Stabilized chimeric synthetic proteins and therapeutic uses thereof

Abstract

FIELD OF THE DISCLOSURE This disclosure relates to compositions and methods for treating disease. More particularly, this disclosure relates to stabilized chimeric synthetic proteins and their use for treating cancer.

Description

[Technical field]

[0001] The present disclosure relates to compositions and methods for treating disease. More particularly, the present disclosure relates to stabilized chimeric synthetic proteins and their use for treating cancer. [Background technology]

[0002] According to the World Health Organization, neoplasms (e.g., cancer) are one of the leading causes of death worldwide, responsible for 8.8 million deaths in 2015. The frequency of cancer in the global human population is significant, accounting for nearly 1 in 6 deaths. The most common cancer deaths in 2015 were due to the following types of cancer: lung cancer (about 1.7 million deaths), liver cancer (about 800,000 deaths), colorectal cancer (about 800,000 deaths), gastric cancer (about 800,000 deaths), and breast cancer (about 600,000 deaths).

[0003] Cancer is typically treated by one of a variety of methods, such as surgery, chemotherapy, radiation therapy, and cancer immunotherapy. Unfortunately, many of these methods have toxic / undesirable side effects. For example, standard cancer chemotherapy was developed based on its ability to kill rapidly dividing cells, and many have toxic properties that cause undesirable side effects, such as immunosuppression, nausea, and hair loss. A central goal of cancer research over the past two decades has been to find new treatments with greater efficacy and fewer side effects.

[0004] One such treatment is encompassed by cancer immunology, which is the study of the interactions between the immune system and cancer cells, such as tumors or malignant tumors. The initiation of immune responses, such as the recognition of cancer-specific antigens expressed by human tumors and not in normal tissues, is of particular interest. In general, methods to control the division and growth of malignant cells have focused on isolating these antigens and presenting them so that they are recognized by the immune system as non-self antigens and induce a specific immune response (e.g., cancer vaccines). Such cancer vaccines can typically be made either as chemical conjugates or recombinant proteins. Disadvantageously, such cancer vaccines exhibit several significant limitations, mainly resulting from the manufacturing method and the potential lack of homogeneity, activity and homology of the protein product. For example, cancer vaccines produced by chemical conjugation (e.g., with glutaraldehyde) generally contain a mixture of recombinant carrier proteins and polypeptides of human origin. Unfortunately, the use of glutaraldehyde as a cross-linking reagent has an undesirable tendency to form covalent bridges between various chemical groups, generally resulting in a highly heterogeneous product. Thus, the resulting vaccine may not only contain carrier protein molecules with various numbers (e.g., 0, 1, 2, 3, etc.) of target human polypeptides attached thereto, but also the human polypeptides may each be attached to the carrier via different atoms, and thus may be present in different positions and orientations. Furthermore, both the target polypeptides and the carrier protein molecules may be conjugated to themselves, resulting in various homomultimers that may not have clinical efficacy and may not contribute to anti-cancer patient immune responses. Furthermore, cancer vaccines produced by recombinant protein technology suffer from the drawback that the target human polypeptides contained within the recombinant proteins may not be able to fold properly, thereby hindering useful immune responses. Thus, there is an urgent need for new cancer vaccines that overcome these significant existing limitations in the field of cancer immunotherapy. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure relates to chimeric synthetic proteins / molecules and their respective methods of production, characterization of the chimeric synthetic proteins / molecules, and therapeutic methods using the chimeric synthetic proteins / molecules to treat chronic diseases, such as, for example, lung, breast, bladder, prostate, ovarian, vulvar, colon, colorectal, intestinal, lung, brain, esophageal, other cancers, and other diseases.

[0006] The present disclosure provides chimeric synthetic proteins that can be used as therapeutics to treat diseases such as, for example, cancer. In an exemplary embodiment, the present disclosure provides chimeric synthetic proteins / molecules that include one or more protein domains from a synthetic growth factor (e.g., VEGF), one or more linker regions, and one or more immunogenic domains. In one aspect, the present disclosure provides chimeric synthetic proteins that include a chimeric polypeptide sequence, at least one linker, and a polypeptide sequence. Advantageously, the chimeric synthetic proteins / molecules described herein have the ability to function as a stabilizing scaffold that favors (e.g., properly folds) the native conformation of the human protein (e.g., growth factor, e.g., VEGF, EGF, TGF, etc.) that is incorporated into the protein / molecule when expressed. Furthermore, the chimeric synthetic proteins / molecules described herein have the ability to generate stabilized chimeric synthetic proteins / molecules that have a long shelf life.

[0007] In certain embodiments, the polypeptide sequence comprises an immunogenic polypeptide sequence.

[0008] In certain embodiments, the polypeptide sequence comprises a cholera toxin B (CT-B) protein.

[0009] In certain embodiments, the at least one linker comprises a first linker that separates the chimeric polypeptide sequence from the polypeptide sequence.

[0010] In certain embodiments, the first linker is selected from the group consisting of SSG, GSSG, SSGGG, SGG, GGSGG, GGGGS, SSGGGSGG, SSGGGGSGGG, TSGGGSG, TSGGGGSGG, SSGGSGGGSG, SSGGGSGGSSG, GGSGGTSGGGSG, SGGTSGGGGSGG, GGSGGTSGGGGSGG, SSGGGGSGGGSSG, SSGGGSGGSSGGG, and SSGGGGSGGGSSGGG.

[0011] In certain embodiments, the first linker is SSGGSGGGSG.

[0012] In certain embodiments, the chimeric polypeptide sequence comprises a vascular endothelial growth factor (VEGF) sequence.

[0013] In certain embodiments, the chimeric polypeptide sequence comprises a VEGF sequence selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and combinations thereof.

[0014] In certain embodiments, the chimeric polypeptide sequence comprises a first VEGF domain and a second VEGF domain.

[0015] In certain embodiments, the first VEGF domain comprises VEGF-D or a portion thereof and the second VEGF domain comprises VEGF-A or a portion thereof.

[0016] In a specific embodiment, the first VEGF domain is TFYDIETLKVIDEEWQRTQ and the second VEGF domain is CHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0017] In certain embodiments, the chimeric polypeptide sequence binds to a vascular endothelial growth factor receptor (VEGFR) selected from the group consisting of VEGFR-1, VEGFR-2, VEGFR-3, and combinations thereof.

[0018] In certain embodiments, the chimeric polypeptide sequence binds to VEGFR-1, VEGFR-2 and VEGFR-3.

[0019] In certain embodiments, the chimeric synthetic protein initially has the amino acid sequence: MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0020] In a particular embodiment, the initial chimeric synthetic protein is processed to have the amino acid sequence TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0021] In another aspect, the disclosure provides an immunogenic composition comprising a chimeric polypeptide sequence, at least one linker, and a polypeptide sequence.

[0022] In certain embodiments, the polypeptide sequence comprises an immunogenic polypeptide sequence.

[0023] In certain embodiments, the polypeptide sequence comprises a cholera toxin B (CT-B) protein.

[0024] In certain embodiments, the at least one linker comprises a first linker that separates the chimeric polypeptide sequence from the polypeptide sequence.

[0025] In certain embodiments, the first linker is selected from the group consisting of SSG, GSSG, SSGGG, SGG, GGSGG, GGGGS, SSGGGSGG, SSGGGGSGGG, TSGGGSG, TSGGGGSGG, SSGGSGGGSG, SSGGGSGGSSG, GGSGGTSGGGSG, SGGTSGGGGSGG, GGSGGTSGGGGSGG, SSGGGGSGGGSSG, SSGGGSGGSSGGG, and SSGGGGSGGGSSGGG.

[0026] In certain embodiments, the first linker is SSGGSGGGSG.

[0027] In certain embodiments, the chimeric polypeptide sequence comprises a vascular endothelial growth factor (VEGF) sequence.

[0028] In certain embodiments, the chimeric polypeptide sequence comprises a VEGF sequence selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and combinations thereof.

[0029] In certain embodiments, the chimeric polypeptide sequence comprises a first VEGF domain and a second VEGF domain.

[0030] In certain embodiments, the first VEGF domain comprises VEGF-D or a portion thereof and the second VEGF domain comprises VEGF-A or a portion thereof.

[0031] In a specific embodiment, the first VEGF domain is TFYDIETLKVIDEEWQRTQ and the second VEGF domain is CHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0032] In certain embodiments, the chimeric polypeptide sequence binds to a vascular endothelial growth factor receptor (VEGFR) selected from the group consisting of VEGFR-1, VEGFR-2, VEGFR-3, and combinations thereof.

[0033] In certain embodiments, the chimeric polypeptide sequence binds to VEGFR-1, VEGFR-2 and VEGFR-3.

[0034] In certain embodiments, the synthetic protein chimeric synthetic protein initially has the amino acid sequence of MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0035] In a particular embodiment, the initial chimeric synthetic protein is processed to have the amino acid sequence TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0036] In certain embodiments, the immunogenic composition further comprises an adjuvant.

[0037] In another aspect, the disclosure provides a method of treating a patient in need thereof comprising administering to the patient an immunogenic composition as described above on the same day or on a different day or at a different time within a vaccination period.

[0038] In certain embodiments, the patient has cancer.

[0039] definition "Epidermal growth factor receptor (EGFR) nucleic acid molecule" refers to a polynucleotide that encodes an EGFR polypeptide. An exemplary EGFR nucleic acid molecule is provided under NCBI Accession No. NM_005228.4, reproduced below (SEQ ID NO: 3). JPEG0007631240000001.jpg104147 JPEG0007631240000002.jpg230149 JPEG0007631240000003.jpg165148

[0040] By "epidermal growth factor receptor (EGFR) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_005219.2 and having epidermal growth factor (EGF) binding activity, reproduced below (SEQ ID NO: 4). JPEG0007631240000004.jpg22149 JPEG0007631240000005.jpg86150

[0041] "Epidermal growth factor (EGF) nucleic acid molecule" refers to a polynucleotide that encodes an EGF polypeptide. An exemplary EGF nucleic acid molecule is provided under NCBI Accession No. NM_001963.5, reproduced below (SEQ ID NO: 5). JPEG0007631240000006.jpg123150 JPEG0007631240000007.jpg231149 JPEG0007631240000008.jpg95150

[0042] By "epidermal growth factor (EGF) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001954.2, corresponding to the pre-proprotein form of EGF that is processed to produce the 53 amino acid EGF molecule (shown in bold), and having EGFR binding activity, reproduced below (SEQ ID NO: 6). JPEG0007631240000009.jpg104152

[0043] "Neuregulin 1 (NRG1) nucleic acid molecule" refers to a polynucleotide that encodes an NRG1 polypeptide. An exemplary NRG1 nucleic acid molecule is provided under NCBI Accession No. BC150609.1 and is reproduced below (SEQ ID NO: 7). JPEG0007631240000010.jpg171150

[0044] By "Neuregulin 1 (NRG1) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAI50610.1 and having Neuregulin 1 (NRG1) binding activity, reproduced below (SEQ ID NO: 8). JPEG0007631240000011.jpg58152

[0045] "Neuregulin 1β (NRG1β) nucleic acid molecule" refers to a polynucleotide that encodes an NRG1 polypeptide. An exemplary NRG1β nucleic acid molecule is provided under NCBI Accession No. NM_001322205.1 and is reproduced below (SEQ ID NO: 9): JPEG0007631240000012.jpg150152 JPEG0007631240000013.jpg232150 JPEG0007631240000014.jpg145152

[0046] By "Neuregulin 1β (NRG1β) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001309134.1 and having Neuregulin 1 (NRG1) binding activity, reproduced below (SEQ ID NO: 10). JPEG0007631240000015.jpg65152

[0047] By "NRG-BVN hybrid polypeptide" is meant a polypeptide or fragment thereof having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the following amino acid sequence (SEQ ID NO:11). >NRG-BVN-Hybrid JPEG0007631240000016.jpg13152

[0048] By "TGFα hybrid polypeptide" is meant a polypeptide or fragment thereof having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the following amino acid sequence (SEQ ID NO:12). >TGF-BVN hybrid JPEG0007631240000017.jpg14152

[0049] By "initial IN-02 polypeptide" is meant a fragment thereof having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the following amino acid sequence (SEQ ID NO:13). >Initial_IN-02_Polypeptide JPEG0007631240000018.jpg23152

[0050] By "processed or final IN-02 polypeptide" is meant a fragment thereof having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the following amino acid sequence (SEQ ID NO:14). >Final_IN-02_Polypeptide JPEG0007631240000019.jpg23152

[0051] "Vascular endothelial growth factor A (VEGF-A) nucleic acid molecule" refers to a polynucleotide that encodes a VEGF-A polypeptide. An exemplary VEGF-A nucleic acid molecule is provided under NCBI Accession No. NM_001025366.3 and is reproduced below (SEQ ID NO: 15). JPEG0007631240000020.jpg18152 JPEG0007631240000021.jpg232152 JPEG0007631240000022.jpg45152

[0052] "VEGF-A polypeptide" refers to a polypeptide encoded by a VEGF-A nucleic acid molecule. An exemplary VEGF-A nucleic acid molecule is provided under NCBI Accession No. NP_001020537.2 and is reproduced below (SEQ ID NO: 16). JPEG0007631240000023.jpg41152

[0053] "Vascular endothelial growth factor A (VEGF-D) nucleic acid molecule" refers to a polynucleotide encoding a VEGF-D polypeptide. An exemplary VEGF-D nucleic acid molecule is provided under NCBI Accession No. NM_004469 and is reproduced below (SEQ ID NO: 17). JPEG0007631240000024.jpg77152 JPEG0007631240000025.jpg96152

[0054] "VEGF-D polypeptide" refers to a polypeptide encoded by a VEGF-D nucleic acid molecule. An exemplary VEGF-D nucleic acid molecule is provided under NCBI Accession No. NP_004460.1 and is reproduced below (SEQ ID NO: 18). JPEG0007631240000026.jpg36152

[0055] Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another aspect includes from one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, using the antecedent "about," it is understood that the particular value forms another aspect. It is further understood that the endpoints of each range are significant in relation to the other endpoint and independently of the other endpoint. It is also understood that there are several values ​​disclosed herein, and that each such value is also disclosed herein as "about" that particular value in addition to the value itself. It is also understood that throughout this application, data is provided in several different formats, and that this data represents the endpoints and starting points and ranges of any combination of the data points. For example, when a specific data point "10" and a specific data point "15" are disclosed, it is understood that greater than, greater than, less than, less than, equal to, and equal to 10 and 15, as well as between 10 and 15, are considered to be disclosed. It is also understood that each number between two specific numbers is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0056] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values ​​between the aforementioned integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from either end of the range are specifically contemplated. For example, nested subranges of the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0057] Where applicable or not specifically excluded, it is contemplated that any one of the embodiments described herein may be combined with any other one or more embodiments, even if the embodiments are described under different aspects of the disclosure.

[0058] These and other embodiments are disclosed and / or encompassed by the following detailed description.

[0059] The following detailed description, given by way of example and not intended to limit the disclosure to only the particular embodiments described, can be best understood in conjunction with the accompanying drawings. [Brief description of the drawings]

[0060] [Figure 1]1A-1D show schematic diagrams, recombinant protein sequences, and bar graphs of the two proteins, respectively. FIG. 1A is a ribbon diagram protein schematic showing a chimeric VEGF molecule (VEGF-DA) according to an exemplary embodiment of the present disclosure, comprising an N-terminal region derived from VEGF-D and a "homology domain" derived from VEGF-A (VEGFR-1, VEGFR-2, and VEGFR-3 binding regions are shown). FIG. 1B is a protein schematic showing the structure and organization of IN-02, according to an exemplary embodiment of the present disclosure, in which a chimeric VEGF-DA protein domain is fused to the C-terminus of CTB via a 10 amino acid linker. FIG. 1C shows the protein sequence of IN-02, with color coordinates consistent with FIG. 1A-1B. The initial methionine residue is not present in the mature protein, as it is removed by methionine aminopeptidase. FIG. 1D is a bar graph showing the effects of VEGF-A, VEGF-D, and IN-02, alone and in combination with neutralizing antibody (NAb), on tube development by human endothelial cells (HUVEC). [Diagram 2] FIG. 2 is a bar graph showing an ELISA demonstrating binding of VEGF-A, IN-02 (eg, VEGF-DA) and VEGF-D proteins to VEGF receptors immobilized on a plate. [Diagram 3] FIG. 3 is a graph showing an ELISA showing pre- and post-immunization (BL3) sera from three rabbits immunized with IN-02 protein binding to immobilized immunogen. [Figure 4] FIG. 4 is a graph showing an ELISA showing pre- and post-immunization (BL3) sera (caprylic acid purified) from three rabbits immunized with IN-02 protein binding to immobilized rCTB. [Diagram 5] FIG. 5 is a graph showing an ELISA showing pre- and post-immunization (BL3) sera from three rabbits immunized with IN-02 protein that binds to immobilized VEGF-A. [Figure 6]FIG. 6 is a graph showing an ELISA showing pre- and post-immunization (BL3) sera from three rabbits immunized with IN-02 protein that binds to immobilized VEGF-D. [Figure 7] FIG. 7 is a bar graph depicting the results of a tube formation assay performed with caprylic acid purified serum from rabbits immunized with IN-02 protein. [Figure 8] FIG. 8 is a bar graph depicting the results of a HUVEC tube formation assay performed on IN-02 protein stored at 4° C. for one month. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The present disclosure is based, at least in part, on the discovery that chimeric synthetic proteins / molecules comprising one or more protein domains from growth factors (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, etc.), one or more linker regions, and one or more immunogenic domains can be used as therapeutic molecules to treat various diseases, such as cancer. Chimeric synthetic molecules offer several unexpected advantages over the prior art. For example, unlike prior art human epidermal growth factor (hEGF) molecules (e.g., Davila et al., U.S. Pat. No. 5,984,018), which exist in a heterogeneous mixture containing up to 12 different molecular species, the synthetic proteins / molecules described herein can be produced as single molecules (e.g., homogenous populations of molecules). Furthermore, the synthetic proteins / molecules described herein contain 10 active moieties per molecule (although the active moieties may increase or decrease by multiples of 5, e.g., as part of a pentamer), whereas prior art hEGF molecules (e.g., Davila et al., U.S. Pat. No. 5,984,018) are highly variable in the number of active moieties present per molecule (e.g., Davila's average number of active moieties per molecule is 1.5). Furthermore, the chimeric synthetic proteins / molecules described herein are much easier to manufacture. For example, prior art hEGF molecules (e.g., U.S. Pat. No. 5,984,018) are made by chemically conjugating rP64k and recombinant human EGF (rhEGF) to produce a final molecule consisting of two molecules chemically conjugated to each other. This is in contrast to the synthetic proteins / molecules described herein, which are single synthetic molecules. In addition, the chimeric synthetic proteins / molecules described herein have the ability to function as a stabilizing scaffold that favors (e.g., proper folding of) the native conformation of the human proteins (e.g., growth factors) that are incorporated into the protein / molecule when expressed. Furthermore, the chimeric synthetic proteins / molecules described herein have the ability to generate stabilized chimeric synthetic proteins / molecules that have long shelf lives.Advantageously, the technology herein provides novel chimeric synthetic proteins (e.g., cancer vaccines) that can be used therapeutically to treat diseases such as cancer with higher levels of immunogenic activity than prior art methods (e.g., U.S. Pat. No. 5,984,018).

[0062] overview Cancer immunology is the study of the interaction between the immune system and cancer cells, such as tumors or malignant tumors. Of particular interest is the initiation of immune responses, such as the recognition of cancer-specific antigens that are expressed by human tumors and not expressed by normal tissues. In general, methods for controlling the division and proliferation of malignant cells have focused on isolating these antigens and presenting them so that they are recognized by the immune system as non-self antigens and induce specific immune responses.

[0063] There are a significant number of growth factors currently identified, most, if not all, of which have been shown to be important mediators of cell proliferation in various cancers in addition to being involved in other disease states. In general, growth factors are soluble serum proteins that recognize and bind to a group of growth factor receptors located on the cell surface. A particular growth factor may be specific for a single receptor or may bind to two or more closely related receptors with varying affinities. Similarly, some receptors only bind to a single growth factor ligand, while others can bind to multiple related growth factors, also usually with different affinities. Upon binding to its native receptor, the cytoplasmic domain of the receptor is phosphorylated, which initiates an intracellular signaling cascade that leads to the regulation of transcription of one or more genes, ultimately leading to progression through the cell cycle and cell proliferation.

[0064] Growth factors and their receptors are essential components of the normal processes of growth, development and repair, and their tissue distribution profiles and expression levels tightly regulate cell proliferation. Numerous studies have shown that growth factors can stimulate the proliferation of various cell types both in vitro and in vivo (Cohen S., Carpenter G., PNAS USA 72, 1317, 1975, Witsch E et al.: Physiology: 25(2): 85-101, (2010)). In addition, certain growth factors have been shown to stimulate proliferation in some cancer cell lines. For example, epidermal growth factor (EGF) can stimulate some non-small cell lung cancer cells (Osborne CK et al., Can Res. 40, 2.361 (1980)). Other growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) are important in several oncological diseases such as non-small cell lung cancer (NSCLC) (Ballas MS, Chachoua A., Onco Targets and Therapy: 4, 43-58 (201 1)), prostate cancer (Cox ME et al; Prostate 69(l): 33-40 (2009)), and breast cancer (Law J et al., Cancer Res; 68, 24: 10238-10346 (2008)).

[0065] High levels of various growth factor receptors have been reported in malignant tissues. For example, epidermal growth factor receptor (EGFR) has been detected at abnormally high levels in malignant tumors of epithelial origin, such as lung cancer, breast cancer, bladder cancer, ovarian cancer, vulvar cancer, colon cancer, lung cancer, brain cancer and esophageal cancer. The role that growth factors and their receptors play in regulating tumor growth is unclear, but there are suggestions that growth factor receptor expression in tumor cells provides a mechanism for autocrine growth stimulation that leads to uncontrolled proliferation (Schlessinger J., Schreiber AB, Levi A., Liberman T., Yarden Y. Crit. Rev. Biochem. 1983, 14(2) 93-1 11). Furthermore, Liao Y et al; Hum Pathol 36(1 1):1186-1 196 (2005) and Cox ME et al; Prostate:69(1)33-40 (2009) describe the role of increased insulin receptor and growth factors in metastatic prostate cancer.

[0066] One therapeutic strategy to target growth factor signaling in cancer treatment has been to use passive immunotherapy (e.g., monoclonal antibodies) against the specific receptor / receptor group involved. Such studies have demonstrated that specific recognition by antibodies of receptors that can inhibit the binding of ligands can have an inhibitory effect on the division stimulation of malignant cells (SATO JD et al., Methods in Enzymology, vol. 146 pp63-81, 1987). However, antibodies of mouse origin usually generate a human anti-mouse antibody response (HAMA), and therefore they are limited to single administration.

[0067] Another therapeutic strategy has been to use active immunotherapy with a vaccine containing the growth factor of interest to induce an immune response against the molecule and inhibit the growth factor's proliferative effect on tumors. U.S. Patent No. 5,984,018 by Davila et al., entitled "Vaccine Composition Comprising Autologous Epidermal Growth Factor or a Fragment or a Derivative Thereof having Anti-tumor Activity and use Thereof in the Therapy of Malignant Diseases," for example, discloses the use of a vaccine containing a mixture of growth factor and an immunogenic (i.e., non-human) carrier protein that is chemically conjugated with glutaraldehyde. However, without being bound to any particular theory, it is believed that chemical conjugation impedes the immune response to the vaccine.

[0068] This is a technically challenging approach since it requires that the host generate an immune response against a "self-antigen" and that the vertebrate immune system has evolved to prevent such a response from occurring. Autoimmune disease states usually result when a strong immune response occurs against a self-antigen, such as an autoantigen that includes T-helper cell activation. For many years, it has been hypothesized that some autoimmune disorders, such as lupus, multiple sclerosis (MS), diabetes, etc., can be caused by early exposure to environmental factors that contain immunogenic epitopes (T-cell epitopes) that closely mimic host self-epitopes. This can result in the stimulation of T-helper cells that are cross-reactive with the host epitopes. Subsequent exposure to environmental factors can result in an anti-autoimmune response (Albert, LJ and Inman, RD New England Journal of Medicine, Dec. 30th pp2068-2074, 1999). It has subsequently been demonstrated that viral antigens can indeed generate anti-autoimmune responses against neuronal proteins (Levin, MC et al., Nature Medicine vol 8(5) pp509-513, 2002).

[0069] U.S. Patent Application Publication No. 2006 / 0251654 (the '654 publication) by Casimiro et al., entitled "Method for Treatment of Malignant and Infectious Chronic Diseases," discloses a method of treating a subject with a malignant or infectious chronic disease, comprising immunizing the subject with a vaccine containing an autoantigen associated with the malignant or infectious chronic disease coupled to a carrier protein, treating the subject with an immunomodulatory agent, and re-immunizing the subject with the vaccine of step 1 and a suitable adjuvant selected from aluminum hydroxide and Montanide ISA 51 (Seppic, Paris, France). Unfortunately, preparation of the vaccine by chemical conjugation is believed to hinder the immune response.

[0070] Most of the above vaccines show some limitations mainly resulting from the manufacturing method and the potential lack of homogeneity and homology of the protein product. The above vaccines generally comprise a mixture of recombinant carrier proteins and polypeptides of human origin chemically conjugated using glutaraldehyde. Unfortunately, this reactive reagent can undesirably form covalent cross-links between various chemical groups, generally resulting in a highly heterogeneous product. Thus, the resulting vaccine may not only contain carrier protein molecules with various numbers (e.g., 0, 1, 2, 3, etc.) of target human polypeptides attached thereto, but each human polypeptide may be attached to the carrier through a different atom and therefore in a different position and orientation. Moreover, both the target polypeptide and the carrier protein molecules may be conjugated to themselves, resulting in various homomultimers that may not have clinical efficacy and may not contribute to anti-cancer patient immune responses.

[0071] Synthetic Proteins / Molecules The present disclosure provides homogeneous synthetic proteins / molecules to increase the maximum number of growth factor epitopes, tumor antigen epitopes, and / or receptor binding sites presented as components of the immunogenic synthetic protein / molecule. In one exemplary embodiment, a synthetic protein / molecule expressing all or part of an immunogenic carrier domain (e.g., cholera toxin B (CT-B)), as well as a synthetic epidermal growth factor (sEGF), tumor antigen, and / or receptor is described. In alternative exemplary embodiments, the protein may express other immunogenic synthetic or recombinant proteins / molecules modeled on known immunogenic proteins. It is contemplated within the scope of the present disclosure that such synthetic proteins / molecules may express polypeptides that are highly immunogenic to the human immune system. Preferably, the synthetic protein / molecule confers additional properties to the chimeric protein, such as, for example, high expression yield and ease of manufacture, oral stability and ability to cross from the gut to the bloodstream, and / or prior safe use in humans.

[0072] In exemplary embodiments, the synthetic proteins / molecules disclosed herein may contain or express a high percentage of protein sequences derived from the target autoantigen as a function of total molecular weight. This may be achieved, for example, by using large protein models that contain multiple growth factor epitopes. These growth factor epitopes may be multiple copies of all or part of a single growth factor, or copies of all or part of two or more different growth factors. These growth factor epitopes may be naturally occurring or synthetic (e.g., artificial). For example, BVN22E (also called IN01), an exemplary synthetic protein described herein, may have a molecular weight of about 120 kD. In exemplary embodiments, the growth factor epitopes described herein may correspond to one or more domains within a growth factor (e.g., EGF targeting signaling pathway (TSP) domains). In exemplary embodiments, the EGF domain may include a region that presents or constrains a β-loop, for example, a region defined by about cysteine ​​6 to about cysteine ​​42 of the synthetic protein sequence, a region defined by about cysteine ​​6 to about cysteine ​​31, or a region defined by about cysteine ​​22 to about cysteine ​​33, or a region defined by about cysteine ​​22 to about cysteine ​​31, or a region defined by about cysteine ​​62 to about cysteine ​​14 (e.g., FIG. 1A). Without being bound to any particular theory, it is contemplated within the scope of the present disclosure that different regions or subregions between cysteine ​​6 and cysteine ​​42 may have beneficial effects when incorporated into the synthetic proteins / molecules of the present disclosure. For example, the following regions may have beneficial effects: between cysteine ​​6 and cysteine ​​14, between cysteine ​​6 and cysteine ​​20, between cysteine ​​6 and cysteine ​​31, between cysteine ​​6 and cysteine ​​33, and between cysteine ​​6 and cysteine ​​42. It is also contemplated within the scope of this disclosure that reverse progressive sequences may also be beneficial.For example, the following regions may have beneficial effects: the region between cysteine ​​42 and cysteine ​​33, the region between cysteine ​​42 and cysteine ​​31, the region between cysteine ​​42 and cysteine ​​20, the region between cysteine ​​42 and cysteine ​​14, and the region between cysteine ​​42 and cysteine ​​6. It is further contemplated within the scope of the present invention that certain sections within the region between cysteine ​​6 and cysteine ​​42 may provide beneficial effects when incorporated into the synthetic proteins / molecules of the present disclosure (e.g., the region between cysteine ​​6 and cysteine ​​14, the region between cysteine ​​14 and cysteine ​​20, the region between cysteine ​​20 and cysteine ​​31, and the region between cysteine ​​33 and cysteine ​​42).

[0073] According to the present disclosure, expression of growth factor epitopes must be folded such that their native conformation is substantially preserved and can be presented to components of the host immune system to elicit a robust host immune response to the epitope. Examples of suitable natural protein models for modeling the epitope-bearing domains of synthetic proteins / molecules include, but are not limited to, cholera toxin B subunit, E. coli heat labile LT and LT-II enterotoxin B subunit, veratoxin, pertussis toxin, C. jejuni enterotoxin, shiga toxin, listeria toxin, tetanus toxoid, diphtheria toxoid, N. meningitidisl outer membrane protein, bacteriophage coat proteins, adenovirus, and other viral coat proteins. Alternatively, the non-self component of the protein may be small. At a minimum, the non-self sequence must have a length of about 9, 10, 11 or more amino acids and contain at least one human T-cell epitope, either in whole or in part. As described herein, non-naturally occurring synthetic polypeptides (e.g., BVN22E, IN01) can be used that meet the requirements of conferring immunogenicity to the entire protein and allowing proper presentation of growth factors, receptors, tumor antigens or epitopes thereof to the host immune system.

[0074] According to the present disclosure, the synthetic proteins / molecules provided herein, whether growth factors or parts thereof, cell receptors or parts thereof, or tumor antigens or parts thereof, are associated with a wide range of cellular pathways involved in chronic diseases, growth factor-based or receptor-based cancers, and / or solid tumors for use as tumor antigens within the synthetic proteins. The proteins are in the form of synthetic proteins / molecules and may be useful in treating chronic diseases, such as breast cancer, lung cancer, bladder cancer, ovarian cancer, vulvar cancer, colon cancer, lung cancer, brain cancer, colorectal cancer, intestinal cancer, head and neck cancer, and esophageal cancer. Since different tumor antigens may be expressed and multiple cell receptors and growth factors may be overexpressed in the above diseases, the proteins described herein 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 the disease. These proteins are referred to as multivalent.

[0075] In an exemplary embodiment, a protein is disclosed that is composed of a homogenous synthetic protein / molecule expressing one or more epidermal growth factor (EGF) neutralizing domains (e.g., TSP domains). The protein may be in the form of a synthetic protein / molecule and may be useful in treating chronic diseases, such as breast cancer, lung cancer, bladder cancer, ovarian cancer, vulvar cancer, colon cancer, lung cancer, brain cancer, colorectal cancer, head and neck cancer, and esophageal cancer. In an exemplary embodiment, the protein is a synthetic protein / molecule that expresses or includes a synthetic EGF sequence and a CT-B sequence, as shown in FIG. 1A. In an exemplary embodiment, the growth factor component of the synthetic protein sequence may include a sequence that has less than 80% identity to EGF. For example, the growth factor component may include an EGF sequence with 11 amino acid substitutions that may increase the immunogenicity of the growth factor portion of the synthetic protein sequence. Without wishing to be bound by theory, it is believed that regions of EGF that "present" or constrain the β-loop (e.g., the region defined by Cys6 to Cys31) may be important to include in the synthetic protein and may be suitable targets for amino acid modification. In exemplary embodiments, regions outside of Cys6 to Cys31 may also be targeted for modification (e.g., E11 and A12).

[0076] In an exemplary embodiment, the TSP1 and TSP2 domains of hEGF may be modified as shown in FIG. 1B to create the synthetic EGF (sEGF) region included in the synthetic proteins / molecules herein.

[0077] In exemplary embodiments, the synthetic proteins / molecules disclosed herein may comprise all or a portion of a growth factor, including, but not limited to, neuregulin 1β (NRG1β), transforming growth factor α (TGFα), vascular endothelial growth factor (VEGF), and the like.

[0078] In other exemplary embodiments, the synthetic proteins / molecules described herein may include one or more linkers or spacers. One or more of the above embodiments include sEGF fused to CT-B such that the sEGF portion of the synthetic molecule is separated from the CT-B portion by a GGSGGTSGGGGGSG linker. These resulting recombinant or chimeric proteins essentially contained sEGF fused directly to CT-B. In other exemplary embodiments, the EGF and CT-B components of the chimeric protein are effectively separated by 3 to 14 amino acids, which form a flexible spacer or linker between the two domains. It is contemplated within the scope of the present disclosure that this linker may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acids long. When the growth factor has a larger size (e.g., human growth factor), it may be useful to use a longer linker sequence. The following exemplary linkers can be used and include, but are not limited to, the following: SSG, SSGGG, SGG, GSSG, GGSGG, GGGGS, SSGGGSGG, SSGGGGSGGG, TSGGGSG, TSGGGGSGG, SSGGGSGGSSG, GGSGGTSGGGSG, SGGTSGGGGSGG, GGSGGTSGGGGSGG, SSGGGGSGGGSSG, SSGGGSGGSSGGG, and SSGGGGSGGGSSGGG. One of skill in the art will appreciate that there are many other sequences / combinations of primarily "G" and "S" that will also function as useful linker sequences.

[0079] Without being bound to any particular theory, it is believed that the synthetic proteins / molecules disclosed herein provide significant clinical benefits. For example, the synthetic proteins / molecules disclosed herein can be expressed in bacterial systems at commercial scale and purity while producing stable polypeptides that are correctly folded and functional. Furthermore, the synthetic proteins / molecules disclosed herein can form pentamers. Furthermore, the synthetic proteins / molecules disclosed herein have the advantageous property of requiring significantly less carrier than prior art methods (e.g., Davila et al., U.S. Pat. No. 5,984,018), thus requiring much lower levels of protein for vaccination. In this regard, the synthetic proteins / molecules disclosed herein can deliver more growth factors to patients with much less vaccine.

[0080] Adjuvants Certain exemplary embodiments provided herein include synthetic proteins / molecules according to the present disclosure in vaccine compositions and immune adjuvant compositions, including pharmaceutical compositions that contain, in addition to the synthetic proteins / molecules, at least one adjuvant, which refers to a component of such compositions that has adjuvant activity. Such adjuvants with adjuvant activity include compositions that can alter (i.e., increase or decrease in a statistically significant manner, and in certain preferred embodiments, enhance or increase) the potency and / or longevity of an immune response 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 with a recognized immune system. In certain exemplary embodiments disclosed herein, the desired antigen and / or antigens are contained within a protein carrier, and optionally one or more adjuvants may so alter (e.g., induce or enhance) the desired antigen and / or immune response to the antigen, and administration thereof may be simultaneous or separated in time and / or space (e.g., at different anatomical sites), although certain exemplary embodiments are not intended to be so limited and thus also contemplate administering the synthetic protein / molecule in the form of a composition that does not include a particular antigen, but may also include one or more co-adjuvants, including but not limited to, imidazoquinoline immune response modifiers.

[0081] Thus, as noted above, adjuvants include compositions having adjuvant effect, 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, WO 95 / 17210), plant alkaloids such as alum, tomatine, detergents such as (but not limited to) saponins, 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), imidazoquinoline immune response modifiers, and dual stem loop immunomodulators (dSLIMs, e.g., Weeratna et al., 2005 Vaccine 23:5263).

[0082] Saponin-containing detergents are taught, for example, in 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. Particulate structures called immune stimulating complexes (ISCOMS), which contain a fraction of Quil A (saponin), are hemolytic and have been used in the manufacture of vaccines (Morein, B., EP 0 109 942). These structures have been reported to have adjuvant activity (EP 0 109 942; WO 96 / 1 1711). The hemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A) have been described as potent systemic adjuvants and their preparation methods are disclosed in US Pat. No. 5,057,540 and EP 0 362 279. These references also describe the use of QS7 (a non-hemolytic fraction of Quil-A) to act as a potent adjuvant for systemic vaccines. The use of QS21 is further described in Kensil et al. (1991. J. Immunology 146:431-437). Combinations of QS21 with polysorbates or cyclodextrins are also known (WO 99 / 10008). Particulate adjuvant systems containing fractions of Quil A such as QS21 and QS7 are described in WO 96 / 33739 and WO 96 / 1 1711. 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 detergent related to saponins for use in the adjuvant compositions of the embodiments disclosed herein. Escin is described in the Merck Index (12. Edition 3737) as a mixture of saponins occurring in the seeds of horse chestnut (Aesculus hippocastanum).Its isolation has been 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). A fraction of escin (also known as escin) has 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 listed as a saponin in the Merck Index (12th ed. entry 3204), derived from the seeds of Digitalis purpurea and purified by the procedures described in J. Am. Pharm. Assoc., 1934, 23, 664, and Rubenstroth-Bauer, Physiol. Chem., 1955, 301, 621.

[0083] Other adjuvants or coadjuvants for use with certain embodiments disclosed herein include block copolymers or biodegradable polymers, which refer to classes of polymeric compounds familiar to those skilled in the art. Examples of block copolymers or biodegradable polymers that can be included in vaccine compositions or immune adjuvants include Pluronic.RTM.L121 (BASF Corp., Mount Olive, NJ; see, for example, Yeh et al., 1996 Pharm.Res.13:1693).

[0084] Certain further exemplary embodiments contemplate immunological adjuvants that include, but are not limited to, oils that may contribute to co-adjuvant activity in some such embodiments and may additionally or alternatively provide a pharma- ceutically acceptable carrier or excipient in other such embodiments. A number of suitable oils are known and may be selected for inclusion in vaccine compositions and immunoadjuvant compositions based on the present disclosure. Examples of such oils include, by way of illustration and not limitation, squalene, squalane, mineral oil, olive oil, cholesterol, and mannide monooleate.

[0085] Immune response modifiers, such as imidazoquinoline immune response modifiers, are also known in the art and may be included as adjuvants or coadjuvants in certain presently disclosed embodiments.

[0086] Also as mentioned above, one type of adjuvant or coadjuvant for use in the vaccine compositions according to the present disclosure described herein can be an aluminum coadjuvant, commonly referred to as "alum." Alum coadjuvants are based on aluminum oxyhydroxide, aluminum hydroxyphosphate, or various proprietary salts. Alum coadjuvants are advantageous because they have a good safety record, boost antibody 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).

[0087] Pharmaceutical Compositions In certain exemplary embodiments, the pharmaceutical composition is a vaccine composition comprising both a synthetic protein / molecule according to the present disclosure, and may further comprise one or more components provided herein selected from a TLR agonist, a co-adjuvant (e.g., a cytokine, an imidazoquinoline immune response modifier and / or a dSLIM, etc.), and / or a recombinant expression construct, in combination with a pharma- ceutically acceptable carrier, excipient or diluent.

[0088] Exemplary carriers are nontoxic to recipients at the dosages and concentrations employed. For vaccines containing synthetic proteins / molecules, about 0.01 μg / kg body weight to about 100 mg / kg body weight are typically administered via intradermal, subcutaneous, intramuscular or intravenous routes, or via other routes.

[0089] It will be clear to those skilled in the art that the number and frequency of administration will depend on the host's response. "Pharmaceutically acceptable carriers" for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985). For example, sterile saline and phosphate-buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes, and even flavoring agents may be added to 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.

[0090] The pharmaceutical composition may be in any form that allows 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, but are not limited to, oral, topical, parenteral (e.g., sublingual or buccal), sublingual, rectal, vaginal, and intranasal (e.g., as a spray). The term parenteral as used herein includes iontophoresis, sonophoresis, passive transdermal, microneedle administration, and also subcutaneous injection, intravenous, intramuscular, intrasternal, intracavity, intrathecal, intrathecal, intraurethral injection or infusion techniques. In certain embodiments, the compositions described herein (including vaccines and pharmaceutical compositions) are administered intradermally by a technique selected from iontophoresis, microcavitation, sonophoresis, or microneedle.

[0091] The pharmaceutical composition is formulated so that the active ingredient contained therein is bioavailable when the composition is administered to a patient. The composition administered to a patient may take the form of one or more dosage units, for example, a tablet may be a single dosage unit, and a container of one or more compounds of the present invention in aerosol form may hold multiple dosage units.

[0092] For oral administration, excipients and / or binders may be present. Examples are sucrose, kaolin, glycerin, starch dextrin, sodium alginate, carboxymethylcellulose and ethylcellulose. Coloring and / or flavoring agents may be present. Coating shells may be used.

[0093] The composition may be in the form of a liquid, such as 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, a preferred composition contains one or more of a sweetener, a preservative, a dye / colorant and a flavor enhancer. A composition intended for administration by injection may contain one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer and an isotonic agent.

[0094] The liquid pharmaceutical composition used herein, whether in solution, suspension or other similar form, may contain one or more of the following carriers or excipients: water for injection, physiological saline, preferably physiological saline, Ringer's solution, sterile diluent such as isotonic sodium chloride, fixed oil such as squalene, squalane, mineral oil, mannide monooleate, cholesterol, and / or synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol or other solvents that can function as a solvent or suspension medium, antibacterial agents such as benzyl alcohol or methylparaben, 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 isotonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes or multiple dose vials. Injectable pharmaceutical compositions are preferably sterile.

[0095] In certain embodiments, the 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, fluorinated lipids, and the like.

[0096] It may be desirable to include other components in the vaccine or pharmaceutical composition, such as delivery vehicles including, but not limited to, aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules, and liposomes. Examples of additional immune stimulants (coadjuvants) for use in such vehicles have been described above and may include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), glucan, IL-12, GM-CSF, gamma interferon, and IL-12.

[0097] Any suitable carrier known to those skilled in the art can be used in the pharmaceutical composition of the present invention, but the type of carrier varies depending on the mode of administration and whether sustained release is desired.For parenteral administration such as subcutaneous injection, the carrier preferably comprises water, physiological saline, alcohol, fat, wax or buffer.For oral administration, any of the above carriers or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose and magnesium carbonate can be used.Biodegradable microspheres (e.g., polylactic galactide) can also be used as carriers for the pharmaceutical composition of the present invention.

[0098] The pharmaceutical composition 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 dextrin, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are typical suitable diluents. Preferably, the product can be formulated as a lyophilizate using suitable excipient solutions (e.g., sucrose) as diluents.

[0099] In an exemplary embodiment, the epitope or receptor-bearing domain of the synthetic protein / molecule, whether derived from a natural or synthetic polypeptide sequence, must have the ability to self-assemble into oligomeric multimers under appropriate chemical / environmental conditions, or to be reduced to monomers under alternative conditions. Ideally, the multimerization domain assembles into stable multimers with a discrete number of subunits, e.g., dimers, trimers, tetramers, pentamers, etc., such that a homogeneous size product is generated. Examples of natural polypeptides include, but are not limited to, leucine zippers, lac repressor proteins, streptavidin / avidin, cholera toxin B subunit, Pseudomonas trimerization domains, and viral capsid proteins.

[0100] In an exemplary embodiment, a method of preparing a multivalent molecule is disclosed, which comprises assembling a multimer from monomeric subunits to form a synthetic protein comprising one or more tumor antigens, receptors, and / or growth factors or portions thereof.

[0101] In another exemplary embodiment, a method of preparing a vaccine formulation is disclosed, which comprises mixing one or more single monovalent multimers together to prepare a multivalent vaccine comprising one or more tumor antigens, receptors, and / or synthetic proteins / molecules comprising growth factors or portions thereof.

[0102] In yet another exemplary embodiment, a method for treating a patient is disclosed, which comprises administering one or more monovalent tumor antigens, receptors, and / or growth factors, recombinant proteins to a patient separately on the same day or on different days or at different times during a vaccination period.

[0103] Although the synthetic proteins / molecules are described as comprising or expressing one or more of a tumor antigen, growth factor, and / or receptor, and all or a portion of at least one of the sequences of the CT-B sequence, the synthetic proteins / molecules may comprise a native CT-B sequence or a sequence substantially similar to a native CT-B sequence, and / or a synthetic sequence. Although the synthetic proteins / molecules are described as comprising or expressing a CT-B sequence, the synthetic proteins / molecules may comprise or express a derivative of the CT-B sequence or a sequence substantially similar to the CT-B sequence.

[0104] Although the homogeneous synthetic proteins / molecules expressing or incorporating one or more tumor antigens, synthetic growth factors, and / or receptors have been described and illustrated with reference to specific embodiments, 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 disclosure. Thus, the disclosure should not be limited to the precise details of the methods or constructions described above, as such variations and modifications are intended to be included within the scope of the disclosure. EXAMPLES

[0105] The present disclosure is further illustrated by the following examples, which should not be construed as limiting. All references, GenBank accession numbers and gene numbers, and the contents of published patents and patent applications cited throughout this application are incorporated herein by reference. Those skilled in the art will understand that the present disclosure can be implemented with modifications to the disclosed structures, materials, compositions and methods, and such modifications are considered to be within the scope of the present disclosure.

[0106] Example 1: Bispecific Chimeric Antigens A problem that arises when combining human proteins (e.g., growth factors) or portions thereof with immunogenic carrier molecules, such as cholera toxin B subunit (CTB), to produce recombinant proteins is that human proteins (e.g., human growth factors) do not always fold into the correct native conformation. The ability of human proteins to fold correctly within recombinant proteins can vary greatly from protein to protein, even between closely related molecules. For example, epidermal growth factor (EGF) can be easily folded correctly from insoluble inclusion bodies and is very stable thereafter, whereas the EGF-like domains of transforming growth factor alpha (TGFα) and neuregulin are both more difficult to produce in properly folded forms and are significantly less stable.

[0107] Vascular endothelial growth factors (VEGFs) comprise four structurally related proteins, VEGF-A, VEGF-B, VEGF-C, and VEGF-D, that mediate signal transduction through three receptors, VEGFR-1, VEGFR-2, and VEGFR-3. VEGF-A and VEGF-D transmit signals through VEGFR-1, while VEGF-A, VEGF-B, and VEGF-C can bind to VEGFR-2. VEGF-C and VEGF-D bind to VEGFR-3, and as a result, show both similarities and differences in their receptor binding properties. All VEGF growth factors share a common structural "homology domain," which is involved in the recognition and binding of VEGFR-1 and VEGFR-2 (VEGF-A, VEGF-B, and VEGF-C), and includes the sequence downstream of the first cysteine ​​residue. The N-terminus of VEGF-A and VEGF-B upstream of the first cysteine ​​residue is not directly involved in receptor binding. In contrast, the N-terminus of VEGF-C and VEGF-D is involved in binding to VEGFR-3.

[0108] VEGF-A can be expressed in E. coli as insoluble inclusion bodies and subsequently denatured, solubilized, and refolded into a fully functional protein. VEGF-D is similarly amenable to refolding from inclusion bodies, but is much less stable, showing visible signs of degradation after only one week at 4°C, making it unsuitable for use in therapeutic applications in its native form. VEGF-C is very difficult to fold correctly when expressed from inclusion bodies or as a soluble protein in bacteria.

[0109] The N-terminal regions of VEGF-C and VEGF-D (sequences upstream of the first cysteine ​​residue) form an alpha helix in the native protein that interacts with VEGFR-3. This structure also requires interactions with other parts of the VEGF molecule to adopt and maintain this configuration. When expressed alone or as a genetic fusion with an "unrelated" carrier, the resulting protein does not show any binding to VEGFR-3. However, as shown in Figure 1A, when the N-terminal domain of VEGF-A is replaced with the N-terminus of VEGF-D, the resulting protein is able to bind to all three VEGF receptors and regulate three separate signaling pathways. Thus, the VEGF-A domain acts as a stabilizing scaffold, allowing the N-terminal domain of VEGF-D to adopt and maintain its native structure. A stabilized bispecific chimeric VEGF containing sequences derived from both VEGF-D and VEGF-A was further fused to the C-terminus of CTB, separated by a 10 amino acid glycine / serine-rich flexible linker. This molecule was designated IN-02 and is shown diagrammatically in Figure 1B. The protein sequence of IN-02 is shown in Figure 1C.

[0110] Two assays were used to analyze the functional characteristics of VEGF-based molecules: tube formation assay (TFA) and ELISA. TFA involved culturing human umbilical vein endothelial cells (HUVEC) and observing the development of "tubes" over time, which represent the formation of capillaries. Tube formation by cells cultured with stimulatory (growth factors) and inhibitory (neutralizing antibodies) modulators was then compared to untreated. For ELISA, recombinant VEGFR (the extracellular domain of the VEGF receptor fused to the human IgG Fc region) was coated onto ELISA plates. The plates were incubated with VEGF protein, and bound VEGF was detected with a protein-specific antibody.

[0111] Figure ID shows the effect of growth factors and neutralizing antibodies (Nab) on tube development (vascularization) by human endothelial cells (HUVEC). Both VEGF-A and VEGF-D can stimulate tube formation independently (Figure ID, white and grey bars), and this stimulation was blocked by the addition of neutralizing antibodies to each growth factor. Chimeric VEGF-DA protein can also stimulate tube formation (first hatched bar). This stimulation can be partially inhibited by neutralizing antibodies against VEGF-A and VEGF-D separately, and more completely inhibited by the addition of both antibodies.

[0112] FIG. 2 shows ELISA data showing binding of VEGF proteins to VEGF receptors immobilized on plates. Recombinant human VEGF-A bound to receptor-1 and receptor 2 (left bar). VEGF-D bound to receptor 3 and receptor 2 (right bar). Chimeric IN-02 protein bound to receptors 1, 2 and 3. Binding of IN-02 to receptor 2 could only be detected using anti-VEGF-A antibody (gray bar) because both receptor-2 and anti-VEGF-D antibody bound to the same region of the chimeric protein. Three rabbits were immunized with 100 μg of IN-02 (subcut) on days 0, 14, 28 and 56. Bleeds were taken on days 0 (pre-immunization), 2, 3 and 56. For clarity, only pre-immunization and bleed 3 data are shown.

[0113] FIG. 3 shows ELISA data showing pre- and post-immunization (BL3) sera from three rabbits immunized with IN-02 protein bound to immobilized immunogen, clearly demonstrating that all three rabbits mounted an immune response to the immunogen and that there was no pre-immunization reactivity.

[0114] Figure 4 shows ELISA data showing pre- and post-immunization (BL3) sera (caprylic acid purified) from three rabbits immunized with IN-02 protein bound to immobilized rCTB. All three rabbits mounted an immune response to the CTB domain of the immunogen and reacted with rCTB. There was no pre-immunization reactivity.

[0115] Figure 5 shows ELISA data showing pre- and post-immunization (BL3) sera from three rabbits immunized with IN-02 protein bound to immobilized VEGF-A. All three rabbits mounted an immune response to the VEGF-A domain of the immunogen, which also reacted with rhVEGF-A. There was no pre-immunization reactivity.

[0116] Figure 6 shows an ELISA showing pre- and post-immunization (BL3) sera from three rabbits immunized with IN-02 protein bound to immobilized VEGF-D. All three rabbits mounted an immune response to the VEGF-D domain of the immunogen, which also reacted with rhVEGF-D. There was no pre-immunization reactivity. After immunization of the rabbits with IN-02 protein, all individuals developed an immune response to the immunizing antigen that was capable of recognizing full-length native rhVEGF-A and rhVEGF-D, in addition to the immunogenic CTB "carrier" domain.

[0117] To determine the effectiveness of immune responses in neutralizing signaling induced by VEGF-A and VEGF-D, HUVEC tube formation assays were performed as described above. Figure 7 shows the results of tube formation assays performed using caprylic acid purified sera from rabbits immunized with IN-02 protein. All three sera significantly inhibited tube formation induced by simultaneous co-stimulation with both VEGF-A and VEGF-D (black bars).

[0118] Figure 8 shows the results of a HUVEC tube formation assay performed on IN-02 protein stored for 1 month at 4° C. Despite storage, IN-02 protein still stimulated tube formation to the same extent as VEGF-A or VEGF-D, and this stimulation could be effectively inhibited by co-incubation with antibodies capable of neutralizing VEGF-A and VEGF-D.

[0119] Incorporation by Reference All documents cited or referenced herein, and all documents cited or referenced in the documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any documents incorporated by reference herein, are hereby incorporated by reference and may be used in the practice of the present disclosure.

[0120] Equivalent It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only and are not to be considered as limiting the present disclosure in any way. In light of this, various modifications or changes will be suggested to those skilled in the art and are considered to be within the spirit and scope of the present application and within the scope of the appended claims. Additional advantageous features and functions associated with the systems, methods, and processes of the present disclosure will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed within the scope of the following claims.

Claims

1. a chimeric polypeptide sequence comprising the sequences of vascular endothelial growth factors (VEGFs), VEGF-A and VEGF-D; At least one linker; an immunogenic polypeptide sequence or a polypeptide sequence comprising a cholera toxin B (CT-B) protein; A chimeric synthetic protein comprising:

2. The chimeric synthetic protein of claim 1 , wherein the at least one linker comprises a first linker separating the chimeric polypeptide sequence from the polypeptide sequence.

3. 3. The chimeric synthetic protein of claim 2, wherein the first linker is selected from the group consisting of SSG, GSSG, SSGGG, SGG, GGSGG, GGGGS, SSGGGGSGG, SSGGGGSGGG, TSGGGGSG, TSGGGGGSGG, SSGGSGGGSG, SSGGGSGGGSSG, GGSGGTSGGGGSG, SGGTSGGGGGSGG, GGSGGTSGGGGGSGG, SSGGGGSGGGSSG, SSGGGSGGGSSGGG, and SSGGGGSGGGSSGGGG, or wherein the first linker is SSGGSGGGSG.

4. The chimeric synthetic protein of claim 1 , wherein the chimeric polypeptide sequence comprises a first VEGF domain and a second VEGF domain.

5. 5. The chimeric synthetic protein of claim 4, wherein the first VEGF domain comprises VEGF-D and the second VEGF domain comprises VEGF-A, or the first VEGF domain is TFYDIETLKVIDEEWQRTQ and the second VEGF domain is CHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

6. 5. The chimeric synthetic protein of claim 4, wherein the chimeric polypeptide sequence binds to a vascular endothelial growth factor receptor (VEGFR) selected from the group consisting of VEGFR-1, VEGFR-2, VEGFR-3, and combinations thereof, or the chimeric polypeptide sequence binds to VEGFR-1, VEGFR-2 and VEGFR-3.

7. 2. The chimeric synthetic protein of claim 1, wherein the chimeric synthetic protein has the amino acid sequence of MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

8. 8. The chimeric synthetic protein of claim 7, wherein the chimeric synthetic protein is processed to have the amino acid sequence of TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

9. a chimeric polypeptide sequence comprising the sequences of vascular endothelial growth factors (VEGFs), VEGF-A and VEGF-D; At least one linker; an immunogenic polypeptide sequence or a polypeptide sequence comprising a cholera toxin B (CT-B) protein; 20. An immunogenic composition comprising:

10. The immunogenic composition of claim 9 , wherein the at least one linker comprises a first linker separating the chimeric polypeptide sequence from the polypeptide sequence.

11. 10. The immunogenic composition of claim 9, wherein the first linker is selected from the group consisting of SSG, GSSG, SSGGG, SGG, GGSGG, GGGGS, SSGGGGSGG, SSGGGGSGGG, TSGGGGSG, TSGGGGGSGG, SSGGSGGGSG, SSGGGSGGGSSG, GGSGGTSGGGGSG, SGGTSGGGGGSGG, GGSGGTSGGGGGSGG, SSGGGGSGGGSSG, SSGGGGSGGGSSG, and SSGGGGSGGGSSGGGG, or wherein the first linker is SSGGSGGGSG.

12. The immunogenic composition of claim 9 , wherein the chimeric polypeptide sequence comprises a first VEGF domain and a second VEGF domain.

13. 13. The immunogenic composition of claim 12, wherein the first VEGF domain comprises VEGF-D and the second VEGF domain comprises VEGF-A, or the first VEGF domain is TFYDIETLKVIDEEWQRTQ and the second VEGF domain is CHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

14. 14. The immunogenic composition of claim 13, wherein the chimeric polypeptide sequence binds to a vascular endothelial growth factor receptor (VEGFR) selected from the group consisting of VEGFR-1, VEGFR-2, VEGFR-3, and combinations thereof, or wherein the chimeric polypeptide sequence binds to VEGFR-1, VEGFR-2, and VEGFR-3.

15. 10. The immunogenic composition of claim 9, wherein the chimeric polypeptide sequence has the amino acid sequence of MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

16. 10. The immunogenic composition of claim 9, wherein the chimeric polypeptide sequence is processed to have the amino acid sequence: TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

17. The immunogenic composition of claim 9, wherein the chimeric polypeptide sequence comprises a VEGF-A sequence in which the N-terminal domain has been replaced with the N-terminus of VEGF-D.

18. A cancer therapeutic composition comprising the immunogenic composition described in claim 9, characterized in that it is administered to a patient on the same day or on a different day or at a different time during a vaccination period.

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