Stable chimeric synthetic proteins and therapeutic uses thereof

Abstract

The present disclosure relates to stable chimeric synthetic proteins and therapeutic uses thereof. More particularly, the present disclosure relates to stable chimeric synthetic proteins and their use for the treatment of cancer.

Description

[0001] This application is a divisional application of PCT patent application number PCT / CN2020 / 096339 entered into China on June 24, 2020, entitled “Stable Chimeric Synthetic Proteins and Therapeutic Uses Thereof,” Chinese patent application number 202080058207.8. TECHNICAL FIELD

[0002] The present disclosure relates to compositions and methods for treating disease. More particularly, the present disclosure relates to stable chimeric synthetic proteins and their use for treating cancer. BACKGROUND

[0003] According to the World Health Organization, tumors (e.g., cancers) are a leading cause of death worldwide, causing 8.8 million deaths in 2015. Cancer is very prevalent in the global population: accounting for nearly one sixth of all deaths. In 2015, the most common cancer deaths occurred in the following types of cancer: lung cancer (about 1,700,000 deaths), liver cancer (about 800,000 deaths), colorectal cancer (about 800,000 deaths), stomach cancer (about 800,000 deaths), and breast cancer (about 600,000 deaths).

[0004] Cancers are typically treated by any of a variety of methods, e.g., surgery, chemotherapy, radiation, cancer immunotherapy, etc. Unfortunately, most of these methods have toxic / undesirable side effects. For example, standard cancer chemotherapy is based on its ability to kill cells that are dividing rapidly, and most have toxic, undesirable side effects such as immunosuppression, nausea, hair loss, etc. Over the past two decades, a central goal of cancer research has been to identify new therapies with greater efficacy and fewer side effects.

[0005] One such therapy is encompassed by cancer immunology, which studies the interaction between the immune system and cancer cells, such as tumors or malignancies. Of particular interest is the initiation of an immune response, such as the recognition of cancer-specific antigens expressed by human tumors but not in normal tissues. Generally, approaches to control malignant cell division and proliferation focus on isolating these antigens and presenting them so that they are recognized by the immune system as non-self antigens to induce a specific immune response (e.g., cancer vaccines). Such cancer vaccines can typically be created as chemical conjugates or recombinant proteins. Disadvantageously, such cancer vaccines exhibit a number of significant limitations, which primarily stem from the manufacturing process and potential lack of uniformity, activity, and homogeneity of the protein product. For example, cancer vaccines generated by chemical conjugation (e.g., by glutaraldehyde) generally comprise a mixture of recombinant carrier proteins and human-derived polypeptides. Unfortunately, the use of glutaraldehyde as a cross-linking reagent has the undesirable tendency to form covalent cross-links between a variety of chemical groups and generally results in a highly heterogeneous product. Thus, the resulting cancer vaccine can not only comprise carrier protein molecules with a number of target human polypeptides (e.g., 0, 1, 2, 3, etc.) attached, but the human polypeptides can each be attached to the carrier through different atoms and thus be present in different positions and different orientations. Furthermore, both the target polypeptides and the carrier protein molecules can be conjugated to themselves, resulting in a variety of homomultimers that can have no clinical efficacy and can not contribute to an anti-cancer patient immune response. Moreover, cancer vaccines generated by recombinant protein technology have the disadvantage that the target human polypeptides included within the recombinant protein can not fold properly, thereby preventing a useful immune response. Accordingly, there is a great need for new cancer vaccines that overcome these existing significant limitations in the field of cancer immunotherapy. SUMMARY

[0006] The present disclosure relates to chimeric synthetic proteins / molecules and their respective methods of making; the characterization of the chimeric synthetic proteins / molecules and methods of treatment using the chimeric synthetic proteins / molecules to treat chronic diseases such as lung cancer, breast cancer, bladder cancer, prostate cancer, ovarian cancer, vulvar cancer, colon cancer, colorectal cancer, intestinal cancer, lung cancer, brain cancer, esophageal cancer, other cancers, and other diseases.

[0007] The present disclosure provides chimeric synthetic proteins that can be used as a therapeutic method to treat diseases, such as cancer. In illustrative embodiments, the present disclosure provides a chimeric synthetic protein / molecule that includes one or more protein domains from a synthetic growth factor (e.g., VEGF), one or more linker sequence regions, and one or more immunogenic domains. In one aspect, the present disclosure provides a chimeric synthetic protein that includes a chimeric polypeptide sequence; at least one linker sequence; and a polypeptide sequence. Advantageously, the chimeric synthetic proteins / molecules described herein have the ability to function to stabilize the scaffold, better enabling the human protein (e.g., growth factor such as VEGF, EGF, TGF, etc.) incorporated into the protein / molecule to adopt a native conformation (e.g., properly fold) when expressed. Further, the chimeric synthetic proteins / molecules described herein have the ability to generate stable chimeric synthetic proteins / molecules with a longer shelf life.

[0008] In some embodiments, the polypeptide sequence includes an immunogenic polypeptide sequence.

[0009] In some embodiments, the polypeptide sequence includes a Cholera Toxin B (CT-B) protein.

[0010] In some embodiments, the at least one linker sequence includes a first linker sequence that separates the chimeric polypeptide sequence from the polypeptide sequence.

[0011] In some embodiments, the first linker sequence is selected from the group consisting of SSG, GSSG, SSGGG, SGG, GGSGG, GGGGS, SSGGGSGG, SSGGGGSGGG, TSGGGSG, TSGGGGSGG, SSGGSGGGSG, SSGGSGGSSG, GGSGGTSGGGSG, SGGTSGGGGSGG, GGSGGTSGGGGSGG, SSGGGGSGGGSSG, SSGGSGGSSGGG, and SSGGGGSGGGSSGGG.

[0012] In some embodiments, the first linker sequence is SSGGSGGGSG.

[0013] In some embodiments, the chimeric polypeptide sequence includes a vascular endothelial growth factor (VEGF) sequence.

[0014] In some embodiments, the chimeric polypeptide sequence includes a VEGF sequence selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and combinations thereof.

[0015] In some embodiments, the chimeric polypeptide sequence includes a first VEGF domain and a second VEGF domain. In some embodiments, the chimeric polypeptide sequence includes a first VEGF domain and a second VEGF domain.

[0016] In some embodiments, the first VEGF domain comprises VEGF-D or a portion thereof, and the second VEGF domain comprises VEGF-A or a portion thereof.

[0017] In some embodiments, the first VEGF domain is TFYDIETLKVIDEEWQRTQ and the second VEGF domain is CHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0018] In some 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.

[0019] In some embodiments, the chimeric polypeptide sequence binds to VEGFR-1, VEGFR-2, and VEGFR-3.

[0020] In some embodiments, the chimeric synthetic protein initially has the amino acid sequence of MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0021] In some embodiments, the initial chimeric synthetic protein is processed to have the amino acid sequence of TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0022] In another aspect, the present disclosure provides an immunogenic composition comprising a chimeric polypeptide sequence; at least one linker sequence; and a polypeptide sequence.

[0023] In some embodiments, the polypeptide sequence comprises an immunogenic polypeptide sequence.

[0024] In some embodiments, the polypeptide sequence comprises a Cholera toxin B (CT-B) protein.

[0025] In some embodiments, the at least one linker sequence comprises a first linker sequence separating the chimeric polypeptide sequence from the polypeptide sequence.

[0026] In some embodiments, the first linker sequence is selected from the group consisting of SSG, GSSG, SSGGG, SGG, GGSGG, GGGGS, SSGGGSGG, SSGGGGSGGG, TSGGGSG, TSGGGGSGG, SSGGSGGGSG, SSGGGSGGSSG, GGSGGTSGGGSG, SGGTSGGGGSGG, GGSGGTSGGGGSGG, SSGGGGSGGGSSG, SSGGSGGSSGGG, and SSGGGGSGGGSSGGG.

[0027] In some embodiments, the first linker sequence is SSGGSGGGSG.

[0028] In some embodiments, the chimeric polypeptide sequence comprises a vascular endothelial growth factor (VEGF) sequence.

[0029] In some 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.

[0030] In some embodiments, the chimeric polypeptide sequence comprises a first VEGF domain and a second VEGF domain.

[0031] In some embodiments, the first VEGF domain comprises VEGF-D or a portion thereof, and the second VEGF domain comprises VEGF-A or a portion thereof.

[0032] In some embodiments, the first VEGF domain is TFYDIETLKVIDEEWQRTQ, and the second VEGF domain is CHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0033] In some 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.

[0034] In some embodiments, the chimeric polypeptide sequence binds to VEGFR-1, VEGFR-2, and VEGFR-3.

[0035] In some embodiments, the synthetic protein chimeric synthetic protein initially has the amino acid sequence of MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0036] In some embodiments, the initial chimeric synthetic protein is treated to have the amino acid sequence of TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0037] In some embodiments, the immunogenic composition further comprises an adjuvant.

[0038] In another aspect, the present application provides a method of treating a patient in need thereof, the method comprising the step of administering to the patient the above-described immunogenic composition on the same day of vaccination or every other day or at a time period within the vaccination phase.

[0039] In some embodiments, the patient has cancer.

[0040] Definitions

[0041] By "Epidermal Growth Factor Receptor (EGFR) nucleic acid molecule" is meant a polynucleotide that encodes an EGFR polypeptide. One exemplary EGFR nucleic acid molecule is provided at NCBI Accession No. NM_005228.4, and reproduced below (SEQ ID NO: 3):

[0042] > NM_005228.4

[0043]

[0044] 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):

[0045] >NP_005219.2

[0046]

[0047] By "Epidermal Growth Factor (EGF) nucleic acid molecule" is meant a polynucleotide that encodes an EGF polypeptide. An exemplary EGF nucleic acid molecule is provided at NCBI Accession No. NM_001963.5, and reproduced below (SEQ ID NO: 5):

[0048] > NM_001963.5

[0049]

[0050] 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 and corresponding to the prepro-form of EGF, which is processed to generate a 53 amino acid EGF molecule (shown in bold) and having EGFR binding activity, reproduced below (SEQ ID NO: 6):

[0051] >NP_001954.2

[0052]

[0053] A“neuregulin 1 (NRG1) nucleic acid molecule” means a polynucleotide that encodes an NRG1 polypeptide. An exemplary NRG1 nucleic acid molecule is provided at NCBI Accession No. BC150609.1, and reproduced below (SEQ ID NO: 7):

[0054] >BC150609.1

[0055]

[0056] “Neuregulin 1 (NRG1) polypeptide” means 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):

[0057] > AAI50610.1

[0058] MSERKEGRGKGKGKKKERGSGKKPESAAGSQSPALPPQLKEMKSQESAAGSKLVLRCETSSEYSSLRFKWFKNGNELNRKNKPQNIKIQKKPGKSELRINKASLADSGEYMCKVISKLGNDSASANITIVESNEIITGMPASTEGAYVSSESPIRISVSTEGANTSSSTSTSTTGTSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCQPGFTGARCTENVPMKVQNQEKAEELYQKRVLTITGICIALLVVGIMCLVAYCKTKKQRKKLHDRLRQSLRSERNNMMNIANGPHHPNPPPENVQLVNQYVSKNVISSEHIVEREAETSFSTSHYTSTAHHSTTVTQTPSHSWSNGHTESILSESHSVIVMSSVENSRHSSPTGGPRGRLNGTGGPRECNSFLRHARETPDSYRDSPHSERYVSAMTTPARMSPVDFHTPSSPKSPPSEMSPPVSSMTVSMPSMAVSPFMEEERPLLLVTPPRLREKKFDHHPQQFSSFHHNPAHDSNSLPASPLRIVEDEEYETTQEYEPAQEPVKKLANSRRAKRTKPNGHIANRLEVDSNTSSQSSNSESETEDERVGEDTPFLGIQNPLAASLEATPAFRLADSRTNPAGRFSTQEEIQARLSSVIANQDPIAV

[0059] “Neuregulin 1 beta (NRG1 beta) nucleic acid molecule” means a polynucleotide encoding an NRG1 polypeptide. One exemplary NRG1 beta nucleic acid molecule is provided by NCBI Accession No. NM_001322205.1 and reproduced below (SEQ ID NO: 9):

[0060] >NM_001322205.1

[0061]

[0062] "Neuregulin 1 beta (NRG1 beta) polypeptide" means 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): >NP_001309134.1 MEIYSPDMSEVAAERSSSPSTQLSADPSLDGLPAAEDMPEPQTEDGRTPGLVGLAVPCCACLEAERLRGCLNSEKICIVPILACLVSLCLCIAGLKWVFVDKIFEYDSPTHLDPGGLGQDPIISLDATAASAVWVSSEAYTSPVSRAQSESEVQVTVQGDKAVVSFEPSAAPTPKNRIFAFSFLPSTAPSFPSPTRNPEVRTPKSATQPQTTETNLQTAPKLSTSTSTTGTSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKHLGIEFMEAEELYQKRVLTITGICIALLVVGIMCVVAYCKTKKQRKKLHDRLRQSLRSERNNMMNIANGPHHPNPPPENVQLVNQYVSKNVISSEHIVEREAETSFSTSHYTSTAHHSTTVTQTPSHSWSNGHTESILSESHSVIVMSSVENSRHSSPTGGPRGRLNGTGGPRECNSFLRHARETPDSYRDSPHSERYVSAMTTPARMSPVDFHTPSSPKSPPSEMSPPVSSMTVSMPSMAVSPFMEEERPLLLVTPPRLREKKFDHHPQQFSSFHHNPAHDSNSLPASPLRIVEDEEYETTQEYEPAQEPVKKLANSRRAKRTKPNGHIANRLEVDSNTSSQSSNSESETEDERVGEDTPFLGIQNPLAASLEATPAFRLADSRTNPAGRFSTQEEIQARLSSVIANQDPIAV

[0063] "NRG-BVN hybrid polypeptide" means 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):

[0064] >NRG-BVN-hybrid

[0065] GTSHLVKCPLSHEAYCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASF

[0066] “TGFa hybrid polypeptide” means 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):

[0067] >TGF-BVN-hybrid

[0068] NTENDCPLSHEAYCLHDGVCRFLVQEDKPACVCVVGYVGERCQFRDLRWWDAR

[0069] “Initial IN-02 polypeptide” means 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: 13): >Initial_IN-02_polypeptide MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG

[0070] “Processed or final IN-02 polypeptide” means 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: 14): >Final_IN-02_polypeptide

[0071] TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG

[0072] “Vascular endothelial growth factor A (VEGF-A) nucleic acid molecule” means a polynucleotide that encodes a VEGF-A polypeptide. An exemplary VEGF-A nucleic acid molecule is provided at NCBI Accession No. NM_001025366.3 and reproduced below (SEQ ID NO: 15):

[0073] NM_001025366.3

[0074]

[0075] “VEGF-A polypeptide” means a polypeptide encoded by a VEGF-A nucleic acid molecule. One exemplary VEGF-A nucleic acid molecule is provided at NCBI Accession No. NP_001020537.2, and is reproduced below (SEQ ID NO: 16):

[0076] >NP_001020537.2

[0077] MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGVALKLFVQLLGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEEKEEERGPQWRLGARKPGSWTGEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPPHSPSRRGSASRAGPGRASETMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR

[0078] “Vascular endothelial growth factor D (VEGF-D) nucleic acid molecule” means a polynucleotide that encodes a VEGF-D polypeptide. One exemplary VEGF-D nucleic acid molecule is provided at NCBI Accession No. NM_004469, and is reproduced below (SEQ ID NO: 17):

[0079] >NM_004469

[0080]

[0081] "VEGF-D polypeptide" means a polypeptide encoded by a VEGF-D nucleic acid molecule. An exemplary VEGF-D nucleic acid molecule is provided as NCBI Accession No. NP_004460.1 and reproduced below (SEQ ID NO: 18):

[0082] >NP_004460.1

[0083] MYREWVVVNVFMMLYVQLVQGSSNEHGPVKRSSQSTLERSEQQIRAASSLEELLRITHSEDWKLWRCRLRLKSFTSMDSRSASHRSTRFAATFYDIETLKVIDEEWQRTQCSPRETCVEVASELGKSTNTFFKPPCVNVFRCGGCCNEESLICMNTSTSYISKQLFEISVPLTSVPELVPVKVANHTGCKCLPTAPRHPYSIIRRSIQIPEEDRCSHSKKLCPIDMLWDSNKCKCVLQEENPLAGTEDHSHLQEPALCGPHMMFDEDRCECVCKTPCPKDLIQHPKNCSCFECKESLETCCQKHKLFHPDTCSCEDRCPFHTRPCASGKTACAKHCRFPKEKRAAQGPHSRKNP

[0084] Herein, ranges can be expressed as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Also, when the

[0085] Ranges provided herein are understood to be shorthand for all values within the range. For example, a range from 1 to 50 is understood to include any number, combination or sub-range of integers 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 tenths of a unit between the aforementioned integers, e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9. With respect to "sub-ranges," "nested sub-ranges" extending from either end of the range are specifically contemplated. For example, exemplary nested sub-ranges of the range 1 to 50 can include 1 to 10, 1 to 20, 1 to 30 and 1 to 40 in one direction, and 50 to 40, 50 to 30, 50 to 20 and 50 to 10 in the other direction.

[0086] Any of the embodiments described herein are contemplated to be combinable with any other one or more embodiments, even if the embodiments are described under different aspects of the disclosure, where applicable or not expressly disclaimed.

[0087] These and other embodiments are disclosed and / or encompassed by the following DETAILED DESCRIPTION. BRIEF DESCRIPTION OF DRAWINGS

[0088] The following DETAILED DESCRIPTION is presented in example routes, not intended to limit the disclosure solely to the specific embodiments described, which can be best understood in conjunction with the accompanying drawings, in which:

[0089] Figures 1A to 1D Depicts two protein schematics, a recombinant protein sequence and a bar graph, respectively. Figure 1A is a band graph protein schematic showing a chimeric VEGF molecule (VEGF-DA) according to exemplary embodiments of the disclosure, which includes an N-terminal region derived from VEGF-D, and indicates the "homology domains" (VEGFR-1, VEGFR-2 and VEGFR-3 binding regions) from VEGF-A. Figure 1B is a protein schematic showing the structure and organization of IN-02, in which the chimeric VEGF-DA protein domains are fused to the C-terminus of CTB via a 10 amino acid linker sequence according to exemplary embodiments of the disclosure. Figure 1C Depicts the protein sequence of IN-02, with colors coordinated to match Figures 1A to 1B The initiating methionine residue is removed by methionine amino peptidases, thus missing from the mature protein. Figure 1Dis a bar graph showing the effect of EGF-A, VEGF-D, and IN-02 alone or in combination with neutralizing antibodies (NAb) on the development of tubes formed by human endothelial cells (HUVEC).

[0090] Figure 2 is a bar graph depicting an ELISA showing the binding of VEGF-A, IN-02 (e.g., VEGF-DA), and VEGF-D proteins to VEGF receptors immobilized on a plate.

[0091] Figure 3 is a graph depicting an ELISA showing the binding of serum from three rabbits before immunization and after immunization with IN-02 protein (BL3) to immobilized immunogen.

[0092] Figure 4 is a graph depicting an ELISA showing the binding of serum from three rabbits before immunization and after immunization with IN-02 protein (BL3) to immobilized rCTB (purified with caprylic acid).

[0093] Figure 5 is a graph depicting an ELISA showing the binding of serum from three rabbits before immunization and after immunization with IN-02 protein (BL3) to immobilized VEGF-A.

[0094] Figure 6 is a graph depicting an ELISA showing the binding of serum from three rabbits before immunization and after immunization with IN-02 protein (BL3) to immobilized VEGF-D.

[0095] Figure 7 is a bar graph depicting the results of a tube formation assay performed using serum from rabbits immunized with IN-02 protein purified with caprylic acid.

[0096] Figure 8 is a bar graph depicting the results of a HUVEC tube formation assay performed on IN-02 protein that had been stored at 4°C for one month. DETAILED DESCRIPTION

[0097] The present disclosure is based, at least in part, on the discovery that chimeric synthetic proteins / molecules comprising one or more protein domains from a growth factor (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, etc.), one or more linker sequence regions, and one or more immunogenic domains can be used as therapeutic molecules for treating a variety of diseases, such as cancer. The chimeric synthetic molecules provide several unexpected advantages over prior art. For example, unlike prior art human epidermal growth factor (hEGF) molecules (e.g., U.S. Patent No. 5,984,018 to Davila et al.) that exist as a heterogeneous mixture containing as many as 12 different molecular species, the synthetic proteins / molecules described herein can be produced as a single molecule (e.g., a homogeneous population of molecules). Further, the synthetic proteins / molecules described herein comprise ten active components per molecule (but the active components can be increased or decreased in multiples of five, e.g., as part of a pentamer), whereas the number of active components present per molecule of prior art hEGF molecules (e.g., U.S. Patent No. 5,984,018 to Davila et al.) is highly variable (e.g., Davila has an average of 1.5 active components per molecule). Further, the chimeric synthetic proteins / molecules described herein are easier to manufacture. For example, prior art hEGF molecules (e.g., U.S. Patent No. 5,984,018) are produced by chemically conjugating rP64k with recombinant human EGF (rhEGF) to produce a final molecule that consists of two molecules chemically conjugated to each other. This is in stark contrast to the synthetic proteins / molecules described herein, which are single synthetic molecules. Further, the chimeric synthetic proteins / molecules described herein have the ability to function to stabilize the scaffold, better enabling the human proteins (e.g., growth factors) incorporated into the protein / molecule to adopt a native conformation (e.g., properly fold) when expressed. Further, the chimeric synthetic proteins / molecules described herein have the ability to generate stable chimeric synthetic proteins / molecules with a longer shelf life. Advantageously, the technology herein provides novel chimeric synthetic proteins that can be used therapeutically to treat diseases, such as cancer, at a higher level of immunogenic activity than prior art methods (e.g., U.S. Patent No. 5,984,018).

[0098] SUMMARY

[0099] Cancer immunology is the study of the interactions between the immune system and cancer cells, such as tumors or malignancies. Of particular interest is the initiation of an immune response, such as the recognition of cancer-specific antigens that are expressed by human tumors but not in normal tissues. Generally, approaches to control malignant cell division and proliferation focus on isolating these antigens and presenting them so that they are recognized by the immune system as non-self antigens to induce a specific immune response.

[0100] A large number of growth factors are currently identified, and most, if not all, have been shown to be important mediators of cell proliferation in various cancers in addition to involvement in other disease conditions. Typically, growth factors are soluble serum proteins that recognize and bind to a set of growth factor receptors located on the cell surface. Particular growth factors can be specific for a single receptor, or can bind with varying affinities to more than one closely related receptor. Similarly, some receptors bind only to a single growth factor ligand, while others can bind to multiple related growth factors, again often with different affinities. When bound to their natural receptors, the cytoplasmic domains of the receptors are phosphorylated, and this initiates intracellular signaling cascades leading to the regulation of one or more gene transcription and ultimately progression through the cell cycle and cell proliferation.

[0101] Growth factors and their receptors are essential components of normal processes of growth, development and repair, and their tissue distribution and expression levels are tightly regulated to control cell growth. Numerous studies have demonstrated that growth factors can stimulate 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)). Furthermore, certain growth factors have been shown to stimulate proliferation of some cancer cell lines. For example, epidermal growth factor (EGF) can stimulate some non-small cell lung cancer cells (Osborne C.K. 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 neoplastic 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)).

[0102] It has been reported that high levels of various growth factor receptors are present in malignant tissue. For example, high levels of epidermal growth factor receptor (EGFR) are often detected in malignancies of epidermal origin such as lung, breast, bladder, ovarian, vulvar, colon, lung, brain and esophageal cancers. The role played by growth factors and their receptors in regulating tumor growth is not well understood, but it is thought that expression of growth factor receptors in tumor cells provides a mechanism for autocrine growth stimulation, leading to uncontrolled proliferation (Schlessinger J., Schreiber A.B., Levi A., Liberman T., Yarden Y. Crit. Rev. Biochem. 1983, 14(2) 93-1 1 1 ). Furthermore, Liao Y et al; Hum Pathol 36(1 1 ):1 186-1 196 (2005) and Cox ME et al; Prostate: 69(1 ) 33-40 (2009) describe the role of increased pancreatic islet receptors and growth factors in metastatic prostate cancer.

[0103] One therapeutic strategy to target growth factor signaling in cancer therapy has been to use passive immunotherapy (e.g., monoclonal antibodies) directed against one or more specific receptors implicated. Such studies have demonstrated that specific recognition of antibodies to receptors capable of inhibiting ligand binding can have an inhibitory effect on mitogenic stimulation of malignant cells (SATO J.D., et al. Methods in Enzymology, vol. 146 pp 63-81, 1987). However, antibodies of murine origin will often generate a human anti-murine antibody response (HAMA), thus limiting their use to a single administration.

[0104] Other therapeutic strategies are active immunotherapy using vaccines containing the growth factor of interest to induce an immune response against the molecule to inhibit the proliferative effect of the growth factor on the tumor. U.S. Patent No. 5,984,018 to 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" discloses, for example, the use of vaccines containing a mixture of growth factors chemically conjugated together using glutaraldehyde with immunogenic (i.e., non-human) carrier proteins. However, without being bound to any particular theory, it is believed that chemical conjugation hinders the immune response against the vaccine.

[0105] This is a technically challenging approach because it requires the host to mount an immune response to "self antigens" that the immune system of vertebrates has evolved to prevent. When a strong immune response is mounted against self antigens (e.g., including antigens that involve activation of helper T cells), it often leads to an autoimmune disease state. It has been hypothesized for many years that some autoimmune conditions, such as lupus, multiple sclerosis (MS), diabetes, etc., can be caused by early exposure to environmental agents that include immunogenic epitopes (T cell epitopes) that closely resemble the host's own epitopes. This can lead to stimulation of helper T cells that can cross-react with the host epitopes. Subsequent exposure to the environmental agent would then lead to an anti-self immune response (Albert, L.J., and Inman, R.D New England Journal of Medicine, Dec. 30th pp 2068-2074, 1999). It has been shown that, in fact, viral antigens can generate anti-self immune responses against neural cell proteins (Levin, M.C. et. al, Nature Medicine vol 8(5) pp 509-513, 2002).

[0106] U.S. Patent Publication No. 2006 / 0251654 ('654 Publication) entitled "Method for Treatment of Malignant and Infectious Chronic Diseases" to Casimiro et al. discloses a method of treating a subject suffering from a malignant or infectious chronic disease, comprising a method of immunizing the subject with a vaccine containing a self-antigen associated with the malignant or infectious chronic disease conjugated to a carrier protein; treating the subject with an immunomodulator; and immunizing the subject with the vaccine of step 1 and an appropriate adjuvant selected from the group consisting of aluminum hydroxide and Montanide ISA 51 (Seppic, Paris, France). Unfortunately, the preparation of the vaccine by chemical conjugation is believed to hinder the immune response.

[0107] Most of the above vaccines exhibit a number of limitations, which are primarily derived from the manufacturing process and the potential lack of homogeneity and identity of the protein product. The above vaccines typically comprise a mixture of recombinant carrier proteins and human polypeptides using glutaraldehyde chemical conjugation. Unfortunately, this reagent can have an undesirable tendency to form covalent cross-links between a variety of chemical groups and typically results in a highly heterogeneous product. Thus, the resulting cancer vaccine can not only comprise carrier protein molecules with a number of target human polypeptides (e.g., 0, 1, 2, 3, etc.) attached, but the human polypeptides can each be attached to the carrier through different atoms and thus exist in different positions and different orientations. Furthermore, both the target polypeptides and the carrier protein molecules can be conjugated to themselves, resulting in a variety of homomultimers, which can have no clinical efficacy and can not contribute to an anti-cancer patient immune response.

[0108] Synthetic protein / molecule

[0109] The present disclosure provides a homogenous synthetic protein molecule for improving the maximum number of presentation of growth factor epitopes, tumor antigen epitopes, and / or receptor binding sites as an immunogenic synthetic protein / molecule element. In one exemplary embodiment, a synthetic protein / molecule is described that expresses all or a portion of an immunogenic carrier domain (e.g., Cholera Toxin B (CT-B)) and a synthetic Epidermal Growth Factor (sEGF), tumor antigen, and / or receptor. In alternative exemplary embodiments, the protein can 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 protein / molecules can 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 high expression levels and ease of manufacture, oral stability and ability to cross from the digestive tract to the bloodstream, and / or prior safe use in humans.

[0110] In illustrative embodiments, the synthetic proteins / molecules disclosed herein can include or express a high proportion of protein sequences derived from a target self-antigen as a function of total molecular weight. For example, this can be achieved using a large protein model containing multiple growth factor epitopes. These growth factor epitopes can be multiple copies of a single growth factor, either in whole or in part, or copies of more than one different growth factor, either in whole or in part. These growth factor epitopes can be naturally occurring or synthetic (e.g., artificial). For example, BVN22E (also referred to as IN01) is an illustrative synthetic protein described herein that can have a molecular weight of about 120 kD. In illustrative embodiments, the growth factor epitopes described herein can correspond to one or more domains of the growth factor (e.g., the targeting signaling pathway (TSP) domain of EGF). In illustrative embodiments, the EGF domain can include a region that presents or constrains a beta-loop, for example, a region defined by about cysteine 6 to about cysteine 42, 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. Figure 1A Without being bound by any particular theory, it is contemplated within the scope of the present disclosure that different regions or sub-regions between cysteine 6 and cysteine 42 can have beneficial effects when incorporated within the synthetic proteins / molecules of the present disclosure. For example, the following regions can have beneficial effects: the region between cysteine 6 and cysteine 14, the region between cysteine 6 and cysteine 20, the region between cysteine 6 and cysteine 31, the region between cysteine 6 and cysteine 33, and the region between cysteine 6 and cysteine 42. It is also contemplated within the scope of the present disclosure that reverse progression sequences can also be beneficial. For example, the following regions can 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 disclosure that specific spacing within the region between cysteine 6 and cysteine 42 can provide beneficial effects when incorporated within 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 42).

[0111] According to the present disclosure, expression of growth factor epitopes should be folded such that their native conformation is substantially preserved and presented to components of the host immune system in a manner that elicits a robust host immune response to the epitopes. Examples of suitable natural protein models to model epitope support 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, listeriolysin, tetanus toxoid, diphtheria toxoid, meningococcal outer membrane protein, phage coat protein, adenovirus and other viral coat proteins. Alternatively, the non-self component of the protein can be small. At a minimum, the non-self sequence should comprise a length of about 9, 10, 11, or more amino acids and comprise, in whole or in part, at least one T cell epitope. As described herein, non-natural synthetic polypeptides (e.g., BVN22E, IN01) can be used to satisfy the need to render the complete protein immunogenic and allow for proper presentation of the growth factor, receptor, tumor antigen, or epitope thereof to the host immune system.

[0112] According to the present disclosure, the synthetic proteins / molecules provided herein, whether a growth factor or portion thereof, a cell receptor or portion thereof, or a tumor antigen or portion thereof, are related to a number of cellular pathways implicated in chronic diseases, growth factor- or receptor-based cancers, and / or solid tumors, and are used as the tumor antigen in the synthetic protein. The proteins are in the form of synthetic proteins / molecules and can be used to treat chronic diseases, for example, 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. Because different tumor antigens can be expressed and a variety of cell receptors and growth factors are overexpressed in the disease, the proteins described below can contain one or more different tumor antigens, one or more different receptors, or one or more cellular pathways related to the disease. These proteins are referred to as multivalent.

[0113] In exemplary embodiments, a protein is disclosed that is comprised of a homogenous synthetic protein / molecule expressing one or more epidermal growth factor (EGF) neutralizing domains (e.g., TSP domains). The protein can be in the form of a synthetic protein / molecule and can be used to treat chronic diseases, for example, 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 exemplary embodiments, the protein is a synthetic protein / molecule expressing or comprising a synthetic EGF sequence and a CT-B sequence, such as Figure 1AIn illustrative embodiments, the growth factor component of the synthetic protein sequence can include a sequence that is less than 80% identical to EGF. For example, the growth factor component can include an EGF sequence with 11 amino acid substitutions that increase the immunogenicity of the growth factor portion of the synthetic protein sequence. Without being bound by theory, it is believed that the EGF "presenting" or constraining the beta-loop (e.g., the region defined by Cys6 to Cys31) can be important to include in the synthetic protein and suitable as a target for amino acid modification. In illustrative embodiments, regions outside of Cys6 to Cys31 can also be targets for modification (e.g., El 1 and A12).

[0114] In illustrative embodiments, the TSP1 and TSP2 domains of hEGF can be modified as shown in Figure 1B to create a synthetic EGF (sEGF) region to be included in the synthetic proteins / molecules herein.

[0115] In illustrative embodiments, the synthetic proteins / molecules disclosed herein can include all or a portion of a growth factor including, but not limited to, for example, Neuregulin 1 beta (NRG1 beta), Transforming Growth Factor alpha (TGFa), Vascular Endothelial Growth Factor (VEGF), and the like.

[0116] In other illustrative embodiments, the synthetic proteins / molecules described herein can include one or more linker or spacer sequences. The above-described one or more 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 sequence. These resulting recombinant or chimeric proteins include sEGF directly fused to CT-B. In other illustrative embodiments, the EGF component of the chimeric protein is effectively separated from the CT-B component by 3 to 14 amino acids that form a flexible spacer or linker sequence between the two domains. Within the scope of the present disclosure, it is contemplated that the linker or spacer sequence can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids in length. In some cases, where the growth factor is of a larger size (e.g., human growth factor), it can be useful to use a longer linker sequence. The following exemplary linker sequences can be used, and include, but are not limited to, the following: SSG, SSGG, SGG, GSSG, GGSGG, GGGGS, SSGGGSGG, SSGGGGSGGG, TSGGGSG, TSGGGGSGG, SSGGGSGGSSG, GGSGGTSGGGSG, SGGTSGGGGSGG, GGSGGTSGGGGSGG, SSGGGGSGGGSSG, SSGGSGGSSGGG, and SSGGGGSGGGSSGGG. Those skilled in the art will appreciate that there are many other sequences that are primarily “G” and “S” that can also serve as useful linker sequences.

[0117] Without being bound by any particular theory, it is contemplated 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 the appropriate polypeptides that are correctly folded and functional. Further, the synthetic proteins / molecules disclosed herein are capable of forming pentamers. Further, the synthetic proteins / molecules disclosed herein have a much lower advantageous property of the level of protein needed to vaccinate, as the amount of carrier necessary is significantly lower than prior art methods (e.g., U.S. Patent No. 5,984,018 to Davila et al.). In this regard, the synthetic proteins / molecules disclosed herein are capable of delivering more growth factor to a patient in a significantly lower vaccine volume.

[0118] Adjuvants

[0119] Certain exemplary embodiments as provided herein include synthetic proteins / molecules according to the present disclosure within vaccine compositions and immunoadjuvant compositions, including pharmaceutical compositions, which in addition to containing a synthetic protein / molecule, also contain at least one adjuvant, which refers to a component of such a composition that has adjuvant activity. Adjuvants that have such adjuvant activity include a composition that, when administered to a subject such as a human (e.g., a human patient), a non-human primate, a mammal, or other higher eukaryote having a recognized immune system, is capable of altering (i.e., increasing or decreasing, and in certain preferred embodiments, enhancing or increasing, in a statistically significant manner) the potency and / or longevity of an immune response. In certain exemplary embodiments disclosed herein, a desired antigen or antigens contained within a protein carrier and optionally one or more adjuvants contained can thus alter (e.g., elicit or enhance) an immune response against a desired antigen or antigens that can be administered simultaneously or can be administered separately in time and / or space (e.g., at different anatomical sites), although certain exemplary embodiments do not seek to be so limited and thus also contemplate administration of a synthetic protein / molecule in a composition that does not include a specified antigen but which can include, but is not limited to, one or more ancillary adjuvants (an imidazoquinoline immune response modifier).

[0120] Accordingly and as noted above, adjuvants include compositions having adjuvant effects, such as saponins and saponin mimetics, including QS21 and QS21 mimetics (see, e.g., U.S. Patent No. 5,057,540, EP 0 362 279 Bl, WO 95 / 17210); alum; plant alkaloids such as tomatine; detergents such as, but not limited to, saponin, polysorbate 80, Span 85, and stearyl tyrosine; one or more cytokines (e.g., GM-CSF, IL-2, IL-7, IL-12, TNF-a, IFN-g), imidazoquinoline immune response modifiers, and double stem loop immune modifiers (dSLIMs, e.g., Weeratna et al, 2005 Vaccine 23:5263).

[0121] Detergents including saponins are described in, for example, U.S. Patent No. 6,544,518, Lacaille-Dubois, M and Wagner H. (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 0 362 279 Bl. Particulate structures comprising a fractionation of Quil A (saponin) known as immunostimulating complexes (ISCOMS) are hemolytic and have been used to manufacture vaccines (Morein, B., EP 0 109 942 Bl). These structures have been reported to have adjuvant activity (EP 0 109 942 Bl; WO 96 / 11711). The hemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A) have been described as potential systemic adjuvants, and methods of their production are disclosed in U.S. Patent No. 5,057,540 and EP 0362 279 Bl. The use of QS7 (a non-hemolytic fraction of Quil-A) as a potential adjuvant for systemic vaccination is also described in these references. The use of QS21 is further described by Kensil et al. (1991. J. Immunology 146:431-437). Combinations of QS21 with polysorbate or cyclodextrin are also known (WO 99 / 10008). Particulate adjuvant systems comprising fractions of Quil A 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 those derived from other plant species such as Gypsophila and Saponaria fabrics (Bomford et al, Vaccine, 10(9):572-577, 1992). Aescin is another detergent related to saponins that can be combined with the adjuvant compositions used in the embodiments disclosed herein. Aescin is described in the Merck Index (12th edition, entry 3737) as a mixture of saponins occurring in the seeds of Aesculus hippocastanum, the European horse chestnut. Its isolation and purification were carried out by chromatography (Fiedler, Arzneimittel-Forsch. 4, 213 (1953)) and by ion exchange resins (Erbring et al, U.S. Pat. No. 3,238,190).Fractionated extracts of esculin have been purified and shown to have biological activity (Yoshikawa M, et al. (Chem Pharm Bull (Tokyo) 1996 August; 44(8): 1454-1464)). Digoxin is another detergent, also described as a saponin in the Merck Index (12th edition, entry 3204), which is purified from the seeds of Digitalis purpurea and according to procedures described in Gisvold et al, J. Am. Pharm. Assoc., 1934, 23, 664 and Rubenstroth-Bauer, Physiol. Chem., 1955, 301, 621.

[0122] Other adjuvants or co-adjuvants for use in accordance with the presently disclosed embodiments include block copolymers or biodegradable polymers, which refer to a class of polymeric compounds familiar to those of ordinary skill in the relevant art. Block copolymers or biodegradable polymers that can be included in vaccine compositions or immunological adjuvants include Ribi® SM.L121 (BASF Corp., Mount Olive, N.J.; see, e.g., Yeh et al, 1996 Pharm. Res. 13: 1693).

[0123] Certain further exemplary embodiments contemplate immunological adjuvants including, but not limited to, oils, which in some such embodiments can facilitate co-adjuvant activity, and in other such embodiments can additionally or alternatively provide a pharmaceutically acceptable carrier or excipient. Any number of suitable oils are known in light of the present disclosure, and can be selected for inclusion in vaccine compositions and immunological adjuvant compositions. By way of example but not limitation, examples of such oils include squalene, squalane, mineral oil, olive oil, cholesterol, and mannitol monooleate.

[0124] Immune response modifiers such as pyrazoloquinoline immune response modifiers are also known in the art, and can also be included as adjuvants or co-adjuvants in certain embodiments of the present disclosure.

[0125] As also noted above, one type of adjuvant or co-adjuvant for use in the vaccine compositions according to the disclosure described herein can be an aluminum co-adjuvant, which are generally referred to as "alum." Alum co-adjuvants are based on aluminum hydroxide; aluminum hydroxyphosphate; or various suitable salts. Alum co-adjuvants have advantages because they have a good safety record, increase antibody responses, stabilize antigens, and are relatively simple to produce on a large scale (Edelman 2002 Mol. Biotechnol. 21 : 129-148; Edelman, R. 1980 Rev. Infect. Dis. 2: 370-383).

[0126] Pharmaceutical compositions

[0127] In certain illustrative embodiments, the pharmaceutical composition is a vaccine composition comprising a synthetic protein / molecule according to the disclosure and can further comprise one or more components, as provided herein, selected from TLR agonists, co-adjuvants (including, for example, cytokines, imidazoquinoline immune response modifiers, and / or dSLIM), and the like, and / or recombinant expression constructs, in combination with a pharmaceutically acceptable carrier, excipient, or diluent.

[0128] The illustrative carriers will be nontoxic to the recipient at the dosages and concentrations employed. For vaccine compositions comprising a synthetic protein / molecule, administration will typically be about 0.01 pg / kg to about 100 mg / kg body weight by intradermal, subcutaneous, intramuscular, or intravenous routes, or by other routes.

[0129] It will be apparent to those skilled in the art that the amount and frequency of administration will depend on the response of the host. "Pharmaceutically acceptable carriers" for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, sterile saline and phosphate-buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and a esters of p-hydroxybenzoic acid can be added as preservatives. In addition, antioxidants and suspending agents can be used.

[0130] The pharmaceutical composition can be in any form which allows the composition to be administered to a patient. For example, the composition can be in the form of a solid, liquid, or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral (e.g., sublingual or buccal), sublingual, rectal, vaginal, and intranasal (e.g., as a spray). As used herein, the term parenteral includes iontophoresis, sonophoresis, passive transdermal, microneedle administration, as well as subcutaneous injections, intravenous, intramuscular, intrasternal, intrasinus, intrathecal, intravaginal, intraurethral injections and infusion techniques. In particular embodiments, the compositions described herein (including vaccines and pharmaceutical compositions) are administered by a technique selected from iontophoresis, microcavitation, sonophoresis, or microneedling.

[0131] The pharmaceutical composition is formulated to allow the active ingredient(s) it contains to be bioavailable when the composition is administered to a patient. Compositions that will be administered to a patient are in unit dosage form, one or more dosage units being present in a package, for example, a tablet can be a single dosage unit, and a container of aerosol form of one or more compounds of the invention can hold a plurality of dosage units.

[0132] For oral administration, excipients and / or binders can be present. Examples are sucrose, kaolin, glycerin, starch dextrin, sodium alginate, carboxymethylcellulose, and ethyl cellulose. Colorants and / or flavorants can be present. Coating shells can be employed.

[0133] The composition can be in liquid form, for example, an elixir, syrup, solution, emulsion, or suspension. As two examples, the liquid can be for oral administration or for delivery by injection. When intended for oral administration, preferred compositions contain one or more of a sweetening agent, preservatives, dye / colorant, and flavoring agent. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, and isotonizing agent can be included.

[0134] A liquid pharmaceutical composition, as used herein, whether in the form of a solution, suspension, or other like form, can include, as one or more of the vehicle or excipient, sterile diluent such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as squalene, squalane, mineral oil, mannide oleic ester, cholesterol, and / or synthetic glycerol esters of fatty acids, polyethylene glycols, glycerine, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The injectable pharmaceutical composition is preferably sterile.

[0135] In particular embodiments, the pharmaceutical or vaccine composition of the present application comprises a stable aqueous suspension of at least 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.

[0136] It can also 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. Additional immunostimulatory substances (co-adjuvants) for use in such vehicles are also described above, and can include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), dextran, IL-12, GM-CSF, gamma interferon, and IL-12.

[0137] While any suitable carrier known to those of ordinary skill in the art can be used in the pharmaceutical compositions of the present application, the type of carrier will vary depending on the mode of administration and whether sustained release is desired. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, fats, waxes, or buffers. For oral administration, any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate can be employed. Biodegradable microspheres (e.g., polylactic galactlic acid) can also be employed as carriers for the pharmaceutical compositions of the present application.

[0138] The pharmaceutical compositions can also contain diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates (including glucose, sucrose or dextrins), chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are exemplary appropriate diluents. Preferably, the product can be formulated as a lyophilizate using an appropriate excipient solution (e.g., sucrose) as a diluent.

[0139] In exemplary embodiments, the epitope or receptor supporting domain of the synthetic protein / molecule, whether derived from a natural or synthetic polypeptide sequence, should have the ability to self-assemble into an oligomeric multimer under appropriate chemical / environmental conditions or the ability to reduce to a monomer under alternative conditions. Ideally, the multimerization domain will assemble into stable multimers having a small number of subunits, e.g., dimers, trimers, tetramers, pentamers, etc., such that a product of uniform size 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 coat proteins.

[0140] In exemplary embodiments, a process for making a multivalent molecule is disclosed. In this exemplary embodiment, the process includes assembling a multimer from monomeric subunits to form a synthetic protein including one or more tumor antigens, receptors and / or growth factors or portions thereof.

[0141] In another exemplary embodiment, a process for making a vaccine formulation is disclosed. In this exemplary embodiment, the process includes mixing one or more single monovalent multimers together to make a multivalent vaccine including a synthetic protein / molecule including one or more tumor antigens, receptors and / or growth factors or portions thereof.

[0142] In yet another exemplary embodiment, a process for treating a patient is disclosed. In this exemplary embodiment, the process includes administering to the patient one or more monovalent single tumor antigens, receptors and / or growth factors, recombinant proteins, independently, on the same day or one day apart or multiple times during a vaccination period.

[0143] Although the synthetic protein / molecule is described as including or expressing all or a portion of at least one sequence of a tumor antigen, growth factor and / or receptor and one or more of the CT-B sequences, the synthetic protein / molecule can include a native CT-B sequence or a sequence substantially similar to a native CT-B sequence and / or a synthetic sequence. Although the synthetic protein / molecule is described as including or expressing a CT-B sequence, the synthetic protein / molecule can include or express a derivative sequence of a CT-B sequence or a sequence substantially similar to a CT-B sequence.

[0144] Although the mean synthetic protein / molecule expressing or incorporating one or more tumor antigens, synthetic growth factors and / or receptors has been described and exemplified with respect to certain embodiments, many variations and modifications will be apparent to those skilled in the art and can be made without departing from the spirit and scope of the disclosure. Accordingly, the disclosure is not limited to the precise details of the methods or constructions set forth above, as such variations and modifications are intended to be included within the scope of the disclosure.

[0145] EMBODIMENTS

[0146] The disclosure is further illustrated by the following examples, which should not be considered as limiting. The contents of all references applied throughout this application, GenBank accession numbers and gene numbers, and published patents and patent applications are incorporated herein by reference. Those skilled in the art will appreciate that the disclosure can be practiced with variations of the disclosed structures, materials, compositions, and methods, and such variations are considered to be within the scope of the disclosure.

[0147] Example 1: Bispecific Chimeric Antigens

[0148] One problem that arises when a human protein (e.g., a growth factor) or portion thereof is used in conjunction with an immunogenic carrier molecule, such as the cholera toxin B subunit (CTB), to create a recombinant protein is that the human protein (e.g., a human growth factor) does not always fold into the correct native conformation. The ability of the human protein within the recombinant protein to fold correctly can vary significantly between different proteins, even between closely related molecules. For example, epidermal growth factor (EGF) can be readily folded correctly from insoluble inclusion bodies and is very stable thereafter; however, the EGF-like domain of transforming growth factor alpha (TGFa) and neuregulin are more difficult to produce in the proper folded form and are also significantly less stable.

[0149] Vascular endothelial growth factor (VEGF) comprises four structurally related proteins, VEGF-A, VEGF-B, VEGF-C and VEGF-D, which mediate signaling through three receptors, VEGFR-1, VEGFR-2 and VEGFR-3. VEGF-A and VEGF-D signal 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, thus showing both similarities and differences in their receptor binding characteristics. All VEGF growth factors share a structurally conserved "homology domain" which is involved in recognition and binding to VEGFR-1 and VEGFR-2 (VEGF-A, VEGF-B and VEGF-C) and comprises sequences downstream of the first cysteine residue. The N-terminus of VEGF-A and VEGF-B, i.e. 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.

[0150] 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 has a similar amenability to refolding from inclusion bodies, but, after only one week at 4°C, its stability is much poorer and shows clear signs of degradation, which makes it unsuitable for therapeutic use in its native form. VEGF-C is very difficult to fold correctly, either from inclusion bodies or when expressed as a soluble protein in bacteria.

[0151] In the native proteins, the N-terminal region of VEGF-C and VEGF-D (sequences upstream of the first cysteine residue) forms an alpha-helix which interacts with VEGFR-3. This structure also needs to interact with other parts of the VEGF molecule to adopt and maintain this conformation. When expressed alone or as a fusion with "unrelated" carriers, the resulting proteins do not exhibit any binding to VEGFR-3. However, when the N-terminal domain of VEGF-A is replaced with the N-terminus of VEGF-D, as shown in Figure 1A VEGF-A, the resulting protein can bind to all three VEGF receptors and modulate three independent signaling pathways. Thus, the VEGF-A domain acts as a stabilizing scaffold, making the N-terminal domain of VEGF-D available and maintaining its native structure. The stable bispecific chimeric VEGF, comprising sequences from both VEGF-D and VEGF-A, is further fused to the C-terminus of CTB, separated by a 10 amino acid glycine / serine rich flexible linker sequence. This molecule is designated IN-02 and is shown schematically in Figure 1BThe protein sequence of IN-02 is shown in Figure 1C

[0152] To analyze the functional characteristics of the VEGF-based molecules, two assays were employed: a tube formation assay (TFA) and an ELISA. The TFA involves culturing human umbilical vein endothelial cells (HUVECs) and observing the development of "tubes" that present capillary formation over time. Tube formation by cells cultured with stimulatory modulators (growth factors) and inhibitory modulators (neutralizing antibodies) is then compared to those that were untreated. For the ELISA, recombinant VEGFRs (extracellular domains of VEGF receptors fused to human IgG Fc regions) are coated on ELISA plates. The plates are incubated with VEGF proteins, and bound VEGF is detected using protein-specific antibodies.

[0153] Figure 1D The effect of growth factors and neutralizing antibodies (Nabs) on tube development (angiogenesis) by human endothelial cells (HUVECs) is shown. Both VEGF-A and VEGF-D are able to independently stimulate tube formation (white and gray bars), and this stimulation is prevented by the addition of neutralizing antibodies against each growth factor. The chimeric VEGF-DA protein is also able to stimulate tube formation (first hatched bar). This stimulation can be partially inhibited by the addition of neutralizing antibodies against VEGF-A and VEGF-D alone, and more completely inhibited when both antibodies are added. Figure 1D

[0154] Figure 2 ELISA data is depicted showing the binding of VEGF proteins to VEGF receptors immobilized on plates. Recombinant human VEGF-A binds to receptor 1 and receptor 2 (left bars). VEGF-D binds to receptor 3 and receptor 2 (right bars). The chimeric IN-02 protein binds to receptor 1, receptor 2, and receptor 3. The binding of IN-02 to receptor 2 is only detected when an anti-VEGF-A antibody is used (gray bars), as both receptor 2 and the anti-VEGF-D antibody bind to the same region of the chimeric protein. Three rabbits were immunized with 100 μg of IN-02 (s.c.) on days 0, 14, 28, and 56. Bleeds were taken on day 0 (pre-immune), day 2, day 3, and day 56. For clarity, only pre-immune and bleed 3 data are shown.

[0155] Figure 3 ELISA data is depicted showing pre-immune and post-immunization (BL3) sera from three rabbits immunized with IN-02 protein bound to the immobilized immunogen, which clearly indicates that all three rabbits mounted an immune response to the immunogen and had no reactivity prior to immunization. ​​

[0156] Figure 4 ELISA data is plotted showing binding of sera from three rabbits before and after immunization with IN-02 protein (BL3) to immobilized rCTB. All three rabbits produced an immune response to the CTB domain of the immunogen and reacted with rCTB. There was no reactivity before immunization.

[0157] Figure 5 ELISA data is plotted showing binding of sera from three rabbits before and after immunization with IN-02 protein (BL3) to immobilized VEGF-A. All three rabbits produced an immune response to the VEGF-A domain of the immunogen and also reacted with rhVEGF-A. There was no reactivity before immunization.

[0158] Figure 6 ELISA data is plotted showing binding of sera from three rabbits before and after immunization with IN-02 protein (BL3) to immobilized VEGF-D. All three rabbits produced an immune response to the VEGF-D domain of the immunogen and also reacted with rhVEGF-D. There was no reactivity before immunization. After immunization of the rabbits with IN-02 protein, all animals generated an immune response to the immunizing antigen and this antigen was able to recognize both full-length native rhVEGF-A and rhVEGF-D in addition to the immunogenic CTB "carrier" domain.

[0159] To determine the effectiveness of this immune response in neutralizing signaling induced by VEGF-A and VEGF-D, HUVEC tube formation assays were performed as described earlier. Figure 7 Results of tube formation assays performed using sera from rabbits immunized with IN-02 protein purified with caprylic acid are shown. All three sera significantly inhibited tube formation induced by co-stimulation with VEGF-A and VEGF-D (black bars) performed simultaneously.

[0160] Figure 8 Results of HUVEC tube formation assays performed on IN-02 protein that had been stored at 4°C for one month are shown. Despite the storage, the IN-02 protein still stimulated tube formation to a similar extent as VEGF-A or VEGF-D and this stimulation would be effectively inhibited by incubation with an antibody donor that is able to neutralize both VEGF-A and VEGF-D.

[0161] incorporated by reference

[0162] All documents cited or referenced in this text, and all documents cited or referenced in documents cited or referenced in this text, are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Any manufacturer's instructions, illustrations, or descriptions of any product used in this text are hereby incorporated by reference in their entirety for all purposes.

[0163] equivalents

[0164] It is to be understood that the detailed description and embodiments are merely exemplary for illustrative purposes rather than a limitation on the scope of the disclosure. Various modifications and changes can be made thereto by those skilled in the art which fall within the scope and spirit of the disclosure and it is intended that all such possible modifications and changes be within the scope of the application and be encompassed by the appended claims. Additional advantages and features of the disclosure related to its systems, methods, and processes will become apparent in view of the following detailed description. Moreover, those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described herein. Such equivalents are considered to be within the scope of the application.

Claims

1. A chimeric synthetic protein comprising: a chimeric polypeptide sequence; at least one linker sequence; and a polypeptide sequence.

2. The chimeric synthetic protein of claim 1, wherein the polypeptide sequence comprises an immunogenic polypeptide sequence.

3. The chimeric synthetic protein of claim 1, wherein the polypeptide sequence comprises a cholera toxin B protein.

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

5. The chimeric synthetic protein of claim 4, wherein the first linker sequence is selected from the group consisting of SSG, GSSG, SSGGG, SGG, GGSGG, GGGGS, SSGGGSGG, SSGGGGSGGG, TSGGGSG, TSGGGGSGG, SSGGSGGGSG, SSGGSGGSSG, GGSGGTSGGGSG, SGGTSGGGGSGG, GGSGGTSGGGGSGG, SSGGGSGGGSSG, SSGGSGGSSGGG, and SSGGGGSGGGSSGGG.

6. The chimeric synthetic protein of claim 4, wherein the first linker sequence is SSGGSGGGSG.

7. The chimeric synthetic protein of claim 1, wherein the chimeric polypeptide sequence comprises a vascular endothelial growth factor sequence.

8. The chimeric synthetic protein of claim 1, wherein the chimeric polypeptide sequence comprises a vascular endothelial growth factor sequence selected from the group consisting of vascular endothelial growth factor-A, vascular endothelial growth factor-B, vascular endothelial growth factor-C, vascular endothelial growth factor-D, and combinations thereof.

9. The chimeric synthetic protein of claim 8, wherein the chimeric polypeptide sequence comprises a first vascular endothelial growth factor domain and a second vascular endothelial growth factor domain.

10. The chimeric synthetic protein of claim 9, wherein the first vascular endothelial growth factor domain comprises vascular endothelial growth factor-D or a portion thereof, and the second vascular endothelial growth factor domain comprises vascular endothelial growth factor-A or a portion thereof.

11. The chimeric synthetic protein of claim 10, wherein the first vascular endothelial growth factor domain is TFYDIETLKVIDEEWQRTQ, and the second vascular endothelial growth factor domain is CHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

12. The chimeric synthetic protein of claim 9, wherein the chimeric polypeptide sequence binds to a vascular endothelial growth factor receptor selected from the group consisting of vascular endothelial growth factor receptor- 1, vascular endothelial growth factor receptor-2, vascular endothelial growth factor receptor-3, and combinations thereof.

13. The chimeric synthetic protein of claim 12, wherein the chimeric polypeptide sequence binds to vascular endothelial growth factor receptor- 1, vascular endothelial growth factor receptor-2, and vascular endothelial growth factor receptor-3.

14. The chimeric synthetic protein of claim 1, wherein the chimeric synthetic protein initially has an amino acid sequence of MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

15. The chimeric synthetic protein of claim 14, wherein the initial chimeric synthetic protein is treated to have an amino acid sequence of TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

16. An immunogenic composition comprising a chimeric polypeptide sequence; at least one linker sequence; and a polypeptide sequence.

17. The immunogenic composition of claim 16, wherein the polypeptide sequence comprises an immunogenic polypeptide sequence.

18. The immunogenic composition of claim 16, wherein the polypeptide sequence comprises a cholera toxin B protein.

19. The immunogenic composition of claim 16, wherein the at least one linker sequence comprises a first linker sequence that separates the chimeric polypeptide sequence from the polypeptide sequence.

20. The immunogenic composition of claim 16, wherein the first linker sequence is selected from the group consisting of SSG, GSSG, SSGGG, SGG, GGSGG, GGGGS, SSGGGSGG, SSGGGGSGGG, TSGGGSG, TSGGGGSGG, SSGGSGGGSG, SSGGGSGGSSG, GGSGGTSGGGSG, SGGTSGGGGSGG, GGSGGTSGGGGSGG, SSGGGSGGGSSG, SSGGSGGSSGGG, and SSGGGGSGGGSSGGG.

21. The immunogenic composition of claim 20, wherein the first linker sequence is SSGGSGGSG.

22. The immunogenic composition of claim 16, wherein the chimeric polypeptide sequence comprises a vascular endothelial growth factor sequence. ​ ​ ​ ​ ​ 23. The immunogenic composition of claim 16, wherein the chimeric polypeptide sequence comprises a vascular endothelial growth factor sequence selected from the group consisting of vascular endothelial growth factor-A, vascular endothelial growth factor-B, vascular endothelial growth factor-C, vascular endothelial growth factor-D, and combinations thereof.

24. The immunogenic composition of claim 16, wherein the chimeric polypeptide sequence comprises a first vascular endothelial growth factor domain and a second vascular endothelial growth factor domain.

25. The immunogenic composition of claim 24, wherein the first vascular endothelial growth factor domain comprises vascular endothelial growth factor-D or a portion thereof and the second vascular endothelial growth factor domain comprises vascular endothelial growth factor-A or a portion thereof.

26. The immunogenic composition of claim 25, wherein the first vascular endothelial growth factor domain is TFYDIETLKVIDEEWQRTQ and the second vascular endothelial growth factor domain is CHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

27. The immunogenic composition of claim 26, wherein the chimeric polypeptide sequence binds to a vascular endothelial growth factor receptor selected from the group consisting of vascular endothelial growth factor receptor- 1, vascular endothelial growth factor receptor-2, vascular endothelial growth factor receptor-3, and combinations thereof.

28. The immunogenic composition of claim 27, wherein the chimeric polypeptide sequence binds to vascular endothelial growth factor receptor- 1, vascular endothelial growth factor receptor-2, and vascular endothelial growth factor receptor-3.

29. The immunogenic composition of claim 16, wherein the synthetic protein chimeric synthetic protein initially has the amino acid sequence of MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

30. The immunogenic composition of claim 16, wherein the initial chimeric synthetic protein is processed to have the amino acid sequence of TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG. ​ ​ 31. The immunogenic composition of claim 16, further comprising an adjuvant.

32. A method of treating a patient in need thereof, comprising: administering to the patient the immunogenic composition of claim 16 on the same day of vaccination or every other day or at a time interval within the vaccination phase.

33. The method of claim 32, wherein the patient has cancer.

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