was incubated at approximately 37°C without agitation to ensure infection. Serial dilutions of infected cell samples were applied to small TYE dishes containing 100 μg/ml ampicillin and 1% glucose. The rest of the cells were plated on larger bioassay dishes with the same medium. All were incubated overnight at approximately 30°C. Another 0.75 ml of phage, .about.75 .mu.l were infected as above, .about.1 ml of E. coli HB2151 cells in log phase, and serial dilutions were pelleted and incubated as described above. The rest of the phage was stored at approximately -80°C in the form of a glycerol mixture (~15% glycerol).

The next day, large bioassay dishes were scraped off to collect cells, which were then stored as glycerol mixtures or grown and ‘liberated’ with the M13 K07 phage helper as described previously to obtain an enriched phage population for the next round of selection. Usually two or three consecutive rounds of selection were carried out. After completion of selection, individual colonies from small dishes containing serial dilutions of HB2151 cells were selected and placed on 96-well culture plates containing ~100 µl/well 2 x TY with 100 µg/ml ampicillin and incubated with shaking overnight at 37 °C The next day, approximately 5 μl of culture from each well was inoculated onto a new plate with ~150 μl medium/per well and incubated at approximately 37° C. with shaking for approximately 2 hours. Additional medium containing 1 M IPTG was added to provide a final concentration of 1 mM IPTG and the dish was incubated with stirring overnight at 30°C. In HB2151 cells, induction of IPTG resulted in the expression of soluble mutant proteins that included a detectable c-myc peptide tag, rather than proteins fused to a viral particle component, as in the case of TG1 cells from E. coli. The original 96-well culture plate was stored at approximately -80°C.

The next day, culture supernatants from the induced 96-well plate were analyzed for binding to immobilized carbohydrates using standard ELISA protocols and binding was detected using a readily available HRP-labeled anti-c-Myc antibody (FIG. 1). Clones that were very active when binding to GM1 are shown in shaded boxes. Positive and negative controls are framed.

Culture supernatants of clones that were very active at binding in the ELISA assay were further analyzed by SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and Western blotting to analyze the presence of pentamers and other multimeric states illustrated in Figure 2. Samples were not hydrogenated or not boiled before electrophoresis and the protein was detected using HRP-labeled anti-c-Myc antibodies. In the example shown, clones C1 and C3 are positive controls that rapidly assemble into ~60 kDa pentamers. Clone C2 is a negative control that moves as a 12 kDa monomer under the conditions used. It can be seen that clones A1 and A3 move mainly as pentamers, while a significant part of clone B12 forms monomers. The remaining clones either form a monomer (B l, B7) or form many multimeric forms.

EXAMPLE 4 Summary of Mutation Data

In order to evaluate the ability of the maximum number of amino acid residues to accommodate mutations, several different libraries were produced and screened, each targeting a different residue or region of the template. As a result, a large number of clones were identified that exhibited the desired selection criteria for binding to immobilized carbohydrate derivatives and forming predominantly pentamers, however, each of which included many unique mutations/differences, as illustrated in Figure 3A. In order to create clones with potentially improved performance, it was necessary to combine these different sequences into fewer clones.

Since several positions were identified during the production and selection of mutants where three or more different residues could be placed, a total of four combinations of mutants (A-D) were developed, as illustrated in Figure 3B. Four clones containing the combined mutations were expressed and the synthetic proteins were evaluated for their binding against their respective controls using ELISA as illustrated in Figure 4. Two clones (A and C) were found to have only weak binding, however the other two ( B and D) were able to bind to GM1 very strongly. This indicates that mutations derived from individual clones will combine successfully, however, enhancement or even retention of selective binding characteristics is not guaranteed.

EXAMPLE 5 Stabilization of Homopentamers

In their naturally occurring, fully functional form, bacterial holotoxins, described under the group name 'A1B5', consist of five B subunits and one A subunit. The B subunits are assembled into a pentameric ring with a central ‘pore’. The single A subunit is at the top of this ring and protrudes downward into the space, as illustrated in Figure 5. The A subunit itself consists of 2 separate domains. The A1 domain, which is released when the holotoxin is translocated into the cell, is characterized by enzymatic activity associated with the toxicity of the protein complex. It is held as part of the holotoxin by the A2 domain. The latter has an alpha helical structure in which the A1 domain is at the N-terminus and the C-terminus protrudes into a pore formed by 5 B subunits. Some residues of the C-terminus of A2 form interactions with residues of the B subunits. Since there are confirmed interactions between certain residues of the B subunits and the A2 domain (Zhang R.G., et al. (1995)), it is logical to assume that the presence of the A subunit, and especially the A2 domain, contributes to the stabilization of the pentameric ring of the B subunit. Due to structural and functional differences between the two domains of the A subunit, without being supported by concrete evidence, it is believed that the A2 domain taken alone will confer this stabilization effect on the B subunit pentamer. Similarly, since the B subunits are known to be able to form pentameric rings in the complete absence of the A subunit, it is likely that the A2 domain can bind to it and therefore have a stabilizing effect on the B subunit pentamers, either by being co-expressed with the B subunits, or being added exogenously.

In this regard, it is proposed that if a synthetic or non-natural polypeptide is one that has a sufficiently similar conformation to the B subunits of the holotoxin that is capable of assembling into multimers, including pentameric rings, and in which residues corresponding to those that interact with A2 in B -subunits of holotoxins are the same or have similar biophysical properties, then such a synthetic protein pentamer will experience a similar stabilizing effect. Furthermore, it is proposed that, in the absence of the natural A2 domain, a synthetic polypeptide can be isolated from a suitable polypeptide library using techniques known to those skilled in the art, such as 'phage display', which will bind, at least in part, to the exposed surfaces of the two or several natural or synthetic monomers, when assembled into pentamers, and thus stabilize the structure.

Since the natural A2 domains support a structurally independent A1 domain that does not itself interact with any other component of the holotoxin, then it is likely that the A2 domain, or a functional synthetic equivalent, may provide a means to anchor another polypeptide domain. Without being bound by any particular theory, it is believed that a non-natural polypeptide that has a sufficiently similar conformation to the polypeptide described in Sequence ID: 1 is itself conducive to forming pentameric rings that present a stabilized pentameric ring structure that allows attachment epitopes described in this disclosure.

Although devices, systems and methods have been described and illustrated in connection with certain embodiments of the present invention, many variations and modifications will be apparent to those skilled in the art and may be made without departing from the spirit and scope of the present disclosure. Thus, the present disclosure should not be limited to the precise details of the methodology or design set forth above, as such variations and modifications are intended to be included within the scope of this disclosure.

The terms and expressions that have been used are used as terms of description and not limitation, and there is no intention that the use of such terms and expressions excludes any equivalent feature shown and described or parts thereof, but it is understood that various modifications are possible in within the scope of the present invention. Thus, it is to be understood that although the present invention has been specifically disclosed in terms of preferred embodiments and additional features, modifications and variations on the concepts disclosed herein may be made by those skilled in the art, and that such modifications and variations are contemplated within the scope of the present invention. as defined by the appended claims.

Claims (25)

1. Recombinant synthetic protein for the treatment of chronic diseases selected from the group including cancers of the breast, lungs, bladder, ovaries, vulva, colon, pulmonary artery, brain, rectum, intestines, head, neck and esophagus, including Cholera toxin B-subunit (CTB), wherein the CTB subunit includes mutations T1F, P2T, Q3D, N4I M37I, A38I, I39L, I40V, T41N, T78S, E79N, A80S, A95S, A102V, and N103R, while the CTB subunit is optional additionally includes mutations L8I, A10G, F25L, A32V, N44G, and V82I, with the mutations numbered relative to the following sequence of wild-type CTBs: TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWN NKTPHAIAAISMAN and while mutations contribute to the formation of the pentamer of the CTB subunit; peptide spacer and a polypeptide comprising a tumor antigen or a portion thereof having an immunogenic sequence, or a growth factor or a portion thereof selected from the group consisting of IGF-1, IGF-2, FGF1, FGF2, VEGF-A, VEGF-B, VEGF-C, VEGF -D, PDGF, NGF, EGF, HGF, BMP, PDL1 and IL-1, IL-2, IL-3, IL-4, IL-5, and IL-6, wherein the polypeptide is separated from the synthetic B-subunit by a peptide spacer. 2. The recombinant protein of claim 1 wherein the growth factor or portion thereof is epidermal growth factor (EGF). 3. The recombinant protein according to claim 1, wherein the growth factor or a portion thereof is a human growth factor native protein or a portion thereof. 4. The recombinant protein of claim. 1, where the tumor antigen includes the full length or part of one or more different tumor antigens. 5. The recombinant protein of claim 1 wherein the tumor antigen comprises the entire length or portion of one or more tumor antigens in the synthetic protein as single epitopes or as two or more repeated epitopes. 6. The recombinant protein of claim 1 further comprising a second polypeptide comprising at least a portion of a growth factor. 7. The recombinant protein according to claim 6, where the second polypeptide comprises the full length or part of one or more growth factors selected from the group including IGF-1, IGF-2, FGF1, FGF2, TGF-α, TGF-B, VEGF -A, VEGF-B, VEGF-C, VEGF-D, PDGF, NGF, EGF, HGF, BMP and IL 1-6. 8. The recombinant protein of claim 7, wherein the second polypeptide comprises the full length or neutralizing domain of at least two different growth factors. 9. The recombinant protein of claim 7, wherein the second polypeptide comprises the full length or neutralizing domain of one or more growth factors in said recombinant synthetic protein as single epitopes or as two or more tandem repeats. 10. The recombinant protein of claim 9, wherein the second polypeptide further comprises at least a portion of a receptor, or the full length or portion of two to four different receptors. 11. The recombinant protein of claim 9, further comprising a third polypeptide comprising the full length or portion of one or more of the following growth factors selected from the group consisting of IGF-1, IGF-2, FGF1, FGF2, TGF-α, TGF- B, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PDGF, NGF, EGF, HGF, BMP and IL 1-6. 12. The recombinant protein of claim 11 wherein the third polypeptide comprises the full length or neutralizing domain of one or more growth factors as single epitopes or as two or more tandem repeats. 13. The recombinant protein of claim 1, wherein the polypeptide comprises at least two different growth factors or portions thereof. 14. The recombinant protein of claim. 1, where the polypeptide includes the full length or part of one or more growth factors as one domain or two or more multiple repeats. 15. A recombinant protein according to any of the preceding claims for use in inducing an immune response to a human growth factor or portion thereof in a human subject. 16. Recombinant protein according to claim 1, including SEQ ID NO: 1: FTDIITDICGEYHNTQIHTLNDKILSYTESLVGKREIILVNFKGGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLSNSKIEKLCVWNNKTPHSIAAISMVR, or SEQ ID NO: 2: ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCSGGSGGTSGGGGGSGFTDIITDICGEYHNTQIHTLNDKILSYTESLVGKREIILVNFKGGATFQVEVPGSQHIDSQKKA IERMKDTLRIAYLSNSKIEKLCVWNNKTP HSIAAISMVR.