Stable chimeric synthetic proteins and their therapeutic uses

Abstract

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 in treating cancer.

Description

Technical Field

[0001] 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 in treating cancer. Background Art

[0002] According to the World Health Organization, tumors (e.g., cancer) are a leading cause of death worldwide, causing 8.8 million deaths in 2015. Cancer is highly prevalent in the global population, accounting for nearly one in six deaths. In 2015, the most common cancer deaths occurred in the following types of cancer: lung cancer (approximately 1.7 million deaths), liver cancer (approximately 800,000 deaths), colorectal cancer (approximately 800,000 deaths), stomach cancer (approximately 800,000 deaths), and breast cancer (approximately 600,000 deaths).

[0003] Cancer is typically treated by any of a variety of methods, such as surgery, chemotherapy, radiotherapy, 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 rapidly dividing cells and is mostly toxic, causing 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.

[0004] One such therapy is encompassed by cancer immunology, which studies the interaction between the immune system and cancer cells such as tumors or malignancies. The initiation of an immune response, such as the recognition of cancer-specific antigens expressed by human tumors but not in normal tissues, is of particular interest. Typically, methods for controlling the division and proliferation of malignant cells 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. Unfortunately, such cancer vaccines exhibit many significant limitations, primarily stemming from the manufacturing methods and the potential lack of uniformity, activity, and homology of the protein product. For example, cancer vaccines generated by chemical conjugation (e.g., by glutaraldehyde) typically comprise a mixture of a recombinant carrier protein and a human-derived polypeptide. Unfortunately, the use of glutaraldehyde as a cross-linking agent has an undesirable tendency to form covalent cross-links between a variety of chemical groups and typically results in highly heterogeneous products. Therefore, the resulting cancer vaccine may not only include a carrier protein molecule to which many target human polypeptides (e.g., 0, 1, 2, 3, etc.) are attached, but the human polypeptides may each be attached to the carrier via different atoms and therefore exist in different positions and different orientations. In addition, both the target polypeptide and the carrier protein molecule may be conjugated to themselves, resulting in a variety of homopolymers that may not have clinical efficacy and may not contribute to an anti-cancer patient immune response. In addition, cancer vaccines generated by recombinant protein technology have the following disadvantages: the target human polypeptide included in the recombinant protein may not be properly folded, thereby preventing an available immune response. Accordingly, there is an urgent need for new cancer vaccines that overcome these existing significant limitations in the field of cancer immunotherapy. Summary of the Invention

[0005] The present disclosure relates to chimeric synthetic proteins / molecules and methods of making each thereof; characterization of the chimeric synthetic proteins / molecules and therapeutic methods of 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.

[0006] The present disclosure provides chimeric synthetic proteins that can be used as a therapeutic method to treat diseases such as cancer. In exemplary embodiments, the present disclosure provides a chimeric synthetic protein / molecule comprising 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. On the one hand, the present disclosure provides a chimeric synthetic protein comprising a chimeric polypeptide sequence; at least one linker sequence; and a polypeptide sequence. Advantageously, the chimeric synthetic protein / molecule described herein has the ability to function to stabilize the scaffold, better enabling the human protein (e.g., growth factors such as VEGF, EGF, TGF, etc.) incorporated into the protein / molecule to adopt a natural configuration (e.g., appropriately folded) when expressed. In addition, the chimeric synthetic protein / molecule described herein has the ability to generate a stable chimeric synthetic protein / molecule with a longer shelf life.

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

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

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

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

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

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

[0013] 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.

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

[0015] 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.

[0016] In some embodiments, the first VEGF domain is TFYDIETLKVIDEEWQRTQ and the second VEGF domain is CHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0017] 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.

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

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

[0020] In some embodiments, the initial chimeric synthetic protein is processed to have the amino acid sequence of TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

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

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

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

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

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

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

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

[0028] 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.

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

[0030] 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.

[0031] In some embodiments, the first VEGF domain is TFYDIETLKVIDEEWQRTQ and the second VEGF domain is CHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

[0032] 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.

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

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

[0035] In some embodiments, the initial chimeric synthetic protein is processed to have the amino acid sequence of TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

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

[0037] In another aspect, the present invention provides a method for treating a patient in need thereof, comprising administering the immunogenic composition to the patient on the same day of vaccination or every other day or over a period of time during the vaccination period.

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

[0039] definition

[0040] "Epidermal growth factor receptor (EGFR) nucleic acid molecule" means a polynucleotide encoding an EGFR polypeptide. An exemplary EGFR nucleic acid molecule is provided as NCBI Accession No. NM_005228.4 and is reproduced below (SEQ ID NO: 3):

[0041] >NM_005228.4

[0042]

[0043] "Epidermal Growth Factor Receptor (EGFR) polypeptide" means 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):

[0044] >NP_005219.2

[0045]

[0046] "Epidermal growth factor (EGF) nucleic acid molecule" means a polynucleotide encoding an EGF polypeptide. An exemplary EGF nucleic acid molecule is provided as NCBI Accession No. NM_001963.5 and is reproduced below (SEQ ID NO: 5):

[0047] >NM_001963.5

[0048]

[0049] "Epidermal Growth Factor (EGF) polypeptide" means a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001954.2, and corresponding to the preproprotein form of EGF, which is processed to produce the 53 amino acid EGF molecule (shown in bold) and has EGFR binding activity, reproduced below (SEQ ID NO: 6):

[0050] >NP_001954.2

[0051]

[0052] "Neuregulin 1 (NRG1) nucleic acid molecule" means a polynucleotide encoding an NRG1 polypeptide. An exemplary NRG1 nucleic acid molecule is provided as NCBI Accession No. BC150609.1 and is reproduced below (SEQ ID NO: 7):

[0053] >BC150609.1

[0054]

[0055] "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):

[0056] > AAI50610.1

[0057] MSERKEGRGKGKGKKKERGSGKKPESAAGSQSPALPPQLKEMKSQESAAGSKLVLRCETSSEYSSLRFKWFKNGNELNRKNKPQNIKIQKKPGKSELRINKASLADSGEYMCKVISKLGNDSASANITIVESNEIITGMPASTEGAYVSSESPIRISVST EGANTSSSSTSTTGTSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCQPGFTGARCTENVPMKVQNQEKAEELYQKRVLTITGICIALLVVGIMCLVAYCKTKKQRKKLHDRLRQSLRSERNNMMNIANGPHHPNPPPENVQLVNQYVSKNVISS EHIVEREAETSFSTSHYTSTAHHSTTVTQTPSHSWSNGHTESILSESSHVIVMSSVENSRHSSPTGGPRGRLNGTGGPRECNSFLRHARETPDSYRDSPHSERYVSAMTTPARMSPVDFHTPSSPKSPPSEMSPPVSSMTVSMPSMAVSPFMEEERPLLL VTPPRLREKKFDHHPQQFSSFHHNPAHDSNSLPASPLRIVEDEEYETTQEYEPAQEPVKKLANSRRAKRTKPNGHIANRLEVDSNTSSQSSNSESETEDERVGEDTPFLGIQNPLAASLEATPAFRLADSRTNPAGRFSTQEEIQARLSSVIANQDPIAV

[0058] "Neuregulin 1β (NRG1β) nucleic acid molecule" means a polynucleotide encoding an NRG1 polypeptide. An exemplary NRG1β nucleic acid molecule is provided as NCBI Accession No. NM_001322205.1 and is reproduced below (SEQ ID NO: 9):

[0059] >NM_001322205.1

[0060]

[0061] "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):

[0062] >NP_001309134.1

[0063] MEIYSPDMSEVAAERSSSPSTQLSADPSLDGLPAAEDMPEPQTEDGRTPGLVGLAVPCCACLEAERLRGCLNSEKICIVPILACLVSLCLCIAGLKWVFVDKIFEYDSPTHLDPGGLGQDPIISLDATAASAVWVSSEAYTSPVSRAQSESEVQVTVQGDKAVVSFEPSAAPTPK NRIFAFFSFLPSTAPSFPSPTRNPEVRTPKSATQPQTTETNLQTAPKLSTSTSTTGTSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKHLGIEFMEAEELYQKRVLTITGICIALLVVGIMCVVAYCKTKKQRKKLHDRLRQSLRSERNNMM NIANGPHHPNPPPENVQLVNQYVSKNVISSEHIVEREAETSFSTSHYTSTAHHSTTVTQTPSHSWSNGHTESILSESHSVIVMSSVENSRHSSPTGGPRGRLNGTGGPRECNSFLRHARETPDSYRDSPHSERYVSAMTTPARMSPVDFHTPSSPKSPPSEMSPPVSSMTVSMPS MAVSPFMEEERPLLLVTPPRLREKKFDHHPQQFSSFHHNPAHDSNSLPASPLRIVEDEEYETTQEYEPAQEPVKKLANSRRAKRTKPNGHIANRLEVDSNTSSQSSNSESETEDERVGEDTPFLGIQNPLAASLEATPAFRLADSRTNPAGRFSTQEEIQARLSSVIANQDPIAV

[0064] "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):

[0065] >NRG-BVN-hybrid

[0066] GTSHLVKCPLSHEAYCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASF

[0067] 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):

[0068] >TGF-BVN-hybrid

[0069] NTENDCPLSHEAYCLHDGVCRFLVQEDKPACVCVVGYVGERCQFRDLRWWdar

[0070] By "original IN-02 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: 13):

[0071] >Initial_IN-02_PolypeptideMTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG

[0072] "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):

[0073] >Final_IN-02_Peptide

[0074] TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG

[0075] "Vascular endothelial growth factor A (VEGF-A) nucleic acid molecule" means a polynucleotide encoding a VEGF-A polypeptide. An exemplary VEGF-A nucleic acid molecule is provided as NCBI Accession No. NM_001025366.3 and is reproduced below (SEQ ID NO: 15):

[0076] >NM_001025366.3

[0077]

[0078] "VEGF-A polypeptide" means a polypeptide encoded by a VEGF-A nucleic acid molecule. An exemplary VEGF-A nucleic acid molecule is provided as NCBI Accession No. NP_001020537.2 and is reproduced below (SEQ ID NO: 16):

[0079] >NP_001020537.2

[0080] MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGB APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCE CRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR

[0081] "Vascular endothelial growth factor D (VEGF-D) nucleic acid molecule" means a polynucleotide encoding a VEGF-D polypeptide. An exemplary VEGF-D nucleic acid molecule is provided as NCBI Accession No. NM_004469 and is reproduced below (SEQ ID NO: 17):

[0082] >NM_004469

[0083]

[0084] "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 is reproduced below (SEQ ID NO: 18):

[0085] >NP_004460.1

[0086] MYREWVVVNVFMMLYVQLVQGSSNEHGPVKRSSQSTLERSEQQIRAASSLEELLRITHSEDWKLWRCRLRLKSFTSMDSRSASHRSTRFAATFYDIETLKVIDEEWQRTQCSPRETCVEVASELGKSTNTFFKPPCVNVFRCGGCCNEESLICMNTTSSYISKQLFEIISVPLTSVPE LVPVKVANHTGCKCLPTAPRHPYSIIRRSIQIPEEDRCSHSKKLCPIDMLWDSNKCKCVLQEENPLAGTEDHSHLQEPALCGPHMMFDEDRCECVCKTPCPKDLIQHPKNCSCFECKESLETCCQKHKLFHPDTCSCEDRCPFHTRPCASGKTACAKHCRFPKEKRAAQGPHSRKNP

[0087] Herein, ranges can be expressed as from "about" a specific value and / or to "about" another specific value. When such a range is expressed, another aspect includes from the specific value and / or to the other specific value. Similarly, when a numerical value is expressed as an approximate number by the prefix "about", it should be understood that the specific numerical value forms another aspect. It should also be understood that the endpoints of each range are clearly related to the other endpoint and are independent of the other endpoint. It should also be understood that a large number of values are disclosed herein, and each value, in addition to its own value, is also disclosed herein as "about" the specific value. It should also be understood that throughout this application, data is provided in different formats, and this data represents the range of any combination of endpoints and starting points and the data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is understood that values greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as values between 10 and 15, are also considered to be disclosed. It should also be understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are revealed, then 11, 12, 13, and 14 are also revealed.

[0088] 45, 46, 47, 48, 49, or 50 and all intervening tenths 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," specifically contemplated are "nested subranges" extending from either end of the range. For example, nested subranges of the exemplary range 1 to 50 could 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.

[0089] It is contemplated that any embodiment described herein can be combined with any other embodiment or embodiments, where applicable or not expressly denied, even if the embodiments are described under different aspects of the disclosure.

[0090] These and other embodiments are disclosed and / or encompassed by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] The following detailed description is given by way of example and is not intended to limit the disclosure solely to the specific embodiments described and can be best understood in conjunction with the accompanying drawings, in which:

[0092] Figures 1A to 1D Two protein schematics, recombinant protein sequences and bar graphs are depicted respectively. Figure 1A is a ribbon diagram protein schematic illustrating a chimeric VEGF molecule (VEGF-DA) according to an exemplary embodiment of the present disclosure, which includes an N-terminal region derived from VEGF-D and indicates the "homologous domains" (VEGFR-1, VEGFR-2, and VEGFR-3 binding regions) from VEGF-A. Figure 1B is a protein schematic illustrating the structure and organization of IN-02, in which a chimeric VEGF-DA protein domain is fused to the C-terminus of CTB via a 10 amino acid linker sequence according to an exemplary embodiment of the present disclosure. Figure 1C Depicting the protein sequence of IN-02, with colors coordinated to match Figure 1A to Figure 1B The initiating methionine residue is removed by methionine aminopeptidase and is thus absent from the mature protein. Figure 1Dis a bar graph showing the effects of EGF-A, VEGF-D, and IN-02 alone or in combination with neutralizing antibodies (NAbs) on the development of tubes formed by human endothelial cells (HUVECs).

[0093] Figure 2 is a bar graph depicting an ELISA showing binding of VEGF-A, IN-02 (eg, VEGF-DA), and VEGF-D proteins to VEGF receptors immobilized on a plate.

[0094] Figure 3 is a graph depicting an ELISA showing binding of sera from three rabbits before immunization and after immunization with IN-02 protein (BL3) to the immobilized immunogen.

[0095] Figure 4 is a graph depicting an ELISA showing binding of sera (purified with caprylic acid) from three rabbits before immunization and after immunization with IN-02 protein (BL3) to immobilized rCTB.

[0096] Figure 5 is a graph depicting an ELISA showing binding of sera from three rabbits before immunization and after immunization with IN-02 protein (BL3) to immobilized VEGF-A.

[0097] Figure 6 is a graph depicting an ELISA showing binding of sera from three rabbits before immunization and after immunization with IN-02 protein (BL3) to immobilized VEGF-D.

[0098] Figure 7 is a bar graph depicting the results of a tube formation assay performed using caprylic acid-purified serum from rabbits immunized with IN-02 protein.

[0099] 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

[0100] 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 sequence regions, and one or more immunogenic domains can be used as therapeutic molecules to treat a variety of diseases, such as cancer. Chimeric synthetic molecules offer several unexpected advantages over the prior art. For example, unlike the human epidermal growth factor (hEGF) molecule of the prior art (e.g., U.S. Patent No. 5,984,018 to Davila et al.), which exists as a heterogeneous mixture containing up to 12 different molecular species, the synthetic proteins / molecules described herein can be produced as a single molecule (e.g., a homogeneous population of molecules). In addition, the synthetic protein / molecule described herein includes ten active components per molecule (but the active components can be increased or decreased by a factor of 5, for example, as part of a pentamer), while the number of active components present in each molecule of the hEGF molecule of the prior art (e.g., U.S. Patent No. 5,984,018 to Davila et al.) is highly variable (e.g., the average number of active components per molecule of Davila is 1.5). In addition, the chimeric synthetic protein / molecule described herein is easier to manufacture. For example, the hEGF molecule of the prior art (e.g., U.S. Patent No. 5,984,018) is chemically conjugated to rP64k with recombinant human EGF (rhEGF) to produce a final molecule consisting of two molecules chemically conjugated to each other. This is in stark contrast to the synthetic protein / molecule described herein, which is a single synthetic molecule. In addition, the chimeric synthetic protein / molecule described herein has the ability to function to stabilize the scaffold, better enabling the human protein (e.g., growth factor) incorporated into the protein / molecule to adopt a natural configuration (e.g., properly folded) when expressed. In addition, the chimeric synthetic proteins / molecules described herein have the ability to generate stable chimeric synthetic proteins / molecules with a long shelf life. Advantageously, the technology herein provides novel chimeric synthetic proteins 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. Patent No. 5,984,018).

[0101] Overview

[0102] Cancer immunology is the study of the interaction between the immune system and cancer cells, such as tumors or malignancies. The initiation of an immune response, such as the recognition of cancer-specific antigens expressed by human tumors but not in normal tissues, is of particular interest. Typically, 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, thereby inducing a specific immune response.

[0103] There are currently a large number of identified growth factors, and most (if not all) have been shown to be important mediators of cell proliferation in various cancers, in addition to being involved in other disease conditions. Typically, 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 more than one closely related receptor with different affinities. Similarly, some receptors only bind to a single growth factor ligand, while others may bind to a variety of related growth factors (once again, often with different affinities). When bound to its natural receptor, the cytoplasmic domain of the receptor is phosphorylated, and this initiates an intracellular signaling cascade, leading to regulation of one or more gene transcriptions and ultimately progression through the cell cycle and cell proliferation.

[0104] Growth factors and their receptors are essential components of the normal processes of growth, development, and repair, and their tissue distribution and expression levels closely regulate cell growth. 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, it has been shown that certain growth factors stimulate the proliferation of some cancer cell lines. For example, epidermal growth factor (EGF) can stimulate some non-small cell lung cancer cells (Osborne C Ke 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 tumor 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)).

[0105] It has been reported that malignant tumor tissues contain high levels of multiple growth factor receptors. For example, high levels of epidermal growth factor receptor (EGFR) are often detected in malignant tumors of epidermal origin, such as lung cancer, breast cancer, bladder cancer, ovarian cancer, vulvar cancer, colon cancer, lung cancer, brain cancer, and esophageal cancer. The role of growth factors and their receptors in regulating tumor growth is still unclear, but some believe that the expression of growth factor receptors in tumor cells provides a mechanism for autocrine growth stimulation, leading to uncontrolled proliferation (Schlessinger J., Schreiber AB, Levi A., Liberman T., Yarden Y. Crit. Rev. Biochem. 1983, 14(2)93-111). 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) described the role of increased islet receptors and growth factors in metastatic prostate cancer.

[0106] One therapeutic strategy for targeting growth factor signaling in cancer therapy has been to use passive immunotherapy (e.g., monoclonal antibodies) directed against one or more specific receptors involved. Such studies have demonstrated that specific recognition of receptors capable of inhibiting ligand binding by antibodies may 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-mouse antibody response (HAMA), thus limiting them to single administration.

[0107] Another treatment strategy is active immunotherapy using a vaccine containing the growth factor of interest to induce an immune response against a molecule that inhibits the proliferative effects of the growth factor on the tumor. U.S. Patent No. 5,984,018, 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," issued to Davila et al., discloses, for example, the use of a vaccine containing a mixture of a growth factor and an immunogenic (i.e., non-human) carrier protein chemically conjugated together using glutaraldehyde. However, without being bound by any particular theory, it is believed that the chemical conjugation hinders the immune response to the vaccine.

[0108] This is a technically challenging approach because it requires the host to generate an immune response to "self-antigens", and the immune system of vertebrates has evolved to prevent the occurrence of such a response. When a strong immune response is generated against self-antigens (e.g., antigens that activate helper T cells), an autoimmune disease state is usually caused. For many years, it has been assumed that some autoimmune diseases, such as lupus, multiple sclerosis (MS), diabetes, etc., may be caused by early exposure to environmental agents that include immunogenic epitopes (T cell epitopes) that are closely similar to the host's own epitopes. This may lead to the stimulation of helper T cells that can cross-react with the host epitopes. Subsequently, subsequent exposure to environmental agents will lead to an anti-autoimmune response (Albert, LJ, and Inman, RD New England Journal of Medicine, Dec. 30th pp 2068-2074, 1999). It has been shown that viral antigens can actually generate anti-autoimmune responses against nerve cell proteins (Levin, MC et.al, Nature Medicine vol 8 (5) pp 509-513, 2002).

[0109] U.S. Patent Publication No. 2006 / 0251654 (the '654 publication), entitled "Method for Treatment of Malignant and Infectious Chronic Diseases" to Casimiro et al., discloses a method for treating a subject suffering from a malignant or infectious chronic disease, comprising immunizing the subject with a vaccine comprising an autoantigen associated with the malignant or infectious chronic disease coupled to a carrier protein; treating the subject with an immunomodulatory agent; and 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, preparing the vaccine by chemical conjugation is believed to hinder the immune response.

[0110] Most of the above-mentioned vaccines show many limitations, which are mainly derived from the potential lack of uniformity and homology of the manufacturing method and protein products. The above-mentioned vaccines generally comprise a mixture of a recombinant carrier protein and a human polypeptide chemically conjugated using glutaraldehyde. Unfortunately, this reaction reagent may not undesirably form a trend of covalent cross-linking bonds between a variety of chemical groups, and generally result in a highly heterogeneous product. Therefore, the resulting cancer vaccine may not only comprise a carrier protein molecule with many target human polypeptides (e.g., 0, 1, 2, 3 species, etc.), but also the human polypeptide may each be attached to the carrier by different atoms and therefore exist in different positions and different orientations. In addition, both the target polypeptide and the carrier protein molecule can be conjugated to themselves, resulting in a variety of homopolymers, which may not have clinical efficacy and may not contribute to an anti-cancer patient immune response.

[0111] Synthetic proteins / molecules

[0112] The present disclosure provides a kind of homogeneous synthetic protein molecule, for improving the maximum number presentation of growth factor epitopes, tumor antigen epitopes and / or receptor binding sites as immunogenic synthetic protein / molecule elements. In an exemplary embodiment, a synthetic protein / molecule expressing all or part of an immunogenic carrier domain (e.g., cholera toxin B (CT-B)) and synthetic epidermal growth factor (sEGF), tumor antigens and / or receptors is described. In alternative exemplary embodiments, proteins can express other immunogenic synthetic or recombinant proteins / molecules based on known immunogenic protein modeling. Within the scope of the present disclosure, it is envisioned that such synthetic proteins / molecules can express polypeptides that are highly immunogenic for the human immune system. Preferably, the synthetic protein / molecule gives chimeric proteins additional properties, for example, high expression levels and ease of manufacture, oral stability and the ability to cross from the digestive tract to the bloodstream, and / or previous safe uses in the human body.

[0113] In exemplary embodiments, the synthetic proteins / molecules disclosed herein can include or express a high proportion of protein sequences derived from target autoantigens as a function of total molecular weight. For example, this can be achieved by using a large protein model containing multiple growth factor epitopes. These growth factor epitopes can be multiple copies of a single growth factor, complete or partial, or copies of more than one different growth factor, complete or partial. These growth factor epitopes can be naturally occurring or synthetic (e.g., artificial). For example, BVN22E (also referred to as IN01) is an exemplary synthetic protein described herein that can have a molecular weight of about 120 kD. In exemplary embodiments, the growth factor epitopes described herein can correspond to one or more domains of the growth factor (e.g., a signaling pathway (TSP) domain targeting EGF). In exemplary embodiments, the EGF domain may include a region that presents or constrains the β-loop, e.g., 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 of the synthetic protein sequence ( Figure 1A Without being bound by any particular theory, it is contemplated within the scope of this disclosure that different regions or subregions between Cysteine 6 and Cysteine 42 may have a beneficial effect when incorporated into the synthetic proteins / molecules of the present disclosure. For example, the following regions may have a beneficial effect: the region between Cysteine 6 and Cysteine 14, the region between Cysteine 6 and Cysteine 20, the region between Cysteine 6 and Cysteine 31, Cysteine 6 and Cysteine 33, and the region 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 a beneficial effect: the region between Cysteine 42 and Cysteine 33, the region between Cysteine 42 and Cysteine 31, the region between Cysteine 42 and Cysteine 20, 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 specific spacing 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 42).

[0114] According to the present disclosure, the expression of growth factor epitopes should be folded so that their native conformation is substantially retained and presented to the components of the host immune system in a manner that exerts a robust host immune response to the epitope. Examples of suitable natural protein models used to model the epitope support domain of a synthetic protein / molecule include, but are not limited to, cholera toxin B subunit, E. coli heat-labile LT and LT-II enterotoxin B subunit, aflatoxin (veratoxin), pertussis toxin, Campylobacter jejuni (C.jejuni) enterotoxin, Shiga toxin, Listeria toxin, tetanus toxoid, diphtheria toxoid, meningococcal outer membrane proteins, phage coat proteins, adenovirus and other viral coat proteins. Alternatively, the non-self components of the protein may be very small. At least, 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 human T cell epitope. As described herein, non-natural synthetic polypeptides (e.g., BVN22E, INO1) can be used to address the need to confer immunogenicity to the intact protein and allow for proper presentation of the growth factor, receptor, tumor antigen, or epitope thereof to the host immune system.

[0115] According to the present disclosure, the synthetic proteins / molecules provided herein, whether growth factors or portions thereof, cell receptors or portions thereof, or tumor antigens or portions thereof, are related to a large number of cellular pathways involved in chronic diseases, growth factor-based or receptor-based cancers, and / or solid tumors, and are used as tumor antigens in the synthetic proteins. 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 multiple cell receptors and growth factors can be overexpressed in the diseases, the proteins described below can contain one or more different tumor antigens, one or more different receptors, or one or more cellular pathways associated with the disease. These proteins are referred to as multivalent.

[0116] In an exemplary embodiment, a protein is disclosed that is composed of a homogenous synthetic protein / molecule that expresses 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, 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, such as Figure 1AAs shown in . In exemplary embodiments, the growth factor component of the synthetic protein sequence may include a sequence that is less than 80% identical to EGF. For example, the growth factor component may include an EGF sequence having 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 "presentation" or constraint of EGF's β-loop (e.g., the region defined by Cys6 to Cys31) may be important to include in the synthetic protein and suitable as a target for amino acid modification. In exemplary embodiments, regions outside of Cys6 to Cys31 may also be targets for modification (e.g., E11 and A12).

[0117] In exemplary embodiments, the TSP1 and TSP2 domains of hEGF can be as follows Figure 1B Modifications were made as shown in to create the synthetic EGF (sEGF) region to be included in the synthetic proteins / molecules herein.

[0118] In exemplary embodiments, the synthetic proteins / molecules disclosed herein may include all or part of a growth factor, including but not limited to, for example, neuregulin 1β (NRG1β), transforming growth factor alpha (TGFα), vascular endothelial growth factor (VEGF), and the like.

[0119] In other exemplary embodiments, the synthetic protein / molecule described herein can include one or more linker sequences or spacer sequences. One or more of the above embodiments include sEGF fused to CT-B so that the sEGF portion of the synthetic molecule is separated from the CT-B portion by the GGSGGTSGGGGGSG linker sequence. These resulting recombinant or chimeric proteins mainly include sEGF directly fused to CT-B. In other exemplary embodiments, the EGF component of the chimeric protein is effectively separated from the CT-B component by 3 to 14 amino acids, which form flexible spacer sequences or linker sequences between the two domains. Within the scope of the present disclosure, it is envisioned that the linker sequence 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 has a larger size (e.g., human growth factor), it may be useful to use a longer linker sequence. The following exemplary linker sequences can be used, and include, but are not limited to, SSG, SSGGG, SGG, GSSG, GGSGG, GGGGS, SSGGGSGG, SSGGGGSGGG, TSGGGSG, TSGGGGSGG, SSGGGSGGSSG, GGSGGTSGGGSG, SGGTSGGGGSGG, GGSGGTSGGGGSGG, SSGGGGSGGGSSG, SSGGGSGGSSGGG, and SSGGGGSGGGSSGGG. Those skilled in the art will appreciate that many other sequences consisting primarily of "G" and "S" can also serve as useful linker sequences.

[0120] Without being bound by any particular theory, it is envisioned 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 correctly folded and functional suitable polypeptides. In addition, the synthetic proteins / molecules disclosed herein can form pentamers. In addition, the synthetic proteins / molecules disclosed herein have the advantageous property of being much lower in the level of protein required for vaccination, because the amount of necessary carrier is significantly lower than in 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 can deliver more growth factors to patients with significantly lower vaccine volumes.

[0121] adjuvant

[0122] Certain exemplary embodiments as provided herein include synthetic proteins / molecules according to the present disclosure within vaccine compositions and immunoadjuvant compositions, including pharmaceutical compositions containing, in addition to the synthetic proteins / molecules, at least one adjuvant, an adjuvant being a component of such compositions that has adjuvant activity. Adjuvants having such adjuvant activity include compositions that, when administered to a subject such as a human (e.g., a human patient), a non-human primate, a mammal, or other higher eukaryotic organism with a recognized immune system, are capable of altering (i.e., increasing or decreasing in a statistically significant manner, and in certain preferred embodiments, enhancing or increasing) the potency and / or longevity of an immune response. In certain exemplary embodiments disclosed herein, the desired antigen(s) and optionally one or more adjuvants contained within a protein carrier can thereby modify (e.g., elicit or enhance) an immune response against the desired antigen(s) that can be administered simultaneously or can be separated in time and / or space (e.g., at different anatomical sites), but certain exemplary embodiments are not intended to be limited thereto and thus also contemplate administration of the synthetic protein / molecule in a composition that does not include the specified antigen(s) but which can include, but is not limited to, one or more auxiliary adjuvants (imidazoquinoline immune response modifiers).

[0123] Accordingly, and as described above, adjuvants include compositions having an 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 Bl, WO 95 / 17210); alum; plant alkaloids such as 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-α, IFN-γ), imidazoquinoline immune response modifiers, and double stem-loop immune modifiers (dSLIM, e.g., Weeratna et al, 2005 Vaccine 23:5263).

[0124] 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. Particle structures comprising Quil A (saponin) fractions, called immunostimulating complexes (ISCOMS), have hemolytic properties and have been used to make vaccines (Morein, B., EP 0 109 942 Bl). These structures have adjuvant activity (EP 0 109 942 Bl; WO 96 / 1 1711). Hemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A) have been described as potential systemic adjuvants, and their production methods are disclosed in U.S. Patent No. 5,057,540 and EP 0362 279 B1. These references also describe the use of QS7 (a non-hemolytic fraction of Quil-A) as a potential adjuvant for systemic vaccination. The use of QS21 is further described by 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 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 the saponins used in the adjuvant compositions of the embodiments disclosed herein. Aescin is described in The Merck Index (12th edition, entry 3737) as a mixture of saponins that occur in the seeds of chestnut trees, horse chestnut trees (Aesculus hippocastanum). Its separation and purification are carried out by chromatography (Fiedler, Arzneimittel-Forsch.4, 213 (1953)) and by ion exchange resins (Erbring et al, US Pat. No. 3,238,190).Fractions of aescin have been purified and shown to be biologically active (Yoshikawa M, et al. (Chem Pharm Bull (Tokyo) 1996 August; 44(8): 1454-1464)). Digitonin is another detergent, also described as a saponin in the Merck Index (12th edition, entry 3204), which is derived from the seeds of Digitalis purpurea and purified according to the procedures described by Gisvold et al, J. Am. Pharm. Assoc., 1934, 23, 664 and Rubenstroth-Bauer, Physiol. Chem., 1955, 301, 621.

[0125] Other adjuvants or co-adjuvants for use according to the embodiments disclosed herein include block copolymers or biodegradable polymers, which refer to a class of polymeric compounds familiar to those skilled in the relevant art. Block copolymers or biodegradable polymers that can be included in vaccine compositions or immunoadjuvants include RTM.L121 (BASF Corp., Mount Olive, NJ; see, e.g., Yeh et al, 1996 Pharm. Res. 13: 1693).

[0126] Some further exemplary embodiments envision including but not limited to the immunoadjuvant of oil, this immunoadjuvant can promote auxiliary adjuvant activity in some such embodiments, and can provide pharmaceutically finalizable carrier or excipient additionally or alternatively in other such embodiments.Based on the disclosure, any number of suitable oils are known and can be selected for inclusion in vaccine compositions and immunoadjuvant compositions.For example, but not limiting, the example of this type of oil includes squalene, squalane, mineral oil, olive oil, cholesterol and anhydrous mannitol monooleate.

[0127] Immune response modifiers such as pyrazoloquinoline immune response modifiers are also known in the art and may also be included as adjuvants or co-adjuvants in certain embodiments of the present disclosure.

[0128] As also mentioned above, one type of adjuvant or co-adjuvant for use in the vaccine compositions disclosed herein can be an aluminum co-adjuvant, which is generally referred to as "alum". Alum co-adjuvants are based on the following: aluminum oxyhydroxide; aluminum hydroxyphosphate; or a variety of appropriate 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).

[0129] Pharmaceutical composition

[0130] In certain exemplary embodiments, the pharmaceutical composition is a vaccine composition comprising a synthetic protein / molecule according to the present disclosure and may further comprise one or more components, as provided herein, selected from TLR agonists, auxiliary adjuvants (including, for example, cytokines, imidazoquinoline immune response modifiers and / or dSLIM), etc., and / or recombinant expression constructs, used in combination with a pharmaceutically acceptable carrier, excipient or diluent.

[0131] Exemplary carriers will be nontoxic to recipients at the dosages and concentrations employed. For vaccine compositions comprising synthetic proteins / molecules, about 0.01 μg / kg to about 100 mg / kg body weight will typically be administered intradermally, subcutaneously, intramuscularly or intravenously, or by other routes.

[0132] It will be apparent to those skilled in the art that the dosage and frequency will depend on the host's response. "Pharmaceutically acceptable carriers" for therapeutic use are well known in the pharmaceutical field 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 fragrances can be provided in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid, and parabens can be added as preservatives. In addition, antioxidants and suspending agents can be used.

[0133] The pharmaceutical composition can be any form that allows the composition to be administered to the 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, ultrasonic introduction, passive transdermal, microneedle administration, and also includes subcutaneous injection, intravenous, intramuscular, intrasternal, intracavernous, intrathecal, intrachannel, intraurethral injection and infusion technology. In specific embodiments, compositions as described herein (including vaccines and pharmaceutical compositions) are administered by a technology selected from iontophoresis, microcavitation, ultrasonic introduction or microneedle.

[0134] The pharmaceutical composition is formulated to allow the active ingredient contained therein to be bioavailable when the composition is administered to a patient. The composition to be administered to a patient is in the form of one or more dosage units, wherein, for example, a tablet can be a single metered unit, and a container of one or more compounds of the invention in aerosol form can hold multiple metered units.

[0135] For oral administration, excipients and / or binders may be present. Examples are sucrose, kaolin, glycerol, starch dextrin, sodium alginate, carboxymethyl cellulose, and ethyl cellulose. Colorants and / or fragrances may be present. A coating shell may be employed.

[0136] The composition can be in liquid form, for example, elixir, syrup, solution, emulsion or suspension. As two examples, liquid can be used for oral administration or for delivery by injection. When it is expected that it is used for oral administration, the preferred composition contains one or more of a sweetener, a preservative, a dye / colorant and a flavoring agent. In the composition for administration by injection, it is expected that one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer and an isotonic agent can be included.

[0137] As used herein, whether in the form of a solution, suspension or other similar form, a liquid pharmaceutical composition may include one or more of the following carriers or excipients: a 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, anhydrous mannitol monooleate, cholesterol, and / or synthetic monoglycerides or diesters that can serve as solvents or suspending media, polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for regulating osmotic pressure such as sodium chloride or glucose. Parenteral formulations may be sealed in ampoules, disposable syringes or multiple dose bottles made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0138] In certain embodiments, the pharmaceutical or vaccine composition of the present invention comprises a stable aqueous suspension of at least 0.2 μm and further comprises at least one component selected from the group consisting of phospholipids, fatty acids, surfactants, detergents, saponins, fluorinated lipids, and the like.

[0139] It may 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 immunostimulating substances (auxiliary adjuvants) for use in such vehicles are also described above and may include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), dextran, IL-12, GM-CSF, gamma interferon and IL-12.

[0140] Although any suitable carrier known to those of ordinary skill in the art can be used in pharmaceutical composition of the present invention, the type of carrier will change according to mode of administration and whether to wish sustained release. For parenteral administration such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, fat, wax or buffer. For oral administration, any of the above-mentioned carriers or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose and magnesium carbonate can be adopted. Biodegradable microspheres (for example, polylactic acid galactoside) can also be adopted as carrier for pharmaceutical composition of the present invention.

[0141] 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 nonspecific serum albumin is an exemplary suitable diluent. Preferably, the product can be formulated as a lyophilisate using a suitable excipient solution (e.g., sucrose) as a diluent.

[0142] In exemplary embodiments, the epitope or receptor support domain of the synthetic protein / molecule, whether derived from a natural or synthetic polypeptide sequence, should 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 will assemble into stable multimers with a small number of subunits, e.g., dimers, trimers, tetramers, pentamers, etc., so that products of uniform size are generated. Examples of natural polypeptides include, but are not limited to, leucine zippers, lac repressor proteins, streptavidin / avidin, cholera toxin B subunits, Pseudomonas trimerization domains, and viral capsid proteins.

[0143] In an exemplary embodiment, a process for preparing a multivalent molecule is disclosed. In this exemplary embodiment, the process 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.

[0144] In another exemplary embodiment, a process for preparing a vaccine formulation is disclosed. In this exemplary embodiment, the process comprises mixing together one or more single monovalent multimers to prepare a multivalent vaccine comprising a synthetic protein / molecule comprising one or more tumor antigens, receptors and / or growth factors or portions thereof.

[0145] In another exemplary embodiment, a process for treating a patient is disclosed. In this exemplary embodiment, the process comprises independently administering to the patient one or more monovalent tumor antigens, receptors and / or growth factors, recombinant proteins, on the same day or on alternate days, or multiple times during the vaccination period.

[0146] Although a synthetic protein / molecule is described as comprising or expressing all or part of at least one sequence of a tumor antigen, growth factor, and / or receptor and one or more of a CT-B sequence, the synthetic protein / molecule may comprise a natural CT-B sequence or a sequence substantially similar to a natural CT-B sequence and / or a synthetic sequence. Although a synthetic protein / molecule is described as comprising or expressing a CT-B sequence, the synthetic protein / molecule may comprise or express a derivative of a CT-B sequence or a sequence substantially similar to a CT-B sequence.

[0147] While the present invention has been described and exemplified with respect to certain embodiments of synthetic proteins / molecules expressing or incorporating one or more tumor antigens, synthetic growth factors, and / or receptors, 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 invention. Therefore, the present disclosure is not limited to the precise details of the methods or configurations set forth above, as such variations and modifications are intended to be encompassed by the scope of the present disclosure.

[0148] Example

[0149] The present disclosure is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, GenBank accession numbers and gene numbers, and published patents and patent applications used throughout this application are incorporated herein by reference. It will be appreciated by those skilled in the art that the present disclosure may be practiced with variations in the disclosed structures, materials, compositions, and methods, and such variations are considered to be within the scope of the present invention.

[0150] Example 1: Bispecific chimeric antigen

[0151] One problem that arises when human proteins (e.g., growth factors) or portions thereof are used in combination with immunogenic carrier molecules such as cholera toxin B subunit (CTB) to create recombinant proteins is that human proteins (e.g., human growth factors) do not always fold into the correct native conformation. The ability of a human protein within a 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, transforming growth factor alpha (TGFα) and the EGF-like domain of neuregulin are more difficult to produce in a properly folded form and are also significantly more unstable.

[0152] Vascular endothelial growth factor (VEGF) comprises four kinds of structurally related proteins, i.e. VEGF-A, VEGF-B, VEGF-C and VEGF-D, which mediate signal transduction by three kinds of receptors VEGFR-1, VEGFR-2 and VEGFR-3. VEGF-A and VEGF-D carry out signal transduction by VEGFR-1, while VEGF-A, VEGF-B and VEGF-C can be combined with VEGFR-2. VEGF-C and VEGF-D are combined with VEGFR-3, therefore showing both similarity and difference in their receptor binding characteristics. All VEGF growth factors share a total " homeodomain " on structure, this domain is relevant with recognition and in conjunction with VEGFR-1 and VEGFR-2 (VEGF-A, VEGF-B and VEGF-C) and comprises the sequence of the first cysteine residue downstream. The N-terminal of VEGF-A and VEGF-B, i.e. the upstream of the first cysteine residue, is not directly involved in receptor binding. In contrast, the N-termini of VEGF-C and VEGF-D are involved in binding to VEGFR-3.

[0153] 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 receptive to refolding from inclusion bodies, but after only one week at 4°C, it is much less stable and shows significant signs of degradation, making it unsuitable for therapeutic applications in its native form. VEGF-C is very difficult to fold correctly, both from inclusion bodies and when expressed in bacteria as a soluble protein.

[0154] In the native protein, the N-terminal region of VEGF-C and VEGF-D (the sequence upstream of the first cysteine residue) forms an alpha helix that 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 an "unrelated" vector, the resulting protein does not show any binding to VEGFR-3. However, when the N-terminal domain of VEGF-A is replaced with the N-terminal domain of VEGF-D, as shown in Figure 1A As shown in , the resulting protein can bind to all three VEGF receptors and regulate 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 (including sequences derived 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 named IN-02 and is schematically shown in Figure 1BThe protein sequence of IN-02 is shown in Figure 1C middle.

[0155] In order to analyze the functional characteristics of the molecule based on VEGF, two kinds of determinations were adopted: tube formation determination (TFA) and ELISA. TFA includes cultivating human umbilical vein endothelial cells (HUVEC) and observing the development of " tube ", and over time presents the formation of capillaries. Then the tube formation carried out by the cells cultivated together with stimulating regulatory factors (growth factors) and inhibitory regulatory factors (neutralizing antibodies) is compared with those of untreated ones. For ELISA, recombinant VEGFR (the extracellular domain of the VEGF receptor fused to human IgG Fc region) is coated on ELISA plates. Plates are incubated with VEGF protein, and protein-specific antibodies are used to detect the VEGF in combination.

[0156] Figure 1D The effects of growth factors and neutralizing antibodies (Nabs) on tube development (angiogenesis) by human endothelial cells (HUVECs) are shown. Both VEGF-A and VEGF-D are able to stimulate tube formation independently ( Figure 1D , white and gray bars), and this stimulation was blocked by the addition of neutralizing antibodies against each growth factor. The chimeric VEGF-DA protein was also able to stimulate tube formation (first incubation tube). This stimulation was partially inhibited by the addition of neutralizing antibodies against VEGF-A and VEGF-D alone, and more completely inhibited when both antibodies were added.

[0157] Figure 2 Describe ELISA data, which shows the binding of VEGF protein to the VEGF receptor fixed on the plate. Recombinant human VEGF-A binds to receptor 1 and receptor 2 (column on the left). VEGF-D binds to receptor 3 and receptor 2 (column on the right). Chimeric IN-02 protein binds to receptor 1, receptor 2, and receptor 3. The binding of IN-02 to receptor 2 was only detected when using anti-VEGF-A antibodies (grey columns) because both receptor 2 and anti-VEGF-D antibodies bind to the same region of the chimeric protein. Three rabbits were immunized using 100 μg IN-02 (subcutaneously) on day 0, day 14, day 28, and day 56. Bleeding was performed on day 0 (before immunization), day 2, day 3, and day 56. For clarity, only the pre-immunization and bleed 3 data are shown.

[0158] Figure 3 Depicted are ELISA data showing sera from three rabbits before immunization and after immunization (BL3) with IN-02 protein bound to immobilized immunogen, the figure clearly indicating that all three rabbits mounted an immune response to the immunogen and were unreactive prior to immunization.

[0159] Figure 4 Depicts ELISA data showing binding of sera (purified with caprylic acid) from three rabbits before immunization and after immunization with IN-02 protein (BL3) to immobilized rCTB. All three rabbits developed an immune response to the CTB domain of the immunogen and reacted with rCTB. There was no reactivity before immunization.

[0160] Figure 5 Depicts ELISA data showing binding of sera from three rabbits before immunization and after immunization with IN-02 protein (BL3) to immobilized VEGF-A. All three rabbits developed an immune response to the VEGF-A domain of the immunogen and also reacted with rhVEGF-A. There was no reactivity before immunization.

[0161] Figure 6 Depicted is an ELISA showing binding of sera from three rabbits (BL3) to immobilized VEGF-D before and after immunization with IN-02 protein. All three rabbits mounted 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 rabbits with IN-02 protein, all animals mounted an immune response to the immunizing antigen, which recognized full-length native rhVEGF-A and rhVEGF-D in addition to the immunogenic CTB "carrier" domain.

[0162] To determine the effectiveness of this immune response in neutralizing signaling induced by VEGF-A and VEGF-D, a HUVEC tube formation assay was performed as described earlier. Figure 7 Shown are the results of a tube formation assay performed using caprylic acid-purified serum from rabbits immunized with IN-02 protein. All three sera significantly inhibited tube formation induced by simultaneous co-stimulation with VEGF-A and VEGF-D (black bars).

[0163] Figure 8 Shown are the results of a HUVEC tube formation assay performed on IN-02 protein that had been stored for one month at 4° C. Despite storage, IN-02 protein still stimulated tube formation to a similar extent as VEGF-A or VEGF-D, and this stimulation was effectively inhibited by incubation with an antibody donor capable of neutralizing VEGF-A and VEGF-D.

[0164] Incorporated by Reference

[0165] All documents cited or referenced herein, and all documents cited or referenced in documents cited herein, are incorporated herein by reference, along with any manufacturer's instructions, descriptions, product descriptions, and product data sheets for any products mentioned herein or any documents incorporated herein by reference, and may be used to practice the present disclosure.

[0166] Equivalent

[0167] It should be understood that the detailed examples and embodiments described herein are given as examples only for illustrative purposes and are not to be construed as limiting the present disclosure. Various modifications and changes made thereto by those skilled in the art are suggested, and such modifications and changes are included within the spirit and scope of this application and are deemed to be 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 clearly understood from the appended claims. Furthermore, those skilled in the art will recognize, or will be able to learn using no more than routine experimentation, a variety of equivalents to the specific embodiments disclosed herein. Such equivalents are encompassed by the appended claims.

Claims

1. A chimeric synthetic protein comprising: A chimeric polypeptide sequence, wherein the chimeric polypeptide sequence comprises a homology domain of vascular endothelial growth factor-A and an N-terminal region of vascular endothelial growth factor-D; at least one linking sequence; and comprising a polypeptide sequence of cholera toxin B protein, The chimeric synthetic protein is an initial chimeric synthetic protein consisting of the following amino acid sequence: MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGS QHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTF YDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

2. 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.

3. The chimeric synthetic protein of claim 2, wherein the first linker sequence is SSGGSGGGSG.

4. The chimeric synthetic protein of claim 1, wherein the chimeric polypeptide sequence comprises vascular endothelial growth factor-A, the N-terminal domain of which is substituted with the N-terminus of vascular endothelial growth factor-D.

5. The chimeric synthetic protein of claim 1 , wherein the N-terminal region of the vascular endothelial growth factor-D is TFYDIETLKVIDEEWQRTQ, and the homology domain of the vascular endothelial growth factor-A is CHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

6. The chimeric synthetic protein of claim 1, 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.

7. The chimeric synthetic protein of claim 6, 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.

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

9. An immunogenic composition comprising A chimeric synthetic protein comprising A chimeric polypeptide sequence, wherein the chimeric polypeptide sequence comprises a homology domain of vascular endothelial growth factor-A and an N-terminal region of vascular endothelial growth factor-D; at least one linking sequence; and comprising a polypeptide sequence of cholera toxin B protein, wherein the chimeric synthetic protein is The initial chimeric synthetic protein composed of MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

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

11. The immunogenic composition of claim 9, wherein the first linker sequence is SSGGSGGGSG.

12. The immunogenic composition of claim 9, wherein the chimeric polypeptide sequence comprises vascular endothelial growth factor-A, the N-terminal domain of which is substituted with the N-terminus of vascular endothelial growth factor-D.

13. The immunogenic composition of claim 9, wherein the N-terminal region of vascular endothelial growth factor-D is TFYDIETLKVIDEEWQRTQ, and the homology domain of vascular endothelial growth factor-A is CHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

14. The immunogenic composition 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.

15. The immunogenic composition of claim 14, 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.

16. The immunogenic composition of claim 9, wherein the initial chimeric synthetic protein is processed to create The amino acid sequence of TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSSGGSGGGSGTFYDIETLKVIDEEWQRTQCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEG.

17. The immunogenic composition of claim 9, further comprising an adjuvant.

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