RU2777918C2 - Muc16-targeting anticancer vaccines and application thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular, to new compositions of nucleic acid molecules encoding the modified consensus antigen MUC16, and can be used in medicine. Disclosed is a new modified consensus antigen MUC16, and a possibility of application thereof as a tumour marker is demonstrated.

EFFECT: production of safe and effective combined vaccines applicable against MUC16-expressing tumours thereby providing treatment and contributing to the survival of subjects affected by such types of cancer.

31 cl, 30 dwg, 28 tbl, 6 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority and advantage over U.S. Provisional Application No. 62/598,314, filed December 13, 2017, U.S. Provisional Application No. 62/599,513, filed December 15, 2017, the entire contents of which are incorporated herein through a link.

SEQUENCE LIST

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The specified ASCII copy, created on December 12, 2018, is named l04409_000446_sequence_listing.txt and is 54,027 bytes in size.

FIELD OF TECHNOLOGY

[0003] The present invention relates to MUC16 antigens and nucleic acid molecules that encode them. The present invention also relates to vaccines comprising MUC16 antigens and/or nucleic acid molecules. The present invention further relates to methods for using vaccines to induce immune responses and prevent and/or treat subjects having cancer cells that express MUC16.

BACKGROUND OF THE INVENTION

[0004] Cancer remains the leading cause of death in the US and worldwide. The market for cancer vaccines is growing rapidly. Effective cancer vaccines may be useful in preventing tumor growth and/or may be useful as a more effective, less toxic alternative to standard treatment for patients with advanced cancer. An antigen associated with cancer and hence a target for cancer vaccines is MUC16.

[0005] MUC16 is a member of the mucin family, high molecular weight glycoproteins. Mucins are expressed by specialized epithelial cells surrounding the luminal surface of various organs of the respiratory, gastrointestinal, and reproductive tracts. MUC16 plays a direct and indirect role in maintaining the integrity of the epithelium, as well as in the lubrication and protection of epithelial surfaces.

[0006] The MUC16 tandem repeat domain contains a repetitive peptide epitope, CA125, which has become the gold standard biomarker for a variety of clinical scenarios that arise in the diagnosis and treatment of ovarian cancer, including: 1) screening for early detection, 2) distinguishing between benign and malignant disease in pre- and post-menopausal women with pelvic masses, and 3) monitoring response to therapy. In addition, functional studies have shown that MUC16 promotes the transformation and metastasis of ovarian tumors.

[0007] Because of its high expression in most ovarian cancers, as well as its putative role in potentiating tumorigenesis, MUC16 is an attractive target for a cancer therapeutic vaccine. However, antigen construction is hampered by its large size, complex glycosylation profile, and expression in healthy epithelial tissue.

[0008] Vaccines for the treatment and prevention of cancer are of great interest. Existing vaccines that target tumor cell antigens are often limited by poor expression of the antigen in vivo. Accordingly, there remains a need in the art to develop safe and effective vaccines that are applicable to tumors expressing MUC16, thereby providing treatment and promoting the survival of subjects affected by such cancers.

DISCLOSURE OF THE INVENTION

[0001] Provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-1490 of SEQ ID NO: 2; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-1490 of SEQ ID NO: 2; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-1490 of SEQ ID NO: 2; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to amino acids 19-1490 of SEQ ID NO: 2.

[0002] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: nucleotides 55-4470 of SEQ ID NO: 1; (b) a fragment containing at least 90% of the total length of the nucleic acid molecule, including nucleotides 55-4470 of SEQ ID NO: 1; (c) a fragment that is at least 95% identical to nucleotides 55-4470 of SEQ ID NO: 1; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-4470 of SEQ ID NO: 1.

[0003] Provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 2; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of SEQ ID NO: 2; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 2; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 2.

[0004] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 1; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 1; (c) a fragment that is at least 95% identical to SEQ ID NO: 1; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule contains the nucleic acid sequence specified in SEQ ID NO: one.

[0005] Provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-642 of SEQ ID NO: 4; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-642 of SEQ ID NO: 4; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-642 of SEQ ID NO: 4; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to amino acids 19-642 of SEQ ID NO: 4.

[0006] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: nucleotides 55-1926 of SEQ ID NO: 3; (b) a fragment containing at least 90% of the total length of the nucleic acid molecule, including nucleotides 55-1926 of SEQ ID NO: 3; (c) a fragment that is at least 95% identical to nucleotides 55-1926 of SEQ ID NO: 3; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-1926 of SEQ ID NO: 3.

[0007] Provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 4; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of SEQ ID NO: 4; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 4; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 4.

[0008] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 3; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 3; (c) a fragment that is at least 95% identical to SEQ ID NO: 3; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid molecule contains the nucleic acid sequence indicated in SEQ ID NO: 3.

[0009] Provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-710 of SEQ ID NO: 6; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-710 of SEQ ID NO: 6; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-710 of SEQ ID NO: 6; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to amino acids 19-710 of SEQ ID NO: 6.

[0010] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: nucleotides 55-2130 of SEQ ID NO: 5; (b) a fragment containing at least 90% of the total length of the nucleic acid molecule, including nucleotides 55-2130 of SEQ ID NO: 5; (c) a fragment that is at least 95% identical to nucleotides 55-2130 of SEQ ID NO: 5; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-2130 of SEQ ID NO: 5.

[0011] Provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 6; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of SEQ ID NO: 6; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 6; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 6.

[0012] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 5; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 5; (c) a fragment that is at least 95% identical to SEQ ID NO: 5; and (d) a fragment containing at least 90% of the full length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the nucleic acid molecule contains the nucleic acid sequence indicated in SEQ ID NO: 5.

[0013] Provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-642 and 650-1341 of SEQ ID NO: 8; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-642 and 650-1341 of SEQ ID NO: 8; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8.

[0014] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7; (b) a fragment containing at least 90% of the total length of the nucleic acid molecule, including nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7; (c) a fragment that is at least 95% identical to nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7.

[0015] Provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-1341 of SEQ ID NO: 8; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-1341 of SEQ ID NO: 8; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-1341 of SEQ ID NO: 8; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to amino acids 19-1341 of SEQ ID NO: 8.

[0016] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: nucleotides 55-4023 of SEQ ID NO: 7; (b) a fragment containing at least 90% of the total length of the nucleic acid molecule, including nucleotides 55-4023 of SEQ ID NO: 7; (c) a fragment that is at least 95% identical to nucleotides 55-4023 of SEQ ID NO: 7; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-4023 of SEQ ID NO: 7.

[0017] Provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 8; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of SEQ ID NO: 8; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 8; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 8.

[0018] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 7; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 7; (c) a fragment that is at least 95% identical to SEQ ID NO: 7; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 7. In some embodiments, the nucleic acid molecule contains the nucleic acid sequence indicated in SEQ ID NO: 7.

[0019] One or more of the nucleic acid molecules provided herein may be included in a vector, such as a plasmid or a viral vector. One or more of the nucleic acid molecules provided herein may be included in a vector such as a plasmid or viral vector. In some embodiments, the vector comprises nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-1490 of SEQ ID NO: 2; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-1490 of SEQ ID NO: 2; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-1490 of SEQ ID NO: 2; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of a protein that is at least 95% identical to amino acids 19-1490 of SEQ ID NO: 2. In certain embodiments, the vector includes nucleic acid molecules containing one or more nucleic acid sequences selected from the group consisting of: nucleotides 55-4470 of SEQ ID NO: 1; (b) a fragment containing at least 90% of the total length of the nucleic acid molecule, including nucleotides 55-4470 of SEQ ID NO: 1; (c) a fragment that is at least 95% identical to nucleotides 55-4470 of SEQ ID NO: 1 ; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-4470 of SEQ ID NO: 1. In further embodiments, the vector comprises nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 2; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of SEQ ID NO: 2; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 2; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 2. In still other embodiments, the vector contains nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 1; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 1 ; (c) a fragment that is at least 95% identical to SEQ ID NO: 1; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule contains the nucleic acid sequence indicated in SEQ ID NO: one.

[0020] In further embodiments, the vector comprises one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-642 of SEQ ID NO: 4; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-642 of SEQ ID NO: 4; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-642 of SEQ ID NO: 4; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of a protein that is at least 95% identical to amino acids 19-642 of SEQ ID NO: 4. In certain embodiments, the vector comprises nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: nucleotides 55-1926 of SEQ ID NO: 3; (b) a fragment containing at least 90% of the total length of the nucleic acid molecule, including nucleotides 55-1926 of SEQ ID NO: 3; (c) a fragment that is at least 95% identical to nucleotides 55-1926 of SEQ ID NO: 3; and (d) a fragment containing at least 90% of the full length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-1926 of SEQ ID NO: 3. In some embodiments, the vector comprises nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 4; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of SEQ ID NO: 4; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 4; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 4. In additional embodiments, the vector contains nucleic acid molecules comprising one or more sequences nucleic acids selected from the group consisting of: (a) SEQ ID NO: 3; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 3; (c) a fragment that is at least 95% identical to SEQ ID NO: 3; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid molecule contains the nucleic acid sequence indicated in SEQ ID NO: 3.

[0021] In some embodiments, the vector comprises nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-710 of SEQ ID NO: 6; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-710 of SEQ ID NO: 6; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-710 of SEQ ID NO: 6; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of a protein that is at least 95% identical to amino acids 19-710 of SEQ ID NO: 6. In certain embodiments, the vector comprises nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: nucleotides 55-2130 of SEQ ID NO: 5; (b) a fragment containing at least 90% of the total length of the nucleic acid molecule, including nucleotides 55-2130 of SEQ ID NO: 5; (c) a fragment that is at least 95% identical to nucleotides 55-2130 of SEQ ID NO: 5; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-2130 of SEQ ID NO: 5. In further embodiments, the vector comprises nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 6; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of SEQ ID NO: 6; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 6; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 6. In still other embodiments, the vector contains nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 5; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 5; (c) a fragment that is at least 95% identical to SEQ ID NO: 5; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the nucleic acid molecule contains the nucleic acid sequence indicated in SEQ ID NO: 5.

[0022] In further embodiments, the vector comprises nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-642 and 650-1341 of SEQ ID NO: 8; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-642 and 650-1341 of SEQ ID NO: 8; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8. In certain embodiments, the vector contains molecules nucleic acids comprising one or more nucleic acid sequences selected from the group consisting of: nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7; (b) a fragment containing at least 90% of the total length of the nucleic acid molecule, including nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7; (c) a fragment that is at least 95% identical to nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7; and (d) a fragment containing at least 90% of the full length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7. In additional embodiments, the vector comprises nucleic acid molecules, comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-1341 of SEQ ID NO: 8; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-1341 of SEQ ID NO: 8; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-1341 of SEQ ID NO: 8; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of a protein that is at least 95% identical to amino acids 19-1341 of SEQ ID NO: 8. In certain embodiments, the vector comprises nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: nucleotides 55-4023 of SEQ ID NO: 7; (b) a fragment containing at least 90% of the total length of the nucleic acid molecule, including nucleotides 55-4023 of SEQ ID NO: 7; (c) a fragment that is at least 95% identical to nucleotides 55-4023 of SEQ ID NO: 7; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-4023 of SEQ ID NO: 7. In some embodiments, the vector comprises nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 8; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of SEQ ID NO: 8; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 8; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 8. In some embodiments, the vector contains nucleic acid molecules comprising one or more sequences nucleic acids selected from the group consisting of: (a) SEQ ID NO: 7; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 7; (c) a fragment that is at least 95% identical to SEQ ID NO: 7; and (d) a fragment containing at least 90% of the full length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 7. In some embodiments, the nucleic acid molecule contains the nucleic acid sequence indicated in SEQ ID NO: 7.

[0023] In some embodiments, the nucleic acids provided herein are operably linked to a regulatory element. In some embodiments, the regulatory element is a promoter and/or a polyadenylation signal. In additional embodiments, the promoter is a human cytomegalovirus immediate early promoter (hCMV promoter). In other embodiments, the polyadenylation signal is a bovine growth hormone (bGH poly A) polyadenylation signal.

[0024] Also provided herein are compositions that contain one or more nucleic acid molecules as set forth herein. In some embodiments, these compositions contain a pharmaceutically acceptable carrier.

[0025] Additionally, MUC16 antigenic proteins are provided that contain an amino acid sequence selected from the group consisting of: (a) amino acids 19-1490 of SEQ ID NO: 2; (b) a fragment containing at least 90% of the total length of the protein, including amino acids 19-1490 of SEQ ID NO: 2; (c) an amino acid sequence that is at least 95% identical to amino acids 19-1490 of SEQ ID NO: 2; and (d) a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95% identical to amino acids 19-1490 of SEQ ID NO: 2.

[0026] Additionally, MUC16 antigenic proteins are provided that contain an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 2; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 2; (c) an amino acid sequence that is at least 95% identical to SEQ ID NO: 2; and (c) a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2. In some embodiments, the protein contains the amino acid sequence specified in SEQ ID NO: 2.

[0027] Additionally, MUC16 antigenic proteins are provided comprising an amino acid sequence selected from the group consisting of: (a) amino acids 19-642 of SEQ ID NO: 4; (b) a fragment containing at least 90% of the total length of the protein, including amino acids 19-642 of SEQ ID NO: 4; (c) an amino acid sequence that is at least 95% identical to amino acids 19-642 of SEQ ID NO: 4; and (d) a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95% identical to amino acids 19-642 of SEQ ID NO: 4.

[0028] Additionally, MUC16 antigenic proteins are provided that contain an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 4; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 4; (c) an amino acid sequence that is at least 95% identical to SEQ ID NO: 4; and (c) a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95% identical to SEQ ID NO: 4. In some embodiments, the protein contains the amino acid sequence specified in SEQ ID NO: 2.

[0029] Additionally, MUC16 antigenic proteins are provided that contain an amino acid sequence selected from the group consisting of: (a) amino acids 19-710 of SEQ ID NO: 6; (b) a fragment containing at least 90% of the total length of the protein, including amino acids 19-710 of SEQ ID NO: 6; (c) an amino acid sequence that is at least 95% identical to amino acids 19-710 of SEQ ID NO: 6; and (d) a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95% identical to amino acids 19-710 of SEQ ID NO: 6.

[0030] Additionally, MUC16 antigenic proteins are provided that contain an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 6; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 6; (c) an amino acid sequence that is at least 95% identical to SEQ ID NO: 6; and (c) a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95% identical to SEQ ID NO: 6. In some embodiments, the protein contains the amino acid sequence specified in SEQ ID NO: 6.

[0031] Additionally, MUC16 antigenic proteins are provided comprising an amino acid sequence selected from the group consisting of: (a) amino acids 19-642 and 650-1341 of SEQ ID NO: 8; (b) a fragment containing at least 90% of the total length of the protein, including amino acids 19-642 and 650-1341 of SEQ ID NO: 8; (c) an amino acid sequence that is at least 95% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8; and (d) a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8.

[0032] Additionally, MUC16 antigenic proteins are provided comprising an amino acid sequence selected from the group consisting of: (a) amino acids 19-1341 of SEQ ID NO: 8; (b) a fragment containing at least 90% of the total length of the protein, including amino acids 19-1341 of SEQ ID NO: 8; (c) an amino acid sequence that is at least 95% identical to amino acids 19-1341 of SEQ ID NO: 8; and (d) a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95% identical to amino acids 19-1341 of SEQ ID NO: 8.

[0033] Additionally, MUC16 antigenic proteins are provided that contain an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 8; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 8; (c) an amino acid sequence that is at least 95% identical to SEQ ID NO: 8; and (c) a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95% identical to SEQ ID NO: 8. In some embodiments, the protein contains the amino acid sequence specified in SEQ ID NO: 8.

[0034] Also provided are vaccines containing the MUC16 antigen, wherein the antigen comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the antigen is encoded by SEQ ID NO: 1.

[0035] Also provided are vaccines containing the MUC16 antigen, wherein the antigen comprises the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the antigen is encoded by SEQ ID NO: 3.

[0036] Also provided are vaccines containing the MUC16 antigen, wherein the antigen comprises the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the antigen is encoded by SEQ ID NO: 5.

[0037] Also provided are vaccines containing the MUC16 antigen, wherein the antigen comprises the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the antigen is encoded by SEQ ID NO: 7.

[0038] Additionally, vaccines are provided comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence having at least 95% identity over the entire length of the nucleic acid sequence specified in SEQ ID NO: 1. Vaccines are further provided herein containing a nucleic acid molecule, wherein the nucleic acid molecule encodes a MUC16 antigen comprising an amino acid sequence having at least about 95% identity along the entire length of the amino acid sequence specified in SEQ ID NO: 2.

[0039] Additionally, vaccines are provided comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence having at least about 95% identity over the entire length of the nucleic acid sequence specified in SEQ ID NO: 3. Additionally, this document provides vaccines containing a nucleic acid molecule, wherein the nucleic acid molecule encodes a MUC16 antigen comprising an amino acid sequence having at least about 95% identity along the entire length of the amino acid sequence specified in SEQ ID NO: 4.

[0040] Additionally, vaccines are provided comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence having at least about 95% identity over the entire length of the nucleic acid sequence specified in SEQ ID NO: 5. Additionally, this document provides vaccines containing a nucleic acid molecule, wherein the nucleic acid molecule encodes a MUC16 antigen comprising an amino acid sequence having at least about 95% identity along the entire length of the amino acid sequence specified in SEQ ID NO: 6.

[0041] Additionally, vaccines are provided comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence having at least about 95% identity over the entire length of the nucleic acid sequence specified in SEQ ID NO: 7. Additionally, this document provides vaccines containing a nucleic acid molecule, wherein the nucleic acid molecule encodes a MUC16 antigen comprising an amino acid sequence having at least about 95% identity along the entire length of the amino acid sequence specified in SEQ ID NO: 8.

[0042] In some embodiments, the nucleic acid molecule may comprise an expression vector. Vaccines may additionally contain a pharmaceutically acceptable excipient. In some embodiments, the vaccines may further comprise an adjuvant. In certain embodiments, the adjuvant is IL-12, IL-15, IL-28, or RANTES.

[0043] Also provided herein are vaccines comprising the MUC16 antigen, wherein the antigen comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the antigen is encoded by SEQ ID NO: 1.

[0044] Additionally, vaccines are provided that contain a peptide, wherein the peptide contains an amino acid sequence having at least about 90% identity along the entire length of the amino acid sequence specified in SEQ ID NO: 2.

[0045] Also provided herein are vaccines comprising the MUC16 antigen, wherein the antigen comprises the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the antigen is encoded by SEQ ID NO: 3.

[0046] Additionally, vaccines are provided that contain a peptide, wherein the peptide contains an amino acid sequence having at least about 90% identity along the entire length of the amino acid sequence specified in SEQ ID NO: 4.

[0047] Also provided herein are vaccines comprising the MUC16 antigen, wherein the antigen comprises the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the antigen is encoded by SEQ ID NO: 5.

[0048] Additionally, vaccines are provided that contain a peptide, wherein the peptide contains an amino acid sequence having at least about 90% identity along the entire length of the amino acid sequence specified in SEQ ID NO: 6.

[0049] Also provided herein are vaccines comprising a MUC16 antigen, wherein the antigen comprises the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the antigen is encoded by SEQ ID NO: 7.

[0050] Additionally, vaccines are provided that contain a peptide, wherein the peptide contains an amino acid sequence having at least about 90% identity along the entire length of the amino acid sequence specified in SEQ ID NO: 8.

[0051] Additionally, methods for treating a subject with a cancer cell expressing MUC16 are provided, comprising administering a therapeutically effective amount of the vaccine described herein. In some embodiments, the introduction includes the step of electroporation. In other embodiments, administration occurs at one or more sites on the subject.

[0052] Also provided are methods of vaccinating a subject against a cancer cell expressing MUC16. In some embodiments, the introduction includes the step of electroporation. In other embodiments, administration occurs at one or more sites on the subject.

[0053] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 2; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of SEQ ID NO: 2; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 2; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 2 for use as a drug.

[0054] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 4; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of SEQ ID NO: 4; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 4; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 4 for use as a drug.

[0055] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 6; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of SEQ ID NO: 6; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 6; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 6 for use as a drug.

[0056] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 8; (b) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of SEQ ID NO: 8; (c) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 8; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 8 for use as a drug.

[0057] In some embodiments, the nucleic acid molecules provided herein are for use as a drug in the treatment of cancer. In some embodiments. the nucleic acid molecules provided herein are intended for use in the preparation of a medicament. In some embodiments, the nucleic acid molecules provided herein are for use in the preparation of a medicament for the treatment of cancer.

[0058] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 1; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 1; (c) a fragment that is at least 95% identical to SEQ ID NO: 1; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 1 for use as a drug.

[0059] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 3; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 3; (c) a fragment that is at least 95% identical to SEQ ID NO: 3; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 3 for use as a drug.

[0060] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 5; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 5; (c) a fragment that is at least 95% identical to SEQ ID NO: 5; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 5 for use as a drug.

[0061] Also provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 7; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 7; (c) a fragment that is at least 95% identical to SEQ ID NO: 7; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 7 for use as a drug.

[0062] In some embodiments, the nucleic acid molecules provided herein are for use as a drug in the treatment of cancer. In some embodiments, the nucleic acid molecules provided herein are for use in the preparation of a medicament. In some embodiments, the nucleic acid molecules provided herein are for use in the preparation of a medicament for the treatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIG. 1 illustrates the structure of native human MUC16. The areas targeted by the MUC16 vaccine constructs are indicated.

[0064] FIG. 2 depicts a phylogenetic analysis of native human MUC16 central tandem repeats.

[0065] FIG. 3 illustrates the individual native MUC16 repeat sequences from which each MUC16 microconsensus repeat (RMC) was derived and the percent sequence identity to each other (GenBank: AAL65133.2).

[0066] FIG. 4 shows the amino acid sequence of the carboxy-terminal domain of MUC16. The underlined region (NFSPLARRVDR) is a potential cleavage site, the bold underlined region is the transmembrane domain, and the cytoplasmic tail segment is double underlined. An unspecified sequence above the transmembrane domain is an extracellular ectodomain. Boxes indicate potential S/T/Y phosphorylation sites. The N- and O-glycosylation sites are designated X and O, respectively.

[0067] FIG. 5 illustrates the alignment of the modified consensus ectodomain and transmembrane domain of MUC16 and the ectodomain and transmembrane domain of native human MUC16. Consensus changes are marked in grey.

[0068] FIG. 6A-6B illustrate the assembly strategy of RMC, native repeats, and the synthetic consensus ectodomain and transmembrane domain of MUC16 to reduce the likelihood of off-target epitopes. FIG. 6A shows the preparation of a preliminary synthetic MUC16 immunogen by adding the central tandem repeat and carboxy-terminal domain construction strategies. However, when the RMC4-R61 fusion (last 11 amino acids of RMC4 and first 11 amino acids of native R61) was aligned with all MUC16 native repeat fusions (last 11 amino acids and initial 11 amino acids of adjacent native repeats), the highest sequence identity was only 76.2%. , suggesting that unwanted epitopes may have been introduced in this fusion. FIG. 6B shows that one strategy to avoid RMC4-R61 fusion was to add native R59 between RMC4 and R61.

[0069] FIG. 7 is a schematic representation of four synthetic MUC16 consensus immunogens encoded by plasmids pGXl435, pGXl436, pGXl437, pGXl438 and pGXl439.

[0070] FIG. 8A-8C depict size estimation and comparison modeling of the native and synthetic consensus domain of the human MUC16 central tandem IRC repeat. FIG. 8A is a schematic diagram of a native MUC16 (based on GenBank AAL65133.2) and an IRC synthetic consensus MUC16. FIG. 8B shows comparative modeling to illustrate size differences between native MUC16 (left) and synthetic MUC16 IRC consensus design (right). FIG. 8C is a comparative model of the MUC16 synthetic IRC repeat consensus region shown in cpk format. The microconsensus repeat sequences RMC1 to RMC4 are shown in light grey, the designed furin cleavage site and native repeat sequences R61 to R63 are shown in dark grey.

[0071] FIG. 9 is a diagrammatic representation of the modified backbone of pVAXl (pGX0001).

[0072] FIG. 10 is a diagrammatic representation of the backbone of pGX0003.

[0073] FIG. 11 is a diagrammatic representation of plasmid pGXl435 and IRC+R59 of the synthetic consensus MUC16.

[0074] FIG. 12 is a diagrammatic representation of plasmid pGXl436 and RMC of the synthetic consensus MUC16.

[0075] FIG. 13 is a diagrammatic representation of plasmid pGXl437 and NRC of synthetic consensus MUC16.

[0076] FIG. 14 is a diagrammatic representation of plasmid pGXl438 and RMC synthetic consensus MUC16 and NRC synthetic consensus MUC16.

[0077] FIG. 15 is a diagrammatic representation of plasmid pGXl439 and IRC synthetic consensus MUC16.

[0078] FIG. 16 illustrates the expression of synthetic MUC16 consensus constructs by immunoblotting.

[0079] FIG. 17 is a diagrammatic representation of the peptide pools contained in the peptides related to the MUC16 synthetic consensus antigen proteins used in the embodiments of the present invention.

[0080] FIG. 18A-18D illustrate IFNγ ELISpot responses obtained 3 weeks after 3-fold immunization of CB6F1 females with indicated doses of synthetic consensus MUC16 (pGX1435 (FIG. 18A), pGX1438 (FIG. 18B) and pGX1439 (FIG. 18C), n =8/group) or pGX0001 (empty vector, n=8). Also indicated are the detectable responses to the unique R59 epitope (peptide pool 6) induced by pGXl435 (FIG. 18D).

[0081] FIG. 19A-19D illustrate the results obtained 3 weeks after 3x immunization of CB6F1 females with indicated doses of synthetic consensus MUC16 (pGXl436 (FIG. 19A) and pGXl437 (FIG. 19C), n=8/group) or pGX0001 (empty vector, n=8). Specific IFNγ responses in ELISpot to synthetic consensus MUC16 at indicated dose rates of pGXl436 and pGXl437, and comparison of immune responses to full length constructs (FIG. 19B and FIG. 19D).

[0082] FIG. 20 illustrates a flow cytometry gate strategy used in embodiments of the present invention.

[0083] FIG. 21A-21C illustrate the relative frequency of CD4 ⁺ T cells induced by full-length synthetic MUC16 consensus constructs pGXl435 (FIG. 21A), pGXl438 (FIG. 21B), and pGXl439 (FIG. 21C).

[0084] FIG. 22A-22C illustrate the cytolytic potential of CD4 ⁺ T cells specific for synthetic consensus MUC16 and the cytokine profile induced by pGX1435 (FIG. 22A), PGX1438 (FIG. 22B) and pGX1439 (FIG. 22C).

[0085] FIG. 23A-23C illustrate cellular immune responses induced by pGXl435 (FIG. 23A), pGXl438 (FIG. 23B) and pGXl439 (FIG. 23C) in the CD8 ⁺ T cell compartment.

[0086] FIG. 24A-24C illustrate the cytolytic immune responses and cytokine profile induced by pGXl435 (FIG. 24A), pGXl438 (FIG. 24B), and pGXl439 (FIG. 24C) in the CD8 ⁺ T cell compartment.

[0087] FIG. 25A-25B illustrate cellular immune responses induced by pGXl436 in the CD4 ⁺ T cell compartment (FIG. 25A) and cytolytic immune responses and CD4 ⁺ T cell specific response profile to the pGXl436 partial length construct (FIG. 25B).

[0088] FIG. 26A-26B illustrate cellular immune responses induced by pGXl437 in the CD4 ⁺ T cell compartment (FIG. 26A) and cytolytic immune responses and CD4 ⁺ T cell specific response profile to the pGXl437 partial length construct (FIG. 26B).

[0089] FIG. 27A-27B illustrate cellular immune responses induced by pGXl436 in the CD8 ⁺ T cell compartment (FIG. 27A) and cytolytic immune responses and CD8 ⁺ T cell specific response profile to partial length constructs (FIG. 27B).

[0090] FIG. 28A-28B illustrate cellular immune responses induced by pGXl437 in the CD8 ⁺ T cell compartment (FIG. 28A) and cytolytic immune responses and CD8 ⁺ T cell specific response profile to partial length constructs (FIG. 28B).

[0091] FIG. 29A-29B illustrate comparison of cellular immune responses induced by pGXl436 (FIG. 29A) and pGXl437 (FIG. 29B) against full length constructs in CD4 ⁺ and CD8 ⁺ T cell compartments and cytokine profiles of specific cellular responses of CD4 ⁺ and CD8 T cells. ⁺ .

[0092] FIG. 30A-30C provide a summary of IFNγ data (FIG. 30A) and cellular data for CD4 ⁺ T cells (FIG. 30B) and CD8 ⁺ (FIG. 30C) for the full length constructs disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

[0093] The present invention relates to vaccines containing the MUC16 antigen. MUC16 is expressed in many tumors. Accordingly, the vaccines provide treatment for a cancer or tumor expressing MUC16. The vaccine of the invention may provide any combination of specific cancer antigens for a specific prevention or treatment of cancer in a subject in need of treatment.

[0094] One way to construct a nucleic acid and its encoded amino acid sequence of a recombinant cancer antigen is to introduce mutations that change specific amino acids in the overall amino acid sequence of the native cancer antigen. The introduction of mutations does not change the cancer antigen so much that it cannot be universally applied in a mammalian subject and preferably in a human or canine subject, but changes it so much that the resulting amino acid sequence breaks tolerance or is considered a foreign antigen in the body, in order to generate immune response. Another way would be to create a consensus recombinant cancer antigen that has at least 85% to 99% amino acid sequence identity with the corresponding native cancer antigen; preferably at least 90% and up to 98% sequence identity; more preferably at least 93% and up to 98% sequence identity; or even more preferably at least 95% and up to 98% sequence identity. In some cases, the recombinant cancer antigen is 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to the corresponding native cancer antigen. A native cancer antigen is an antigen usually associated with a particular cancer or cancer. Depending on the cancer antigen, the cancer antigen consensus sequence may occur in different mammalian species, or within subtypes of a species, or among viral strains or serotypes. Some cancer antigens do not differ much from the amino acid sequence of the wild-type cancer antigen. Some cancer antigens have nucleic acid/amino acid sequences that vary so much between species that a consensus sequence cannot be obtained. In these cases, a recombinant cancer antigen is generated that breaks tolerance and generates an immune response that has at least 85% to 99% amino acid sequence identity with the corresponding native cancer antigen; preferably at least 90% and up to 98% sequence identity; more preferably at least 93% and up to 98% sequence identity; or even more preferably at least 95% and up to 98% sequence identity. In some instances, the recombinant cancer antigen is 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to its corresponding native cancer antigen. The above approaches can be combined such that the final recombinant cancer antigen has a percentage similarity to the amino acid sequence of the native cancer antigen, as discussed above.

[0095] The mucin-16 antigen ("MUC16") may be a MUC16 consensus antigen derived in part from MUC16 sequences from different species or from different isoforms within a species, and thus the MUC16 consensus antigen is not native.

[0096] The MUC16 antigen may also include a selection of microconsensus repeat (RMC) or native repeat (R) sequences derived from native RMC or R sequences. In some embodiments, nucleic acid constructs are provided in which two or more RMC and/or R linked to each other by proteolytic cleavage sites. A furin proteolytic cleavage site is an example of a proteolytic cleavage site that can bind the RMC and/or R domains in the fusion protein expressed by the construct.

[0097] Further modifications to the MUC16 antigen may include mutations of asparagine residues in the ectodomains and transmembrane domains and the addition of previous Kozak and/or IgE leader sequences to the N-terminus of the MUC16 antigen.

[0098] Recombinant MUC16 can induce antigen-specific T cells and/or high antibody titers, thereby inducing or eliciting an immune response that is directed to or responds to an antigen-expressing cancer or tumor. In some embodiments, the induced or elicited immune response may be a cellular, humoral, or both cellular and humoral immune response. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon-gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or evoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of an antigen-expressing tumor or cancer, such as, without limitation, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T- cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSCs, MCP-1 and immune checkpoint molecule.

[0099] The vaccine can be further combined with antibodies to checkpoint inhibitors such as PD-1 and PDL-1 to enhance stimulation of both cellular and humoral immune responses. The use of anti-PD-1 or anti-PDL-1 antibodies prevents suppression by T-cell and/or B-cell responses. In general, designing cancer antigens to be recognized by the immune system helps overcome other forms of immunosuppression by tumor cells, and these vaccines can be used in combination with suppression or inhibition therapies (eg, anti-PD-1 antibody and anti-PDL-1 therapy) to further increase in T-cell and/or B-cell responses.

Definitions

[00100] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art. In the event of a conflict, the regulation will be governed by this document, including the definitions. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned in this document are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting. The terminology used herein is intended to describe specific embodiments only and is not meant to be limiting.

[00101] The terms "include(s)", "comprise(s)", "having", "has", "may", "include(s)" and variations thereof, in the context of this document, are intended to serve as transitional open-ended phrases, terms or words that do not exclude the possibility of additional actions or structures. The singular forms "a", "and", and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising", "consisting of", and "consisting essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[00102] To list numerical ranges, this document explicitly provides for each intermediate value having the same degree of accuracy as the minimum and maximum of the listed range. For example, for the range 6-9, the numbers 7 and 8 are considered in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly provided.

[00103] The term "adjuvant", as used herein, means any molecule added to the vaccines described herein to enhance the immunogenicity of MUC16 antigens and/or antigen-encoding nucleic acid molecules as described herein.

[00104] The term "antibody", as used herein, means an antibody of the classes IgG, IgM, IgA, IgD, or IgE, or fragments or derivatives thereof, including Fab, F(ab') 2, Fd and single chain antibodies, diabodies, bispecific antibodies , bifunctional antibodies and their derivatives. The antibody can be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity antibody, or any mixture thereof that exhibits sufficient binding specificity to the desired epitope or sequence derived from it.

[00105] "MUC16 antigen" refers to proteins containing an amino acid sequence selected from the group consisting of: amino acids 19-1490 of SEQ ID NO: 2; amino acids 19-642 SEQ ID NO: 4; amino acids 19-710 SEQ ID NO: 6; amino acids 19-642 and 650-1341 SEQ ID NO: 8; SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; their fragments of the length specified herein, variants, i.e. proteins with sequences having sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 as specified herein, fragments of variants having the lengths indicated herein in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8; their fragments of the length specified herein, variants, i.e. proteins with sequences having sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 as specified herein, fragments of variants having the lengths specified in this document and their combinations. Antigens may optionally include signal peptides such as peptides from other proteins.

[00106] The terms "coding sequence" or "coding nucleic acid", as used herein, means nucleic acids (RNA or DNA molecules) that contain a nucleotide sequence that codes for a protein. The coding sequence may further include initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal capable of directing expression in cells of the subject or mammal to which the nucleic acid is administered.

[00107] The terms "complementarity" or "complementary", as used herein, means that a nucleic acid may involve Watson-Crick base pairing (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules .

[00108] The terms "consensus" or "consensus sequence", as used herein, means a polypeptide sequence based on multiple sequence alignment analysis for the same gene from different organisms. Nucleic acid sequences can be prepared that encode a consensus polypeptide sequence. Vaccines containing proteins that contain consensus sequences and/or nucleic acid molecules that encode such proteins can be used to induce broad immunity against an antigen.

[00109] As used herein, the term "direct current" describes the current that is sensed or experienced by the tissue or cells that define said tissue during the duration of an electrical impulse delivered to that same tissue. An electrical impulse is supplied from the electroporation devices described herein. The current density remains constant in said tissue during the duration of the electrical impulse because the electroporation device provided herein has a feedback element, preferably with instantaneous feedback. The feedback element allows you to measure the resistance of the tissue (or cells) throughout the duration of the pulse and cause the electroporation device to change the output electrical power (for example, increase the voltage) so that the current density in the same tissue remains constant during the entire exposure time electrical impulse (on the order of microseconds) and from impulse to impulse. In some embodiments, the feedback element comprises a controller.

[00110] ] The terms "current feedback" or "feedback", in the context of this document, can be used interchangeably and can mean the active response of the provided electroporation devices, which includes measuring the current in the tissue between the electrodes and the corresponding change in the output power delivered by the device EP, to maintain the current density at a constant level. This constant level is set by the user before initiating the pulse train or electrotherapy. Feedback can be provided by an electroporation component, such as the controller of an electroporation device, since the electrical circuit in it is able to continuously monitor the tissue current between the electrodes and compare this controlled current (or tissue current) with the target current and continuously adjust the output energy to maintain the controlled current. at given levels. The feedback loop can be instantaneous because it is analog closed loop.

[00111] The term "decentralized current" as used herein may refer to the circuitry of electrical currents supplied from the various sets of needle electrodes of the electroporation devices described herein, the circuitry minimizing or preferably eliminating the occurrence of thermal stress associated with electroporation, in any area of tissue undergoing electroporation.

[00112] "Electroporation", "electropermeabilization", or "electrokinetic amplification" ("EP"), used interchangeably herein, means the pulsed action of a transmembrane electric field to induce microscopic pathways (pores) in a biomembrane; their presence allows biomolecules such as plasmids, oligonucleotides, miRNAs, drugs, ions, and water to pass from one side of the cell membrane to the other.

[00113] In the context of this document, the term "fragment", when referring to nucleic acid sequences, means a nucleic acid sequence or portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. The fragments may be fragments of a nucleic acid molecule selected from at least one of the various nucleotide sequences that encode the protein fragments indicated below. Fragments may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% , at least 90% or at least 95% of the total length of one or more of the nucleic acid sequences described herein, with the exception of the addition of any heterologous signal peptide. In some embodiments, fragments may contain at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the total length of one or more of the nucleic acid sequences listed below, excluding any added heterologous signal peptide.

[00114] In some embodiments, the fragments may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60 %, at least 70%, at least 80%, at least 90%, or at least 95% identical to one or more of the nucleic acid sequences described herein, with the exception of any heterologous signal peptide. In some embodiments, fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one or more of the nucleic acid sequences specified below, excluding any added heterologous signal peptide.

[00115] In further embodiments, the fragments may optionally additionally include a sequence encoding a heterologous signal peptide that is not taken into account when calculating percent identity. The fragments may also contain coding sequences for a signal peptide, such as an immunoglobulin signal peptide, for example an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and/or a signal peptide may be linked to a coding sequence fragment.

[00116] In some embodiments, fragments may include at least 1700 nucleotides or more, 1750 nucleotides or more, 1800 nucleotides or more, 1850 nucleotides or more, 1900 nucleotides or more, 1950 nucleotides or more, 2000 nucleotides or more, 2050 nucleotides 2100 nucleotides or more 2150 nucleotides or more 2200 nucleotides or more 2250 nucleotides or more 2300 nucleotides or more 2350 nucleotides or more 2400 nucleotides or more 2450 nucleotides or more 2500 nucleotides or more 2600 nucleotides or more 2650 nucleotides or more 2700 nucleotides or more 2750 nucleotides or more 2800 nucleotides or more 2850 nucleotides or more 2900 nucleotides or more 2950 nucleotides or more 3000 nucleotides or more or more, 3100 nucleotides or more, 3150 nucleotides or more, 3200 nucleotides or more, 3250 nucleotides or more, 3300 nucleotides or more 3350 nucleotides or more 3400 nucleotides or more 3450 nucleotides or more 3500 nucleotides or more 3550 nucleotides or more 3600 nucleotides or more 3650 nucleotides or more 3700 nucleotides or more more than 3850 nucleotides or more 3900 nucleotides or more 3950 nucleotides or more 4000 nucleotides or more 4050 nucleotides or more 4100 nucleotides or more 4150 nucleotides or more 4200 nucleotides or more more than 4350 nucleotides or more, 4400 nucleotides or more, 4450 nucleotides or more, 4500 of at least one of the nucleic acid sequences listed below.

[00117] The terms "fragment" or "immunogenic fragment", when referring to polypeptide sequences, means a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. Fragments may be polypeptide fragments selected from at least one of the various amino acid sequences described herein. The consensus protein fragments may include at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the total length of the consensus protein, excluding any added heterologous signal peptide. In some embodiments, the fragment may contain at least 90%, at least 91%, at least 92.%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% of the length of one or more of the amino acid sequences listed below, excluding any added heterologous signal peptide.

[00118] In some embodiments, the fragments may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60 %, at least 70%, at least 80%, at least 90%, or at least 95% identical to one or more amino acid sequences described herein, with the exception of any heterologous signal peptide. In some embodiments, fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one or more of the amino acid sequences specified in herein, excluding any added heterologous signal peptide.

[00119] In additional embodiments, fragments may optionally further include a sequence encoding a heterologous signal peptide that is not taken into account when calculating percent identity. The fragments may further comprise a signal peptide, such as an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide.

[00120] In some embodiments, consensus protein fragments may be 550 amino acids or more, 600 amino acids or more, 650 amino acids or more, 700 amino acids or more, 750 amino acids or more, 800 amino acids or more, 850 amino acids or more, 900 amino acids or more. more than 950 amino acids or more 1000 amino acids or more 1050 amino acids or more 1100 amino acids or more 1150 amino acids or more 1200 amino acids or more 1250 amino acids or more 1300 amino acids or more 1350 of the protein sequence disclosed herein .

[00121] As used herein, the term "genetic construct" refers to DNA or RNA molecules that contain a nucleotide sequence encoding a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal capable of directing expression in the cells of the subject to which the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a gene construct that contains the necessary regulatory elements operably linked to a protein-coding coding sequence such that, when present in a subject's cell, the coding sequence will be expressed.

[00122] In the context of this document, the term "homology" refers to the degree of complementarity. There may be partial homology or complete homology (that is, identity). A partially complementary sequence that at least partially inhibits the hybridization of a fully complementary sequence to a target nucleic acid refers to the use of the functional term "substantially homologous". When used in relation to a double-stranded nucleic acid sequence, such as a cDNA or genomic clone, the term "substantially homologous" as used herein refers to a probe that can hybridize to a strand of a double-stranded nucleic acid sequence under conditions of low stringency. When used with respect to a single-stranded nucleic acid sequence, the term "substantially homologous", as used herein, refers to a probe that can hybridize (i.e., be sequence-complementary) to a single-stranded template nucleic acid sequence under conditions of low stringency.

[00123] The terms "identical" or "identity", in the context of this document, with respect to two or more nucleic acid or polypeptide sequences, means that the sequences have a certain percentage of residues that are the same in a specified region. The percentage can be calculated by optimally aligning two sequences, comparing the two sequences in the specified region, determining the number of positions where the same remainder occurs in both sequences to get the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to get percent sequence identity. In cases where the two sequences are of different lengths or the alignment results in one or more stepped ends and the specified area of comparison includes only one sequence, the remainders of one sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identification can be done manually or with a computer sequencing algorithm such as BLAST or BLAST 2.0.

[00124] The term "impedance", in the context of this document, can be used when considering the details of the feedback mechanism and can be converted to the current value in accordance with Ohm's law, which allows comparison with a given current.

[00125] The term "immune response", in the context of this document, means the activation of the immune system of the host, for example, a mammal, in response to the introduction of an antigen. The immune response may be in the form of a cellular or humoral response, or both.

[00126] ] The terms "nucleic acid" or "oligonucleotide" or "polynucleotide", as used herein, means that at least two nucleotides are covalently linked to each other. The description of one circuit also determines the sequence of the additional circuit. Thus, the nucleic acid also spans the complementary strand of the single strand represented. Many nucleic acid variants can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also includes substantially identical nucleic acids and their complements. One strand provides a probe that can hybridize to the target sequence under stringent hybridization conditions. Thus, the nucleic acid also includes a probe that hybridizes under stringent hybridization conditions.

[00127] Nucleic acids may be single-stranded or double-stranded, or may contain parts of both a double-stranded and a single-stranded sequence. The nucleic acid may be DNA, either genomic or cDNA, RNA or hybrid nucleic acid, wherein the nucleic acid may contain combinations of deoxyribo- and ribonucleotides and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine , isocytosine and isoguanine. Nucleic acids can be produced by chemical synthesis or by recombinant methods.

[00128] The term "operably linked", as used herein, means that the expression of a gene is under the control of the promoter to which it is spatially linked. A promoter can be located 5' (left) or 3' (right) from the gene under its control. The distance between a promoter and a gene may be about the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, changing this distance can be accommodated without loss of promoter function.

[00129] In the context of this document, the terms "peptide", "protein" or "polypeptide" can mean an associated sequence of amino acids and can be natural, synthetic, or be a modification or combination of natural and synthetic.

[00130] The term "promoter", as used herein, means a synthetic or naturally occurring molecule that is capable of conferring, activating or enhancing the expression of a nucleic acid in a cell. The promoter may contain one or more specific transcriptional regulatory sequences to further enhance the expression and/or change the spatial expression and/or temporal expression of the nucleic acid in the cell. The promoter may also contain distal enhancer or repressor elements, which may be up to several thousand base pairs away from the transcriptional start site. The promoter can be obtained from sources including viral, bacterial, fungal, plant, insect and animal sources. A promoter may regulate the expression of a gene component constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac promoter operator, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter, CMV promoter IE and human cytomegalovirus (hCMV) immediate early promoter. In certain embodiments, the promoter is the hCMV promoter.

[00131] The terms "signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a protein provided herein. Signal peptides/leader sequences typically direct protein localization. As used herein, the signal peptides/leader sequences preferably facilitate the secretion of the protein from the cell in which it is produced. Signal peptides/leader sequences are often cleaved from a remnant of the protein, often referred to as the mature protein, upon secretion from the cell. The signal peptides/leader sequences are linked at the amino-terminus (ie, N-terminus) of the protein.

[00132] The term "stringent hybridization conditions", as used herein, means the conditions under which a first nucleic acid sequence (eg, a probe) will hybridize to a second nucleic acid sequence (eg, a target), such as a complex mixture of nucleic acids. Stringent conditions are sequence dependent and will be different in different circumstances. Stringent conditions can be chosen to be about 5-10° C. lower than the melting point (Tm) for a particular sequence at a particular pH and ionic strength. Tm can be the temperature (at a certain ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium (because the target sequences are present in excess, at Tm, 50% of the probes are occupied in equilibrium state). Stringent conditions may be such that the salt concentration is less than about 1.0 M sodium ion, for example, a concentration of about 0.01-1.0 M sodium ion (or other salts) at pH 7.0-8.3, and the temperature is at least about 30° C. for short probes (eg, about 10-50 nucleotides) and at least about 60° C. for long probes (eg, greater than about 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2-10 times the background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC and 1% SDS, incubated at 42°C or 5x SSC, 1% SDS, incubated at 65°C, with a wash in 0.2x SSC and 0.1% SDS at 65°C.

[00133] The term "subject", as used herein, can mean a mammal that requires, wants, or needs to be immunized with the vaccines provided herein. The mammal may be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

[00134] "Substantially complementary", as used herein, means that the first sequence is at least 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98% or 99% identical to the complementary strand of the second sequence in region 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360 , 450, 540 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[00135] "Substantially identical", as used herein, means that the first and second sequences are at least 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88 , 89, 90, 91, 92, 93, 94, 95, 96, 97, 98% or 99% identical at 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450 , 540 or more nucleotides or amino acids, or in relation to nucleic acids, if the first sequence is essentially complementary to the complement of the second sequence.

[00136] The terms "treat", "treating", or "treating", as used herein, may mean protecting an animal from a disease by means of preventing, suppressing, containing, or eliminating the disease. Prevention of a disease includes administering the vaccine of the present invention to an animal prior to the onset of the disease. Suppression of a disease includes administering the vaccine of the present invention to an animal after the induction of the disease but before its clinical manifestation. Controlling the disease includes administering the vaccine of the present invention to an animal after clinical manifestation of the disease.

[00137] The term "variant", in the context of this document, with respect to a nucleic acid, means (i) a part or fragment of the specified nucleotide sequence; (ii) the complement of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid that is essentially identical to the specified nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to said nucleic acid, its complement, or a sequence substantially identical to it.

[00138] The term "variant", as used herein, in relation to a peptide or polypeptide, means a peptide or polypeptide that differs in amino acid sequence by an amino acid insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. A variant can also mean a protein with an amino acid sequence that is substantially identical to a reference protein with an amino acid sequence that retains at least one biological activity. Conservative substitution of an amino acid, that is, the replacement of an amino acid with another amino acid with similar properties (eg, hydrophilicity, degree and distribution of charged regions), is generally recognized in the art as a minor change. These minor changes can be identified, in part, in terms of the amino acid hydrophobicity index, as is understood in the art. Kyte et al, J. Mol. Biol. 157:105-132 (1982). The hydrophobicity index of an amino acid is based on its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophobicity indices can be substituted and still retain protein function. In one aspect, amino acids having hydrophobicity indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that cause proteins to retain biological function. Considering amino acid hydrophilicity in the context of a peptide allows the calculation of the greatest local average hydrophilicity of that peptide, which is a useful measure that has been reported to correlate well with antigenicity and immunogenicity. US Patent No. 4,554,101 is incorporated herein in its entirety by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, eg immunogenicity, as is understood in the art. Substitutions can be made with amino acids having hydrophilicity values within ±2. Both the hydrophobicity index and the amino acid hydrophilicity value depend on the specific side chain of that amino acid. In accordance with this observation, it is believed that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids and, in particular, on the side chains of these amino acids, which manifests itself in hydrophobicity, hydrophilicity, charge, size and other properties.

[00139] ] A variant may be a nucleic acid sequence that is substantially identical throughout the entire length of the gene sequence or fragment thereof. The nucleic acid sequence may be 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 95, 96, 97, 98, 99%, or 100% identity throughout the length of the gene sequence or its fragment. A variant may be an amino acid sequence that is substantially identical over the entire length of the amino acid sequence or fragment thereof. The amino acid sequence can be 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 95, 96, 97, 98, 99%, or 100% identity over the entire length amino acid sequence or fragment thereof.

[00140] The term "vector", in the context of this document, means a nucleic acid sequence containing a source of replication. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector and is preferably a DNA plasmid. The vector may contain or include one or more heterologous nucleic acid sequences.

Vaccine

Provided herein are vaccines containing a MUC16 antigen or a nucleic acid molecule encoding a MUC16 antigen as described herein. In some embodiments, the vaccines comprise one or more nucleic acid molecules that encode the MUC16 antigen as described herein. In some embodiments, the vaccines contain one or more nucleic acid molecules that contain a nucleic acid sequence encoding amino acids 19-1490 of SEQ ID NO: 2; a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-1490 of SEQ ID NO: 2; a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-1490 of SEQ ID NO: 2; and/or a nucleic acid sequence encoding a fragment containing at least 90% of the total length of a protein that is at least 95% identical to amino acids 19-1490 of SEQ ID NO: 2. In some embodiments, the vaccines contain one or more nucleic acid molecules which include a nucleic acid sequence encoding amino acids 19-642 of SEQ ID NO: 4; a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-642 of SEQ ID NO: 4; a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-642 of SEQ ID NO: 4; and/or a nucleic acid sequence encoding a fragment containing at least 90% of the total length of a protein that is at least 95% identical to amino acids 19-642 of SEQ ID NO: 4. In some embodiments, the vaccines contain one or more nucleic acid molecules which include a nucleic acid sequence encoding amino acids 19-710 of SEQ ID NO: 6; a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-710 of SEQ ID NO: 6; a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-710 of SEQ ID NO: 6; and/or a nucleic acid sequence encoding a fragment containing at least 90% of the total length of a protein that is at least 95% identical to amino acids 19-710 of SEQ ID NO: 6. In some embodiments, the vaccines contain one or more nucleic acid molecules , which include a nucleic acid sequence encoding amino acids 19-642 and 650-1341 of SEQ ID NO: 8; a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-642 and 650-1341 of SEQ ID NO: 8; a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8; and/or a nucleic acid sequence encoding a fragment containing at least 90% of the total length of a protein that is at least 95% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8. In some embodiments, the vaccines contain one or more nucleic acid molecules that include a nucleic acid sequence encoding amino acids 19-1341 of SEQ ID NO: 8; a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, including amino acids 19-1341 of SEQ ID NO: 8; a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-1341 of SEQ ID NO: 8; and/or a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to amino acids 19-1341 of SEQ ID NO: 8.

[00141] In some embodiments, the vaccines comprise one or more nucleic acid molecules that include nucleotides 55-4470 of SEQ ID NO: 1; a fragment containing at least 90% of the total length of the nucleic acid molecule containing nucleotides 55-4470 of SEQ ID NO: 1; a fragment that is at least 95% identical to nucleotides 55-4470 of SEQ ID NO: 1; and/or a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-4470 of SEQ ID NO: 1. In some embodiments, the vaccines contain one or more nucleic acid molecules that include nucleotides 55-1926 SEQ ID NO: 3; a fragment containing at least 90% of the total length of the nucleic acid molecule containing nucleotides 55-1926 of SEQ ID NO: 3; a fragment that is at least 95% identical to nucleotides 55-1926 of SEQ ID NO: 3; and/or a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-1926 of SEQ ID NO: 3. In some embodiments, the vaccines contain one or more nucleic acid molecules that include nucleotides 55-2130 SEQ ID NO: 5; a fragment containing at least 90% of the total length of the nucleic acid molecule containing nucleotides 55-2130 of SEQ ID NO: 5; a fragment that is at least 95% identical to nucleotides 55-2130 of SEQ ID NO: 5; and/or a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-2130 of SEQ ID NO: 5. In some embodiments, the vaccines contain one or more nucleic acid molecules that include nucleotides 55-1926 and 1948-4023 SEQ ID NO: 7; a fragment containing at least 90% of the total length of a nucleic acid molecule containing nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7; a fragment that is at least 95% identical to nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7; and/or a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7. In some embodiments, the vaccines contain one or more nucleic acid molecules acids which include nucleotides 55-4023 of SEQ ID NO: 7; a fragment containing at least 90% of the total length of the nucleic acid molecule, including nucleotides 55-4023 of SEQ ID NO: 7; a fragment that is at least 95% identical to nucleotides 55-4023 of SEQ ID NO: 7; and/or a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to nucleotides 55-4023 of SEQ ID NO: 7.

[00142] In some embodiments, the vaccines contain one or more nucleic acid molecules that include a nucleic acid sequence encoding SEQ ID NO: 2; a nucleic acid sequence encoding a fragment containing at least 90% of the length of SEQ ID NO. 2; a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 2; and/or a nucleic acid sequence encoding a fragment containing at least 90% of the total length of a protein that is at least 95% identical to SEQ ID NO: 2. In some embodiments, the vaccines contain one or more nucleic acid molecules that include the sequence nucleic acid encoding SEQ ID NO: 4; a nucleic acid sequence encoding a fragment containing at least 90% of the length of SEQ ID NO. four; a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 4; and/or a nucleic acid sequence encoding a fragment containing at least 90% of the total length of a protein that is at least 95% identical to SEQ ID NO: 4. In some embodiments, the vaccines contain one or more nucleic acid molecules that include the sequence nucleic acid encoding SEQ ID NO: 6; a nucleic acid sequence encoding a fragment containing at least 90% of the length of SEQ ID NO. 6; a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 6; and/or a nucleic acid sequence encoding a fragment containing at least 90% of the total length of a protein that is at least 95% identical to SEQ ID NO: 6. In some embodiments, the vaccines contain one or more nucleic acid molecules that include the sequence nucleic acid encoding SEQ ID NO: 8; a nucleic acid sequence encoding a fragment containing at least 90% of the length of SEQ ID NO. eight; a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 8; and/or a nucleic acid sequence encoding a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 8.

[00143] In some embodiments, the vaccines contain one or more nucleic acid molecules that include SEQ ID NO: 1; a fragment containing at least 90% of the total length of SEQ ID NO: 1; the fragment is at least 95% identical to SEQ ID NO: 1; and/or a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the vaccines contain one or more nucleic acid molecules that include SEQ ID NO: 3; a fragment containing at least 90% of the total length of SEQ ID NO: 3; the fragment is at least 95% identical to SEQ ID NO: 3; and/or a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 3. In some embodiments, the vaccines contain one or more nucleic acid molecules that include SEQ ID NO: 5; a fragment containing at least 90% of the total length of SEQ ID NO: 5; the fragment is at least 95% identical to SEQ ID NO: 5; and/or a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the vaccines contain one or more nucleic acid molecules that include SEQ ID NO: 7; a fragment containing at least 90% of the total length of SEQ ID NO: 7; the fragment is at least 95% identical to SEQ ID NO: 7; and/or a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 7.

[00144] In some embodiments, the vaccines contain the MUC16 antigen, wherein the antigen comprises amino acids 19-1490 of SEQ ID NO: 2; a fragment containing at least 90% of the total length of the protein, including amino acids 19-1490 of SEQ ID NO: 2; the protein is at least 95% identical to amino acids 19-1490 of SEQ ID NO: 2; and/or a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to amino acids 19-1490 of SEQ ID NO: 2.

[00145] In some embodiments, the vaccine contains a MUC16 antigen, wherein the antigen comprises SEQ ID NO: 2; a fragment containing at least 90% of the length of SEQ ID NO. 2; an amino acid sequence that is at least 95% identical to SEQ ID NO: 2; and/or a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 2.

[00146] In some embodiments, the vaccines contain the MUC16 antigen, wherein the antigen comprises amino acids 19-642 of SEQ ID NO: 4; a fragment containing at least 90% of the total length of the protein, including amino acids 19-642 of SEQ ID NO: 4; a protein that is at least 95% identical to amino acids 19-642 of SEQ ID NO: 4; and/or a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to amino acids 19-642 of SEQ ID NO: 4.

[00147] In some embodiments, the vaccine comprises a MUC16 antigen, wherein the antigen comprises SEQ ID NO: 4; a fragment containing at least 90% of the length of SEQ ID NO. four; an amino acid sequence that is at least 95% identical to SEQ ID NO: 4; and/or a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 4.

[00148] In some embodiments, the vaccines contain the MUC16 antigen, wherein the antigen comprises amino acids 19-710 of SEQ ID NO: 6; a fragment containing at least 90% of the total length of the protein, including amino acids 19-710 of SEQ ID NO: 6; a protein that is at least 95% identical to amino acids 19-710 of SEQ ID NO: 6; and/or a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to amino acids 19-710 of SEQ ID NO: 6.

[00149] In some embodiments, the vaccine contains a MUC16 antigen, wherein the antigen comprises SEQ ID NO: 6; a fragment containing at least 90% of the length of SEQ ID NO. 6; an amino acid sequence that is at least 95% identical to SEQ ID NO: 6; and/or a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 6.

[00150] In some embodiments, the vaccine contains a MUC16 antigen, wherein the antigen comprises amino acids 19-642 and 650-1341 of SEQ ID NO: 8; a fragment containing at least 90% of the total length of the protein, including amino acids 19-642 and 650-1341 of SEQ ID NO: 8; an amino acid sequence that is at least 95% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8; and/or a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8.

[00151] In some embodiments, the vaccine contains a MUC16 antigen, wherein the antigen comprises amino acids 19-1341 of SEQ ID NO: 8; a fragment containing at least 90% of the total length of the protein, including amino acids 19-1341 of SEQ ID NO: 8; an amino acid sequence that is at least 95% identical to amino acids 19-1341 of SEQ ID NO: 8; and/or a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95% identical to amino acids 19-1341 of SEQ ID NO: 8.

[00152] In some embodiments, the vaccine comprises a MUC16 antigen, wherein the antigen comprises SEQ ID NO: 8; a fragment containing at least 90% of the length of SEQ ID NO. eight; an amino acid sequence that is at least 95% identical to SEQ ID NO: 8; and/or a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to SEQ ID NO: 8.

[00153] Vaccines may be capable of eliciting an immune response against an antigen in a subject. The immune response may be a therapeutic or prophylactic immune response. Vaccines can be used to protect against cancer, for example cancer or tumor expressing MUC16. Vaccines can be used to prevent and/or treat a tumor expressing MUC16 in a subject in need thereof. Vaccines can induce cellular and/or antibody responses against MUC16 and against tumors expressing MUC16. In one embodiment, vaccines can be used to protect against MUC16-expressing ovarian cancer cells, to prevent and/or treat, or to induce a cellular and/or antibody response against MUC16-expressing ovarian cancer cells, specifically MUC16-expressing epithelial ovarian cancer cells. more specifically, serous ovarian cancer cells expressing MUC16.

[00154] The development of a cancer vaccine as described herein involves the identification of a cancer antigen, such as MUC16, that is not recognized by the immune system and is an aberrantly expressed autoantigen. The identified cancer antigen is changed from self antigen to foreign antigen for recognition by the immune system. Changing the structure of the nucleic acid and amino acid sequences of the recombinant cancer antigen in the direction from the structure of "own" to a foreign antigen violates the tolerance of the immune system against the antigen. Several restructuring measures can be used to break tolerance to a cancer antigen, as described below.

[00155] The recombinant cancer antigen of the vaccine is not recognized as its own, thereby breaking tolerance. Impaired tolerance can induce antigen-specific T-cell and/or high titer antibody responses, thereby inducing or eliciting an immune response that is directed against the cancer or tumor or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon-gamma (IFN-γ) and/or tumor necrosis factor-alpha (TNF-α). In other embodiments, the induced or induced immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of an antigen-expressing tumor or cancer, for example, but not limited to, factors that suppress MHC presentation, factors that stimulate antigen-specific regulatory T- cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-l, MDSCs, MCP-1 and immune checkpoint molecule.

[00156] The vaccine may be a DNA vaccine. DNA vaccines are disclosed in US Patent Nos. 5,593,972; 5,739,118; 5,817,637; 5,830,876; 5,962,428; The DNA vaccine may additionally contain elements or reagents that prevent its integration into the chromosome.

[00157] The vaccine may be an RNA of one or more cancer antigens. The RNA vaccine can be introduced into the cell.

[00158] The vaccine can be a live attenuated vaccine, a vaccine using recombinant antigen delivery vectors, subunit vaccines, and glycoprotein vaccines, such as, but not limited to, the vaccines presented in US Patent Nos.: 4,510,245; 4,797,368; 4,722,848;4,790,987; 4,920,209; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424; 5,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668; 5,387,744; 5,389,368; 5,424,065; 5,451,499; 5.453.3 64; 5,462,734; 5,470,734; 5,474,935; 5,482,713; 5,591,439; 5,643,579; 5,650,309; 5,698,202; 5,955,088; 6,034,298; 6,042,836; 6,156,319 and 6,589,529, which are incorporated herein by reference in their entirety.

[00159] In some embodiments, the vaccine may further comprise a molecular adjuvant, in some cases the molecular adjuvant may be IL-12, IL-15, IL-28, IL-31, IL-33 and/or RANTES, and in some cases the molecular adjuvant is a checkpoint inhibitor, including anti-cytotoxic T-lymphocyte type 4 antigen (CTLA-4), programmed death receptor-1 (PD-1), and lymphocyte activation gene (LAG-3). The coding sequence for IL-12, IL-15, IL-28, IL-31, IL-33 and/or RANTES may be included in one or more nucleic acid molecules that contain the coding sequence for one or more antigens. The coding sequence for IL-12, IL-15, IL-28, IL-31, IL-33 and/or RANTES can be included in separate nucleic acid molecules, such as a separate plasmid.

[00160] The vaccine of the present invention may have properties required for effective vaccines, such as safety such that the vaccine itself does not cause disease or death; protect against disease; inducing neutralizing antibodies; inducing protective responses of T cells; and providing ease of administration, few side effects, biological stability and low cost per dose. A vaccine may achieve some or all of these characteristics by containing a cancer antigen, as discussed below.

[00161] The vaccine may further comprise one or more inhibitors of one or more immune checkpoint molecules (ie, an immune checkpoint inhibitor). The immune checkpoint molecules are described in more detail below. An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any component in the immune system, such as presentation by MHC classes, presentation and/or differentiation of T cells, presentation and/or differentiation of B cells, any cytokine, chemokine, or transmission signals for the proliferation and/or differentiation of immune cells. As also described in more detail below, the vaccine can be further combined with antibodies to checkpoint inhibitors such as PD-1 and PDL-1 to enhance stimulation of both cellular and humoral immune responses. The use of anti-PD-1 or anti-PDL-1 antibodies prevents T-cell and/or B-cell responses from suppressing PD-1 or PDL-1.

Antigen

[00162] The vaccine described above may contain an antigen or a nucleic acid molecule encoding an antigen. The antigen may be MUC16, a fragment thereof, a variant thereof, or a combination thereof. MUC16 is a member of the mucin family of high molecular weight glycoproteins. Mucins are expressed by specialized epithelial cells surrounding the luminal surface of various organs of the respiratory, gastrointestinal, and reproductive tracts. Mucins play a direct and indirect role in maintaining the integrity of the epithelium and in lubricating and protecting epithelial surfaces.

[00163] MUC16 has been associated with tumor or cancer formation. The MUC16 tandem repeat domain contains a repetitive peptide epitope, CA125, which has become the gold standard biomarker for a variety of clinical scenarios that arise in the diagnosis and treatment of ovarian cancer, including: 1) screening for early detection, 2) distinguishing between benign and malignant disease in women before and postmenopausal with pelvic mass, and 3) monitoring response to therapy. In addition, functional studies have shown that MUC16 promotes the transformation and metastasis of ovarian tumors.

[00164] Accordingly, the vaccine can be used to treat subjects suffering from cancer or tumors expressing MUC16. In some embodiments, the cancer is ovarian cancer. The MUC16 antigen may differ from native "normal" MUC16 and thus provide therapy or prophylaxis against a tumor expressing the MUC16 antigen. Accordingly, provided herein are MUC16 antigen sequences that differ from the native MUC16 sequence, and nucleic acid molecules encoding such MUC16 antigen sequences (ie, recombined or mutated MUC16 genes or sequences).

[00165] Proposed nucleic acid molecules containing the above heterologous sequences. Proposed nucleic acid molecules, consisting of the above heterologous sequences. In some embodiments, nucleic acid molecules comprising one or more nucleic acid sequences are selected from the group consisting of: (a) nucleotides 55-4470 of SEQ ID NO: 1; (b) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of a nucleic acid molecule containing nucleotides 55-4470 of SEQ ID NO: 1; (c) a fragment that is at least 95%, 96%, 97%, 98% or 99% identical to nucleotides 55-4470 of SEQ ID NO: 1; and (d) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total length of the nucleic acid sequence, which is at least 95%, 96%, 97%, 98% or 99% identical to nucleotides 55-4470 of SEQ ID NO: 1. In some embodiments, nucleic acid molecules comprising one or more nucleic acid sequences are selected from the group consisting of: (a) SEQ ID NO: 1; (b) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of SEQ ID NO: 1; (c) a fragment that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule contains the sequence nucleic acid as set forth in SEQ ID NO: 1.

[00166] In some embodiments, nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) nucleotides 55-1926 of SEQ ID NO: 3; (b) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of a nucleic acid molecule comprising nucleotides 55-1926 of SEQ ID NO: 3; (c) a fragment that is at least 9%, 96%, 97%, 98%, or 99% identical to nucleotides 55-1926 of SEQ ID NO: 3; and (d) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total length of the nucleic acid sequence, which is at least 95%, 96%, 97%, 98% or 99% identical to nucleotides 55-1926 of SEQ ID NO: 3. In some embodiments, nucleic acid molecules comprising one or more nucleic acid sequences are selected from the group consisting of: (a) SEQ ID NO: 3; (b) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of SEQ ID NO: 3; (c) a fragment that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid molecule contains the sequence nucleic acid as set forth in SEQ ID NO: 3.

[00167] In some embodiments, nucleic acid molecules comprising one or more nucleic acid sequences are selected from the group consisting of: (a) nucleotides 55-2130 of SEQ ID NO: 5; (b) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of a nucleic acid molecule containing nucleotides 55-2130 of SEQ ID NO: 5; (c) a fragment that is at least 95, 96, 97, 98% or 99% identical to nucleotides 55-2130 of SEQ ID NO: 5; and (d) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total length of the nucleic acid sequence, which is at least 95%, 96%, 97%, 98% or 99% identical to nucleotides 55-2130 of SEQ ID NO: 5. In some embodiments, nucleic acid molecules comprising one or more nucleic acid sequences are selected from the group consisting of: (a) SEQ ID NO: 5; (b) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of SEQ ID NO: 5; (c) a fragment that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5. In some embodiments, the nucleic acid molecule contains the sequence nucleic acid as set forth in SEQ ID NO: 5.

[00168] In some embodiments, nucleic acid molecules comprising one or more nucleic acid sequences are selected from the group consisting of: (a) nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7; (b) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of a nucleic acid molecule containing nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7 ; (c) a fragment that is at least 95%, 96%, 97%, 98%, or 99% identical to nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7; and (d) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total length of the nucleic acid sequence, which is at least 95%, 96%, 97%, 98% or 99% identical to nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7. In some embodiments, nucleic acid molecules comprising one or more nucleic acid sequences are selected from the group consisting of: (a) nucleotides 55-4023 of SEQ ID NO: 7; (b) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of a nucleic acid molecule containing nucleotides 55-4023 of SEQ ID NO: 7; (c) a fragment that is at least 95%, 96%, 97%, 98%, or 99% identical to nucleotides 55-4023 of SEQ ID NO: 7; and (d) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total length of the nucleic acid sequence, which is at least 95%, 96%, 97%, 98% or 99% identical to nucleotides 55-4023 of SEQ ID NO: 7. In some embodiments, nucleic acid molecules comprising one or more nucleic acid sequences are selected from the group consisting of: (a) SEQ ID NO: 7; (b) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of SEQ ID NO: 7; (c) a fragment that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7; and (d) a fragment containing at least 90% of the total length of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the nucleic acid molecule contains the sequence nucleic acid as set forth in SEQ ID NO: 7.

[00169] Provided herein are nucleic acid sequences encoding MUC16 antigens. In some embodiments, nucleic acid molecules comprising one or more nucleic acid sequences are selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-1490 SEQ ID NO: 2; (b) a nucleic acid sequence encoding a fragment containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98% or 99% of the total length of the protein, including amino acids 19-1490 of SEQ ID NO: 2; (c) a nucleic acid sequence encoding a protein that is at least 95%, 96%, 97%, 98%, or 99% identical to amino acids 19-1490 of SEQ ID NO: 2; and (d) a nucleic acid sequence encoding a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of the protein, which is at least 95%, 96%, 97% , 98% or 99% identical to amino acids 19-1490 of SEQ ID NO: 2. In some embodiments, nucleic acid molecules are provided comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 2; (b) a nucleic acid sequence encoding a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of SEQ ID NO: 2; (c) a nucleic acid sequence encoding a protein that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total protein length that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2.

[00170] In some embodiments, nucleic acid molecules are provided comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-642 of SEQ ID NO: 4; (b) a nucleic acid sequence encoding a fragment containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98% or 99% of the total length of the protein, including amino acids 19-642 of SEQ ID NO: 4; (c) a nucleic acid sequence encoding a protein that is at least 95%, 96%, 97%, 98%, or 99% identical to amino acids 19-642 of SEQ ID NO: 4; and (d) a nucleic acid sequence encoding a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of the protein, which is at least 95%, 96%, 97% , 98% or 99% identical to amino acids 19-642 of SEQ ID NO: 4. In some embodiments, nucleic acid molecules are provided comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 4; (b) a nucleic acid sequence encoding a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of SEQ ID NO: 4; (c) a nucleic acid sequence encoding a protein that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total protein length that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4.

[00171] In some embodiments, nucleic acid molecules comprising one or more nucleic acid sequences are selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-710 of SEQ ID NO: 6; (b) a nucleic acid sequence encoding a fragment containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98% or 99% of the total length of the protein, including amino acids 19-710 of SEQ ID NO: 6; (c) a nucleic acid sequence encoding a protein that is at least 95%, 96%, 97%, 98%, or 99% identical to amino acids 19-710 of SEQ ID NO: 6; and (d) a nucleic acid sequence encoding a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of the protein, which is at least 95%, 96%, 97% , 98% or 99% identical to amino acids 19-710 of SEQ ID NO: 6. In some embodiments, nucleic acid molecules are provided comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 6; (b) a nucleic acid sequence encoding a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of SEQ ID NO: 6; (c) a nucleic acid sequence encoding a protein that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total protein length that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6.

[00172] In some embodiments, nucleic acid molecules are provided comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-642 and 650-1341 of SEQ ID NO: 8; (b) a nucleic acid sequence encoding a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of a protein comprising amino acids 19-642 and 650-1341 of SEQ ID NO: 8; (c) a nucleic acid sequence encoding a protein that is at least 95%, 96%, 97%, 98% or 99% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8; and (d) a nucleic acid sequence encoding a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of the protein, which is at least 95%, 96%, 97% , 98% or 99% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8. In some embodiments, nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence , encoding amino acids 19-1341 SEQ ID NO: 8; (b) a nucleic acid sequence encoding a fragment containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98% or 99% of the total length of the protein, including amino acids 19-1341 of SEQ ID NO: 8; (c) a nucleic acid sequence encoding a protein that is at least 95%, 96%, 97%, 98%, or 99% identical to amino acids 19-1341 of SEQ ID NO: 8; and (d) a nucleic acid sequence encoding a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of the protein, which is at least 95%, 96%, 97% , 98% or 99% identical to amino acids 19-1341 of SEQ ID NO: 8. In some embodiments, nucleic acid molecules are provided comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO: 8; (b) a nucleic acid sequence encoding a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of SEQ ID NO: 8; (c) a nucleic acid sequence encoding a protein that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; and (d) a nucleic acid sequence encoding a fragment containing at least 90% of the total protein length that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.

[00173] Isolated nucleic acid molecules containing the heterologous sequences described above can be included in vectors such as plasmids, viral vectors, and other forms of nucleic acid molecules below.

[00174] Proposed protein molecules containing the heterologous amino acid sequences described above. Proposed protein molecules, consisting of the above-described heterologous amino acid sequences. provided herein are proteins and polypeptides having the sequences described above. The proteins and polypeptide may be referred to as MUC16 antigens and MUC16 immunogens. MUC16 antigens are capable of eliciting an immune response against tumors expressing the MUC16 antigen. In some embodiments, proteins comprising an amino acid sequence are selected from the group consisting of: (a) amino acids 19-1490 of SEQ ID NO: 2; (b) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of a protein comprising amino acids 19-1490 of SEQ ID NO: 2; (c) an amino acid sequence that is at least 95, 96, 97, 98%, or 99% identical to amino acids 19-1490 of SEQ ID NO: 2; and (d) a fragment containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98%, or 99% of the total length of an amino acid sequence that is at least 95, 96, 97, 98% or 99% identical to amino acids 19-1490 of SEQ ID NO: 2. In some embodiments, proteins containing an amino acid sequence are selected from the group consisting of: (a) SEQ ID NO: 2; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 2; (c) an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2; and (c) a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the protein contains the amino acid sequence specified in SEQ ID NO: 2.

[00175] In some embodiments, proteins comprising an amino acid sequence are selected from the group consisting of: (a) amino acids 19-642 of SEQ ID NO: 4; (b) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of a protein comprising amino acids 19-642 of SEQ ID NO: 4; (c) an amino acid sequence that is at least 95, 96, 97, 98%, or 99% identical to amino acids 19-642 of SEQ ID NO: 4; and (d) a fragment containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98%, or 99% of the total length of an amino acid sequence that is at least 95, 96, 97, 98% or 99% identical to amino acids 19-642 of SEQ ID NO: 4. In some embodiments, proteins containing an amino acid sequence are selected from the group consisting of: (a) SEQ ID NO: 4; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 4; (c) an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4; and (c) a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the protein contains the amino acid sequence specified in SEQ ID NO: 4.

[00176] In some embodiments, proteins comprising an amino acid sequence are selected from the group consisting of: (a) amino acids 19-710 of SEQ ID NO: 6; (b) a fragment containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98% or 99% of the total length of the protein, including amino acids 19-710 of SEQ ID NO: 6; (c) an amino acid sequence that is at least 95, 96, 97, 98%, or 99% identical to amino acids 19-710 of SEQ ID NO: 6; and (d) a fragment containing at least 90, 91, 92, 93, 94, 95.96, 97, 98% or 99% of the total length of the amino acid sequence, which is at least 95%, 96%, 97%, 98% or 99% identical to amino acids 19-710 of SEQ ID NO: 6. In some embodiments, proteins comprising an amino acid sequence are selected from the group consisting of: (a) SEQ ID NO: 6; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 6; (c) an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6; and (c) a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In some embodiments, the protein contains the amino acid sequence specified in SEQ ID NO: 6.

[00177] In some embodiments, proteins comprising an amino acid sequence are selected from the group consisting of: (a) amino acids 19-642 and 650-1341 of SEQ ID NO:8; (b) a fragment containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98% or 99% of the total length of the protein, including amino acids 19-642 and 650-1341 of SEQ ID NO: 8; (c) an amino acid sequence that is at least 95, 96, 97, 98%, or 99% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8; and (d) a fragment containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98% or 99% of the total length of the amino acid sequence, which is at least 95%, 96%, 97%, 98% or 99% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8. In some embodiments, proteins comprising an amino acid sequence are selected from the group consisting of: (a) amino acids 19-1341 of SEQ ID NO: 8 ; (b) a fragment containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total length of a protein comprising amino acids 19-1341 of SEQ ID NO: 8; (c) an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to amino acids 19-1341 of SEQ ID NO: 8; and (d) a fragment containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98%, or 99% of the total length of an amino acid sequence that is at least 95, 96, 97, 98% or 99% identical to amino acids 19-1341 of SEQ ID NO: 8. In some embodiments, proteins comprising an amino acid sequence are selected from the group consisting of: (a) SEQ ID NO: 8; (b) a fragment containing at least 90% of the total length of SEQ ID NO: 8; (c) an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; and (c) a fragment containing at least 90% of the total length of an amino acid sequence that is at least 95, 96%, 97, 98%, or 99% identical to SEQ ID NO: 8. In some embodiments, the protein contains an amino acid sequence, specified in SEQ ID NO: 8.

[00178] In one aspect of the invention, it is desirable that the consensus antigen provides improved transcription and translation, including the presence of one or more of the following: a low GC leader sequence to increase transcription; mRNA stability and codon optimization and elimination, as far as possible, of cis-acting sequence motifs (i.e. internal TATA boxes).

[00179] The MUC16 antigen can be an antigen (or immunogen) consensus sequence derived from two or more species. In some embodiments, the consensus antigen may comprise a MUC16 ectodomain and a transmembrane domain. The MUC16 consensus antigen may contain one or more mutations in the ectodomain and/or transmembrane domain. One or more mutations may include the replacement of one or more amino acids that are involved in N-glycosylation. One or more mutations may include the replacement of asparagine by alanine. Accordingly, in some embodiments, one or more mutations may replace 1, 2, or 3 amino acids in the ectodomain and/or transmembrane domain of MUC16.

[00180] In some embodiments, the MUC16 antigen may further include any combination of one or more microconsensus repeat (RMC) sequences derived from native RMC sequences, e.g., RMC1, RMC2, RMC3, and/or RMC4. In some embodiments, the MUC16 antigen may include any combination of one or more native repeat (R) sequences, such as R59, R61, R62 and/or R63. In some embodiments, the MUC16 antigen includes the sequences RMC1, RMC2, RMC3, RMC4, R61, R62, and R63. In some embodiments, the MUC16 antigen includes the sequences RMC1, RMC2, RMC3, RMC4, R59, R61, R62, and R63. In some embodiments, the MUC16 antigen includes the sequences RMC1, RMC2, RMC3, and RMC4. In some embodiments, the MUC16 antigen includes the sequences R61, R62, and R63.

[00181] The MUC16 antigen may contain modifications for improved expression. The modification may include codon optimization, RNA optimization, addition of a Kozak sequence (eg, GCC ACC) to increase translation initiation, and/or addition of an immunoglobulin leader sequence to increase the immunogenicity of the MUC16 antigen. The MUC16 antigen may comprise a signal peptide, such as an immunoglobulin signal peptide, for example, without limitation, an immunoglobulin E (IgE) or immunoglobulin G (IgG) signal peptide.

[00182] The MUC16 antigen may contain modifications to optimize the epitope. In some embodiments, a cleavage site may be inserted between RMC sequences, between R sequences, or at the border of RMC or R sequences. The cleavage site may be a furin cleavage site. In certain embodiments, a furin cleavage site may be inserted between RMC4 and R61.

Vaccine in combination with immune checkpoint inhibitor

[00183] The vaccine may further comprise one or more inhibitors of one or more immune checkpoint molecules (ie, an immune checkpoint inhibitor). The immune checkpoint molecules are described in more detail below. An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any component in the immune system, such as presentation by MHC classes, presentation and/or differentiation of T cells, presentation and/or differentiation of B cells, any cytokine, chemokine, or transmission signals for the proliferation and/or differentiation of the immune cell.

[00184] Such an inhibitor may be a nucleic acid sequence, an amino acid sequence, a small molecule, or a combination thereof. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid may also include additional sequences that encode linker or tag sequences that are linked to the immune checkpoint inhibitor by a peptide bond. A small molecule can be a low molecular weight, for example, less than 800 daltons, organic or inorganic compound that can serve as a substrate for an enzyme, a ligand (or analogue thereof) associated with a protein or nucleic acid, or a regulator of a biological process. The amino acid sequence may be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.

[00185] In some embodiments, an immune checkpoint inhibitor may be one or more nucleic acid sequences encoding an antibody, a variant thereof, a fragment thereof, or a combination thereof. In other embodiments, the immune checkpoint inhibitor may be an antibody, a variant thereof, a fragment thereof, or a combination thereof.

a. Immune checkpoint molecule

[00186] An immune checkpoint molecule can be a nucleic acid sequence, an amino acid sequence, a small molecule, or a combination thereof. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid may also include additional sequences that encode linker or tag sequences that are linked to the immune checkpoint inhibitor by a peptide bond. A small molecule can be a low molecular weight, for example, less than 800 daltons, organic or inorganic compound that can serve as a substrate for an enzyme, a ligand (or analogue thereof) associated with a protein or nucleic acid, or a regulator of a biological process. The amino acid sequence may be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.

(1) PD-1 and PD-L1

[00187] The immune checkpoint molecule can be programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), a fragment thereof, a variant thereof, or a combination thereof. PD-1 is a cell surface protein encoded by the PDCDI gene. PD-1 is a member of the immunoglobulin superfamily and is expressed on T cells and pro-B cells and thus promotes recruitment and/or differentiation of these cells. In particular, PD-1 is a type 1 membrane protein of the CD28/CTLA-4 T cell regulator family and negatively regulates T cell receptor (TCR) signals, thereby negatively regulating immune responses. PD-1 can negatively regulate CD8+ T cell responses and thus inhibit CD8-mediated cytotoxicity and promote growth.

[00188] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 is upregulated on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, and on T cells and B cells in TCR signaling and B cell receptors. PD-L1 is expressed in many tumor cell lines, including myelomas, mastocytomas, and melanomas.

b. Antibody against immune checkpoint molecule

[00189] The immune checkpoint inhibitor described above may be an antibody. An antibody may bind or react with an antigen (ie, the immune checkpoint molecule described above). Accordingly, an antibody may be considered to be an antibody against an immune checkpoint molecule or an antibody against an immune checkpoint molecule. An antibody may be encoded by a nucleic acid sequence contained herein.

[00190] An antibody may include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide may include a heavy chain variable region (VH) and/or at least one heavy chain constant region (CH). The at least one heavy chain constant region may include heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3) and/or a hinge region.

[00191] In some embodiments, the heavy chain polypeptide may include a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide may include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.

[00192] A heavy chain polypeptide may include a set of complementarity determining regions ("CDRs"). A CDR set may contain three VH hypervariable regions. Based on the N-terminus of the heavy chain polypeptide, these CDRs are designated "CDR1", "CDR2", and "CDR3", respectively. The CDR1, CDR2, and CDR3 of a heavy chain polypeptide may contribute to antigen binding or recognition.

[00193] The light chain polypeptide may include a variable light chain (VL) region and/or a constant light chain (CL) region. The light chain polypeptide may include a set of complementarity determining regions ("CDRs"). A CDR set may contain three VL hypervariable regions. Based on the N-terminus of the light chain polypeptide, these CDRs are designated "CDR1", "CDR2", and "CDR3", respectively. The CDR1, CDR2, and CDR3 of a light chain polypeptide can contribute to antigen binding or recognition.

[00194] An antibody may contain a set of light chain and heavy chain complementarity determining ("CDR") regions, and accordingly be placed between a set of heavy chain and light chain framework regions ("FR"), which provide support for the CDR and determine the spatial relationship different CDRs relative to each other. A set of CDRs may contain three heavy or light chain V region hypervariable regions. Based on the N-terminus of the heavy or light chain, these regions are designated "CDR1", "CDR2", and "CDR3", respectively. Therefore, an antigen binding site may include six CDRs, including a set of CDRs from each heavy and light chain V region.

[00195] The antibody may be an immunoglobulin (Ig). The Ig may be, for example, IgA, IgM, IgD, IgE and IgG. The immunoglobulin may include a heavy chain polypeptide and a light chain polypeptide. An immunoglobulin heavy chain polypeptide may include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. An immunoglobulin light chain polypeptide may include a VL region and a CL region.

[00196] In addition, the proteolytic enzyme papain preferentially cleaves IgG molecules to form multiple fragments, two of which (F(ab) fragments) contain a covalent heterodimer that includes an intact antigen-binding site. The pepsin enzyme is able to cleave IgG molecules into several fragments, including the F(ab') 2 fragment, which contains both antigen-binding sites. Accordingly, the antibody may be a Fab or F(ab') 2. The Fab may include a heavy chain polypeptide and a light chain polypeptide. The Fab heavy chain polypeptide may include a VH region and a CH1 region. The light chain Fab may include a VL region and a CL region.

[00197] The antibody may be a polyclonal or monoclonal antibody. The antibody can be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. The humanized antibody may be a non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and the framework regions of a human immunoglobulin molecule.

(1) PD-l antibody

[00198] An anti-immune checkpoint antibody can be an anti-PD-1 antibody (also referred to herein as an "anti-PD-1 antibody"), a variant thereof, a fragment thereof, or a combination thereof. The anti-PD-1 antibody may be nivolumab. An anti-PD-1 antibody can inhibit the activity of PD-1, thereby inducing, eliciting or enhancing an immune response against a tumor or cancer and reducing tumor growth.

(2) PD-L1 antibody

[00199] An anti-immune checkpoint antibody can be an anti-PD-L1 antibody (also referred to herein as an "anti-PD-L1 antibody"), a variant thereof, a fragment thereof, or a combination thereof. An anti-PD-L1 antibody can inhibit PD-L1 activity, thereby inducing, eliciting or enhancing an immune response against a tumor or cancer and reducing tumor growth.

Vector

[00200] The vaccine may contain one or more vectors that include a heterologous nucleic acid encoding a MUC16 antigen. One or more vectors may be capable of expressing an antigen in an amount effective to elicit an immune response in a mammal. The vector may contain a heterologous nucleic acid encoding an antigen. The vector may have a nucleic acid sequence containing an origin of replication. The vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector can either be a self-replicating extrachromosomal vector or a vector that integrates into the host's genome.

[00201] One or more vectors may be an expression construct, which is usually a plasmid, which is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein encoded by the gene is produced by the ribosomal complexes of the cellular transcription and translation machinery. The plasmid is often designed to contain regulatory sequences that act in the enhancer and promoter regions and result in efficient transcription of the gene transferred to the expression vector. The vectors of the present invention express large amounts of stable messenger RNA and hence proteins.

[00202] Vectors may have expression signals such as a strong promoter, a strong termination codon, regulation of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and PTIS (portable translation initiation sequence).

[00203] The vectors may contain nucleic acid sequences operably linked to a regulatory element selected from a promoter and a polyadenylation signal. In some embodiments, the promoter is a human cytomegalovirus immediate early promoter (hCMV promoter). In some embodiments, the polyadenylation signal is a bovine growth hormone (bGH poly A) polyadenylation signal.

[00204] The vector can be a circular plasmid or a linear nucleic acid. The circular plasmid and the linear nucleic acid are capable of directing the expression of a particular nucleotide sequence in the corresponding cell of the subject. The vector may have a promoter operably linked to a nucleotide sequence encoding an antigen that may be operably linked to termination signals. The vector may also contain sequences necessary for the correct translation of the nucleotide sequence, as well as sequences for cloning and subcloning of the vector and its fragments. The vector containing the desired nucleotide sequence may be chimeric, which means that at least one of its components is heterologous with respect to at least one of its other components. Expression of a nucleotide sequence in an expression cassette may be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when a particular external stimulus is applied to the host cell. In the case of a multicellular organism, the promoter may also be specific to a particular tissue or organ or developmental stage.

[00205] The vector may be a plasmid. The plasmid may be useful for transfecting cells with an antigen-encoding nucleic acid that is cultured and maintained in transformed host cells under conditions under which the antigen is expressed.

[00206] A plasmid may include a nucleic acid that encodes one or more of the various antigens described above, including coding sequences that encode a synthetic consensus antigen capable of eliciting an immune response against the antigen, fragments of such proteins, variants of such proteins, fragments of variants, or fusion proteins. , which consist of combinations of consensus proteins and/or consensus protein fragments and/or consensus protein variants and/or consensus protein variant fragments.

[00207] In some embodiments, the plasmid may further comprise a coding sequence that encodes CCR20 alone or as part of one of these plasmids. Similarly, plasmids may further contain coding sequences for IL-12, IL-15 and/or IL-28.

[00208] The plasmid may further comprise a start codon, which may be upstream of the coding sequence, and a stop codon, which may be downstream of the coding sequence. The initiation and termination codon may be in reading frame with the coding sequence.

[00209] The plasmid may also contain a promoter that is operably linked to a coding sequence. The promoter operably linked to the coding sequence may be the simian virus 40 (SV40) promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus long terminal repeat (LTR) promoter. cattle (BIV), Moloney virus promoter, avian leukemia virus (ALV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter, Epstein-Barr virus (EBV) promoter or Rous sarcoma virus (RSV) promoter. The promoter may also be a human gene promoter such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter can also be a tissue specific promoter such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are provided in US Patent Application Publication No. US 20040175727, the content of which is incorporated herein in its entirety.

[00210] The plasmid may also contain a polyadenylation signal, which may be located after the coding sequence. The polyadenylation signal can be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be the polyadenylation signal from plasmid pCEP4 (Invitrogen, San Diego, CA).

[00211] The plasmid may also contain an enhancer before the coding sequence. The enhancer can be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as a CMV, FMDV, RSV or EBV enhancer. Amplifications of polynucleotide function are presented in US Pat.

[00212] The plasmid may also contain a mammalian source of replication for maintaining an extrachromosomal plasmid and making multiple copies of the plasmid in a cell. The plasmid may be p V AXI, pCEP4 or pREP4 from Invitrogen (San Diego, CA) which may include an Epstein-Barr virus origin of replication and an EBNA-1 nuclear antigen coding region which may produce high copy number episomal replication without integration . The basis of the plasmid can be pA V0242. The plasmid may be a replication-deficient adenovirus type 5 (Ad5) plasmid.

[00213] The plasmid may also contain a regulatory sequence that may be well suited for gene expression in the cell into which the plasmid is introduced. The coding sequence may contain a codon that can provide more efficient transcription of the coding sequence in the host cell.

[00214] The coding sequence may also contain an Ig leader sequence. The leader sequence may be a sequence encoding 5". Consensus antigens encoded by this sequence may contain an N-terminal leader Ig followed by a consensus antigenic protein. The N-terminal leader Ig may be IgE or IgG.

[00215] The plasmid may be pSE420 (Invitrogen, San Diego, CA), which may be used to produce protein in Escherichia coli (E. coli). The plasmid can also be pYES2 (Invitrogen, San Diego, CA) which can be used for protein production in yeast strains of Saccharomyces cerevisiae. The plasmid can also have the complete MAXBAC™ baculovirus expression system (Invitrogen, San Diego, CA) which can be used to produce the protein in insect cells. The plasmid can also be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells. The plasmid can also be pGX001 (Inovio) modified from pVAX1 (Thermo Fisher Scientific, Waltham, MA).

[00216] The vector can be a circular plasmid that can transform the target cell by integrating into the cellular genome or exist extrachromosomally (eg, an autonomous replicating plasmid with an origin of replication).

[00217] The vector can be pVAX, pcDNA3.0 or provax, or any other expression vector capable of expressing the DNA encoding the antigen and allowing the cell to translate the sequence into an antigen that is recognized by the immune system.

[00218] Also provided herein is a linear nucleic acid vaccine or linear expression cassette ("LEC") that is capable of being efficiently delivered to a subject by electroporation and expression of one or more desired antigens. The LEC can be any linear DNA lacking any phosphate backbone. DNA may encode one or more antigens. The LEC may contain a promoter, an intron, a stop codon, and/or a polyadenylation signal. The expression of an antigen can be controlled by a promoter. The LEC may not contain antibiotic resistance and/or phosphate backbone genes. The LEC may not contain other nucleic acid sequences not associated with the desired expression of the antigen gene.

[00219] LEC can be obtained from any plasmid capable of linearization. The plasmid may be capable of expressing the antigen. The plasmid can be pNP (Puerto Rico/34) or pM2 (New Caledonia/99). The plasmid can be WLV009, pVAX, pcDNA3.0 or provax, or any other expression vector capable of expressing DNA encoding an antigen and allowing the cell to translate the sequence into an antigen that is recognized by the immune system.

[00220] The LEC may be pcrM2. The LEC may be pcrNP. pcrNP and pcrMR can be obtained from pNP (Puerto Rico/34) and pM2 (New Caledonia/99), respectively.

[00221] The vector may have a promoter. The promoter can be any promoter that is capable of directing gene expression and regulating the expression of the isolated nucleic acid. Such a promoter is a cis-acting sequence element required for transcription through a DNA-dependent RNA polymerase that transcribes an antigen sequence as described herein. The choice of promoter used for direct expression of a heterologous nucleic acid depends on the particular application. The promoter can be located approximately the same distance from the transcription start in the vector as it is from the transcription start site in its natural setting. However, changing this distance can be accepted without loss of promoter function.

[00222] A promoter may be operably linked to a nucleic acid sequence encoding an antigen and signals necessary for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.

[00223] The promoter can be a CMV promoter, an SV40 early promoter, an SV40 late promoter, a metallothionein promoter, a mouse mammary tumor virus promoter, a Rous sarcoma virus promoter, a polyhedrin promoter, or another promoter that has been shown to be effective for expression in eukaryotic cells.

[00224] The vector may include an enhancer and an intron with functional splicing donor and acceptor sites. The vector may contain a transcription termination region located upstream of the transcription of the structural gene to ensure efficient termination. The terminal region may be derived from the same gene as the promoter sequence or may be derived from different genes.

Methods for obtaining a vector

[00225] Provided herein are methods for producing a vector that contains a nucleic acid molecule encoding the MUC16 antigen discussed herein. The vector, after the final step of subcloning into a mammalian expression plasmid, can be used to inoculate cell cultures in a large scale fermentation tank using methods known in the art.

[00226] The vector for use with the EPO devices, which are described in more detail below, may be formulated or manufactured using a combination of known devices and techniques, but it is preferred that they be manufactured using an optimized plasmid manufacturing technique as described in a licensed document, concurrently pending U.S. provisional application, serial number 60/939,792, filed May 23, 2007. In some examples, DNA plasmids used in these studies can be prepared at concentrations greater than or equal to 10 mg/ml. Manufacturing techniques also include or cover various devices and protocols that are generally known to those skilled in the art, in addition to those presented in US application, US serial number 60/939792, including those shown in licensed US patent No. 7,238,522, issued July 3, 2007. The above application and the patent, respectively, US Serial No. 60/939,792 and US Patent No. 7,238,522, are incorporated herein in their entirety.

Excipients and other components of the vaccine

[00227] The vaccine may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules such as excipients, carriers or diluents. The pharmaceutically acceptable excipient may be a transfection facilitating agent which may include surface active agents such as immunostimulatory complexes (ISCOMS), incomplete Freund's adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene , hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known agents that facilitate transfection.

[00228] The transfection facilitator is a polyanion, a polycation, including poly-L-glutamate (LGS), or a lipid. The transfection facilitator is poly-L-glutamate, and poly-L-glutamate may be present in the vaccine at a concentration of less than 6 mg/ml. The transfection facilitating agent may also include surface active agents such as immunostimulatory complexes (ISCOMS), incomplete Freund's adjuvant, an LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used in conjunction with a genetic construct. DNA plasmid vaccines may also include a transfection facilitator such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a mixture of DNA liposomes (see e.g. W09324640), calcium ions, viral proteins, polyanions, polycations or nanoparticles or other known transfection facilitating agents. The transfection facilitator is a polyanion, a polycation including poly-L-glutamate (LGS), or a lipid. The transfection agent concentration in the vaccine is less than 4 mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml, less than 0.250 mg/ml. less than 0.100 mg/ml, less than 0.050 mg/ml, or less than 0.010 mg/ml.

[00229] The pharmaceutically acceptable excipient may be one or more adjuvants. The adjuvant may be other genes that are expressed on an alternative plasmid or delivered as proteins in combination with the above plasmid in a vaccine. One or more adjuvants may be selected from the group consisting of: CCL20, α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNR β, GM- CSF, epidermal growth factor (EGF), dermal T-cell chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, , IL-28, MHC, CD80, CD86, IL-l, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-l, MIP-la, MIP-1~, IL-8 , L-selectin, P-selectin, E-selectin, CD34, GlyCAM-l, MadCAM-l, LFA-l, VLA-l, Mac-l, pl50.95, PECAM, ICAM-l, ICAM-2, ICAM -3, CD2, LFA-3, M-CSF, G-CSF, IL-1 mutants 8, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas , TNF receptor, Flt, Apo-l, p55, WSL-l, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, Caspazu ICE, Fos, c -jun, Sp-l, Ap-l, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, S AP K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP2, IL-l 5 having a signal sequence or coding sequence that encodes a deleted signal sequence and optionally including another signal peptide such as from IgE or a coding sequence that encodes another signal peptide, such as from IgE, and functional fragments thereof, or a combination thereof. The adjuvant may be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-l, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-l8, or a combination thereof.

[00230] In some embodiments, the adjuvant may be one or more proteins and/or nucleic acid molecules that encode proteins selected from the group consisting of: CCL-20, IL-12, IL-15, IL-28, CTACK , TECK, MEC or RANTES. Examples of IL-12 constructs and sequences are disclosed in PCT Application No. PCT/US1997/019502 and the corresponding US application, serial number 08/956,865, and US provisional application, serial number 61/569600, filed December 12, 2011, each of which is incorporated herein by reference. Examples of IL-15 constructs and sequences are disclosed in PCT Application No. PCT/US04/18962 and corresponding US application serial number 10/560,650, and in PCT application no. PCT/US07/00886 and corresponding US application serial no. 12/160,766 and PCT application no. PCT/USI0/048827, each of which is incorporated here by reference. Examples of IL-28 constructs and sequences are disclosed in PCT Application No. PCT/US09/039648 and the corresponding US application, serial number 12/936,192, each of which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT Application No. PCT/US 1999/004332 and the corresponding US application, serial number and 09/622452, each of which is incorporated herein by reference. Other examples of RANTES constructs and sequences are disclosed in PCT Application No. PCT/US 11/024098, which is incorporated here by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT Application No. PCT/US1999/004332 and the corresponding US application, serial number 09/622452, each of which is incorporated here by reference. Other examples of RANTES constructs and sequences are disclosed in PCT Application No. PCT/US11/024098, which is incorporated here by reference. Examples of chemokines, constructs and sequences of CTACK, TECK and MEC are disclosed in PCT application no. PCT/US 2005/042231 and the corresponding US application, serial number 11/719646, each of which is incorporated herein by reference. Examples of Ox40 and other immunomodulators are disclosed in US application No. 10/560653, which is incorporated into the present description by reference. Examples of DR5 and other immunomodulators are disclosed in US application No. 09/622452, which is incorporated into the present description by reference.

[00231] Other genes that may be useful as adjuvants include genes encoding: MCP-l, MIP-la, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34 , GlyCAM-l, MadCAM-l, LFA-l, VLA-l, Mac-l, pl50.95, PECAM, ICAM-l, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G -CSF, IL-4, IL-18 mutants, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo -l, p55, WSL-l, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspazu ICE, Fos, c-jun, Sp-l, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-l, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3 , TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

[00232] The vaccine may further include a genetic vaccine stimulating agent as disclosed in US Application Serial No. 021,579, filed April 1, 1994, which is hereby incorporated by reference in its entirety.

[00233] The vaccine may contain antigen and plasmids in an amount of from about 1 nanogram to 100 milligrams; 1 microgram to about 10 milligrams; or preferably from about 0.1 micrograms to about 10 milligrams; or more preferably from about 1 milligram to about 2 milligrams. In some preferred embodiments, the vaccine of the present invention contains from about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the vaccine may contain from about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the vaccine may contain from about 1 to about 350 micrograms of DNA. In some preferred embodiments, the vaccine may contain from about 25 to about 250 micrograms, from about 100 to about 200 micrograms, from about 1 nanogram to 100 milligrams; about 1 microgram to about 10 milligrams; about 0.1 micrograms to about 10 milligrams; about 1 milligram to about 2 milligrams, about 5 nanograms to about 1000 micrograms, about 10 nanograms to about 800 micrograms, about 0.1 to about 500 micrograms, about 1 to about 350 micrograms, about 25 to about 250 micrograms, from about 100 to about 200 micrograms of the antigen or its plasmid.

[00234] The vaccine may be formulated according to the route of administration employed. The pharmaceutical composition for injectable vaccines may be sterile, pyrogen-free, and particulate-free. You can use an isotonic composition or solution. Isotonicity supplements may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine may contain a vasoconstrictor. Isotonic solutions may include phosphate buffered saline. The vaccine may additionally contain stabilizers, including gelatin and albumin. Stabilizers can provide formulation stability at room or ambient temperature for extended periods of time, including LGS or polycations or polyanions.

Vaccine Pharmaceutical Compositions

[00235] The vaccine may be in the form of a pharmaceutical composition. The pharmaceutical composition may contain a vaccine. Pharmaceutical compositions may contain from about 5 nanograms (ng) to about 10 milligrams (mg) of vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions of the present invention contain from about 25 ng to about 5 mg of vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions contain from about 50 ng to about 1 mg of vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions contain from about 0.1 to about 500 micrograms of vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions contain from about 1 to about 350 micrograms of vaccine nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 5 to about 250 micrograms of vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions contain from about 10 to about 200 micrograms of vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions contain from about 15 to about 150 micrograms of vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions contain from about 20 to about 100 micrograms of vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions contain from about 25 to about 75 micrograms of vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions contain from about 30 to about 50 micrograms of vaccine nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 35 to about 40 micrograms of vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions contain from about 100 to about 200 micrograms of vaccine nucleic acid molecules. In some embodiments, pharmaceutical compositions contain from about 10 micrograms to about 100 micrograms of vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions contain from about 20 micrograms to about 80 micrograms of vaccine nucleic acid molecules. In some embodiments, pharmaceutical compositions contain from about 25 micrograms to about 60 micrograms of vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions contain from about 30 ng to about 50 micrograms of vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions contain from about 35 ng to about 45 micrograms of vaccine nucleic acid molecules. In some preferred embodiments, the pharmaceutical compositions contain from about 0.1 to about 500 micrograms of vaccine nucleic acid molecules. In some preferred embodiments, the pharmaceutical compositions contain from about 1 to about 350 micrograms of vaccine nucleic acid molecules. In some preferred embodiments, the pharmaceutical compositions contain from about 25 to about 250 micrograms of vaccine nucleic acid molecules. In some preferred embodiments, the pharmaceutical compositions contain from about 100 to about 200 micrograms of vaccine nucleic acid molecules.

[00236] In some embodiments, the pharmaceutical compositions of the present invention comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng vaccine nucleic acid molecules. In some embodiments, the pharmaceutical compositions may contain at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100 , 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 ,230,235,240,245,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350 , 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475 , 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700 , 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825 830 835 840 845 850 855 860 865 870 875 880 885 890 895 900 905 910 915 920 925 930 935 940 945 950 , 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000 micrograms of n molecules ucleic acid vaccine. In some embodiments, the pharmaceutical composition may contain at least 1.5; 2; 2.5; 3; 3.5; four; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; eight; 8.5; 9; 9.5 or 10 mg or more of vaccine nucleic acid molecules.

[00237] In other embodiments, the pharmaceutical composition may include up to and including 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng vaccine nucleic acid molecules. In some embodiments, the pharmaceutical composition may include up to and including 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 ,230,235,240,245,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350 , 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475 , 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700 ,705,710,715,720,725,730,735,740,745,750,755,760,765,770,775,780,785,790,795,800,805,810,815,820,825 830 835 840 845 850 855 860 865 870 875 880 885 890 895 900 905 910 915 920 925 930 935 940 945 950 , 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000 micrograms of nucleic molecules nic acid vaccine. In some embodiments, the pharmaceutical composition may include up to and including 1.5; 2; 2.5; 3; 3.5; four; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; eight; 8.5; 9; 9.5 or 10 milligrams of vaccine nucleic acid molecules.

[00238] the Pharmaceutical composition may additionally contain other agents for the purposes of inclusion in the composition in accordance with the method of administration used. Where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, non-pyrogenic and free of particulate matter. It is preferable to use an isotonic composition. Typically, isotonicity additives may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

[00239] The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient as described above. For example, a pharmaceutically acceptable excipient may include functional molecules, excipients, adjuvants, carriers, diluents, or transfection aids, as discussed above.

Methods of vaccination

[00240] Provided herein are methods for the treatment and/or prevention of MUC16 expressing cancer, such as but not limited to ovarian cancer, using the pharmaceutical formulations described above. Also described herein are methods of using the pharmaceutical compositions described above for the treatment and/or prevention of MUC16 expressing cancer, such as, without limitation, ovarian cancer, in a subject. This document also provides methods for vaccinating a subject. Also provided herein are methods for administering the pharmaceutical compositions provided herein to a subject in need thereof. The methods provided herein, collectively referred to as the methods of treatment using the pharmaceutical compositions provided herein, may include administering one or more vaccines as described herein to a subject in need thereof to induce a therapeutic and/or prophylactic immune response. The vaccine may be administered to a subject to modulate the activity of the subject's immune system and enhance the immune response. The administration of the vaccine may be the transfection of cancer antigens, as described herein, in the form of a nucleic acid molecule that is expressed in the cell and delivered to the cell surface, whereupon the immune system recognizes them and induces a cellular, humoral or cellular and humoral response. The introduction of the vaccine can be used to induce or elicit an immune response in subjects against MUC16, as described herein, by administering the vaccine to the subject, as described above in this document.

[00241] A vaccine may be administered to a subject to modulate the activity of the subject's immune system, thereby enhancing the immune response. In some embodiments, the subject is a mammal. When a vaccine is administered to a mammal, and thereby the vector is introduced into mammalian cells, the transfected cells will express and secrete one or more cancer antigens as described herein. These secreted proteins or synthetic antigens will be recognized as foreign by the immune system, leading to an immune response that may include: antibodies raised against one or more cancer antigens and a specific T cell response against one or more cancer antigens. In some examples, a mammal vaccinated with the vaccines discussed herein will have a primed immune system, and upon administration of one or more cancer antigens as described herein, the primed immune system will rapidly clear subsequent cancer antigens as described herein, regardless on whether it is produced through humoral, cellular, or both cellular and humoral immune responses.

[00242] Methods for administering a DNA vaccine are presented in US Pat. Nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference in their entirety.

[00243] The vaccine may be administered to a mammal to elicit an immune response in the mammal. The mammal can be human, non-human primate, cow, pig, sheep, goat, antelope, bison, water buffalo, bulls, deer, hedgehogs, elephants, llama, alpaca, mice, rats, and preferably human, cow or pig. The vaccine can also be administered to a non-mammalian subject, such as a chicken, to elicit an immune response.

[00244] The dose of the vaccine may be from 1 μg to 10 mg of active ingredient per kilogram (kg) of body weight over time (component/kg body weight/time) and may be from 20 micrograms to 10 mg component/kg of body weight time. The vaccine can be given every 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 days or 31 days. The number of doses of the vaccine for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses.

Ways to Generate an Immune Response with a Vaccine

[00245] The vaccine can be used to generate an immune response in a mammalian or non-mammalian subject, including a therapeutic or prophylactic immune response. The immune response may generate antibodies and/or killer T cells that are directed to one or more cancer antigens, as described herein. Such antibodies and T cells can be isolated.

[00246] Some embodiments provide methods for generating immune responses against one or more cancer antigens as described herein, which embodiments include administering a vaccine to a subject. Some embodiments provide methods for prophylactically vaccinating a subject against a cancer or tumor expressing one or more MUC16 antigens as described above, which embodiments include administering the vaccine. Some embodiments provide methods for therapeutically vaccinating a subject who is suffering from ovarian cancer or a tumor expressing MUC16, which embodiments include administering the vaccine. Diagnosis of ovarian cancer or a tumor expressing one or more MUC16 antigens as described herein prior to vaccine administration may be carried out in a conventional manner.

Vaccine treatment for cancer

[00247] The vaccine can be used to generate or induce an immune response in a mammal that is reactive or directed against a cancer expressing MUC16, such as, but not limited to, ovarian cancer, more specifically epithelial ovarian cancer, most specifically serous ovarian cancer. The triggered immune response can prevent ovarian cancer or tumor growth.

[00248] The elicited immune response can prevent and/or reduce metastasis of cancer or tumor cells in a subject with ovarian cancer. Accordingly, the vaccine may be used in a method that treats and/or prevents cancer or tumors in a mammal or subject with cancer to which the vaccine is administered.

[00249] In some embodiments, the administered vaccine may mediate clearance or prevent tumor cell growth by inducing (1) humoral immunity through B cell responses to generate antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thereby delaying suppressor cells (MDSC) derived from myeloid cells and causing suppression of tumor growth; (2) by increasing cytotoxic T lymphocytes such as CD8+ (CTL) to attack and kill tumor cells; (3) through an increase in T-helper responses; (4) and enhancing inflammatory responses through IFN-γ and TFN-α, or preferably through all of the above. The vaccine can increase tumor-free survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45%. The vaccine after immunization can reduce the tumor mass by 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59% and 60%. The vaccine can prevent and block the rise of monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells. Vaccine may increase cancer survival by 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59% and 60%.

[00250] In some embodiments, the immune response may generate a humoral immune response and/or an antigen-specific cytotoxic T lymphocyte (CTL) response that does not cause damage or inflammation to various tissues or systems (e.g., brain or neurological system, etc.) in a subject who has been vaccinated.

[00251] In some embodiments, the vaccine can be administered peripherally (as described in more detail below) to mount an antigen-specific immune response directed to cancer or tumor cells or tissue, to clear the cancer or tumor, or to eliminate the cancer or tumor expressing one or more types of cancer antigens without damaging or causing disease or death in the subject to whom the vaccine is administered.

[00252] ] An administered vaccine can enhance a cellular immune response in a subject from about 50-fold to about 6000-fold, from about 50-fold to about 5500-fold, from about 50-fold to about 5000-fold, from about 50 times to about 4500 times, from about 100 times to about 6000 times, from about 150 times to about 6000 times, from about 200 times to about 6000 times, from about 250 to about 6000 times, or from about 300 to about 6000 times the cellular immune response in a subject who did not receive the vaccine. In some embodiments, the administered vaccine may increase the cellular immune response in the subject by about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 , 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 30.3 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5700, 5700, 5700, 5700 5800, 5900 times or 6000 times compared to the cellular immune response in the subject who was not injected with the vaccine.

[00253] The administered vaccine can increase the levels of interferon gamma (IFN-γ) in a subject by about 50 times, about 50 times, about 50 times, about 5000 times, about 50 times, about 4500 times, from about 100 times to about 6000 times, from about 150 times to about 6000 times, from about 200 times to about 6000 times, from about 250 times to about 6000 times, or from about 300 times to about 6000 times times compared to the cellular immune response in a subject who did not receive the vaccine. In some embodiments, the administered vaccine may increase the subject's IFN-γ levels by about 50, 100, l50, 200, 250, 300, 350 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 950, 1000, 1100, L200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5800, 5800 , 5900 times or 6000 times compared to the cellular immune response in the subject who was not injected with the vaccine.

[00254] The dose of the vaccine may be from 1 µg to 10 mg of the active ingredient per kilogram (kg) of body weight in a time period (component/kg of body weight/time) and can be from 20 micrograms to 10 mg of ingredient/kg of body weight/time . The vaccine can be given every 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 or 31 days. The number of doses of the vaccine for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses.

Routes of administration

[00255] The vaccine or pharmaceutical composition may be administered by various routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhaled, buccal, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and /or intra-articular, or combinations thereof. For veterinary use, the composition may be administered as a conveniently acceptable composition in accordance with normal veterinary practice. The veterinarian can easily determine the dosing regimen and route of administration that is most appropriate for a particular animal. The vaccine can be administered using conventional syringes, needleless injection devices, "microparticle bombardment gene guns" or other physical methods such as electroporation ("EP"), "hydrodynamic method" or ultrasound.

[00256] The vaccine vector can be administered to a mammal using several well-known technologies, including DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, liposome-mediated transfection, nanoparticle-stimulated transfection, and the use of recombinant vectors such as as recombinant adenovirus, recombinant adenovirus-associated virus and recombinant vaccination. One or more vaccine cancer antigens can be administered by DNA injection together with in vivo electroporation.

electroporation

[00257] The vaccine or pharmaceutical composition may be administered by electroporation. Vaccine administration by electroporation can be accomplished using electroporation devices that can be configured to deliver to the desired mammalian tissue an energy pulse effective to form reversible pores in cell membranes, and preferably the energy pulse is a direct current corresponding to an incoming current specified by the user. The electroporation device may include an electroporation component and an electrode assembly or manual control assembly. An electroporation component may include and encompass one or more different elements of electroporation devices, including: a controller, a current signal generator, an impedance tester, a signal recorder, an input element, a status message element, a communication port, a memory component, a power supply, and a switch. Electroporation to facilitate transfection of cells with a plasmid can be performed using an in vivo electroporation device such as the CELLECTRA® EP system (Inovio Pharmaceuticals, Inc., Blue Bell, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Inc.).

[00258] Examples of electroporation devices and electroporation methods that can facilitate the administration of DNA vaccines of the present invention include those presented in US Patent No. 7,245,963 to Draghia-Akli, et al, US Patent Publication 2005/0052630 filed by Smith, et al, the contents of which are incorporated herein by reference in their entirety. Other electroporation devices and electroporation methods that can be used to facilitate the administration of DNA vaccines include those disclosed in co-pending and co-owned U.S. Patent Application Serial No. 11/874072, filed Oct. 17, 2007. , which claims benefit under 35 USC 119(e) to U.S. Provisional Applications Serial No. 60/852,149, filed October 17, 2006, and Serial No. 60/978,982, filed October 10, 2007, all of which are incorporated herein in their entirety. volume.

[00259] US Patent No. 7,245,963 authors Draghia-Akli, et al. describes modular electrode systems and their use to facilitate the introduction of a biomolecule into the cells of a selected tissue of an organism or plant. Modular electrode systems may include a plurality of needle electrodes; hypodermic needle; an electrical connector that provides a conductive connection from the DC programmable switching controller to the plurality of needle electrodes; and power supply. The operator can grip the plurality of needle electrodes that are mounted on the support structure and firmly insert them into the selected tissue in the body or in the plant. The biomolecules are then injected through a hypodermic needle into the selected tissue. The programmable DC pulse controller is activated and a DC electrical pulse is applied to the plurality of needle electrodes. An applied direct current electrical pulse facilitates the introduction of the biomolecule into the cell between the electrode array. The entire contents of US Pat. No. 7,245,963 are incorporated herein by reference in their entirety.

[00260] US Patent Publication 2005/0052630, filed by Smith, et al., describes an electroporation device that can be used to efficiently facilitate the introduction of a biomolecule into cells of a selected tissue of an organism or plant. The electroporation device contains an electrokinetic device ("EKD device"), the operation of which is set by external software or firmware. The EKD generates a series of programmable DC pulse trains between the electrodes in the array based on user control and input of pulse parameters and allows the storage and retrieval of current waveform data. The electroporation device also includes a replaceable electrode disc having a row of needle electrodes, a central channel for an injection needle, and a removable guide disc. The entire content of US Patent Publication 2005/0052630 is incorporated by reference in its entirety.

[00261] The electrode arrays and methods presented in US Pat. No. 7,245,963 and US Patent Publication 2005/0052630 can be adapted for deep penetration not only into tissues such as muscle, but also into other tissues or organs. Due to the configuration of the electrode array, the injection needle is also fully inserted into the target organ, and the injection is administered perpendicular to the target problem, in the area that is pre-delineated by the electrodes. The electrodes shown in US Pat. No. 7,244,563 and US Patent Publication 2005/005263 are preferably 20 mm long and 21 gauge.

[00262] In addition, in some embodiments that include electroporation devices and their use, there are electroporation devices that are presented in the following patents: US patent 5,273,525 issued December 28, 1993; August 2000, 6,261,281, issued July 17, 2001, and 6,958,060, issued October 25, 2005, and in U.S. Patent 6,939,862, issued September 6, 2005. covering the subject matter provided in US Pat. . The above patents are incorporated by reference in their entirety.

Ways to get a vaccine

[00263] This document provides methods for preparing the DNA plasmids discussed herein. DNA plasmids after the final step of subcloning into a mammalian expression plasmid can be used to inoculate a cell culture in a large scale fermentation tank using methods known in the art.

[00264] DNA plasmids for use with the EPO devices of the present invention can be formulated or manufactured using combinations of known devices and technologies, but are preferably manufactured using the optimized plasmid production technology as disclosed in U.S. Published Application No. 20090004716, filed May 23, 2007. In some examples, the DNA plasmids used in these studies can be prepared at concentrations greater than or equal to 10 mg/mL. Manufacturing techniques also include or cover various devices and protocols that are widely known to those skilled in the art, in addition to those presented in US patent application serial No. 60/939792, including those shown in licensed US patent No. 7,238,522, issued July 3, 2007. The above application and US Patent, respectively, Serial No. 60/939,792 and US Patent No. 7,238,522 are incorporated herein in their entirety.

[00265] The present invention has many aspects, illustrated by the following non-limiting examples.

EXAMPLES

[00266] The present invention is further illustrated in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are provided by way of illustration only. From the above discussion and these examples, a person skilled in the art can determine the main characteristics of this invention and, without departing from its essence and scope, can make various changes and modifications to the invention in order to adapt it to various applications and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1: MUC16 Tandem Repeat Central Domain Design Strategy

[00267] Native human MUC16 is a large, heavily glycosylated protein that is 22,152 amino acids long. It consists of 3 domains: an N-terminal domain, a central tandem repeat domain, and a carboxy-terminal domain. Because the native N-terminal domain of human MUC16 is very large (12,068 amino acids) and lacks repeat sequences, only the tandem repeat central domain and carboxy-terminal domain were targeted (FIG. 1). Different design strategies were used for the tandem repeat domain and the carboxy-terminal domain.

[00268] Native human MUC16 tandem repeat central domain, which is 9,799 amino acids long, is a series of 63 homologous repeats, each of 156 amino acids, except for the last repeat, which is only 127 amino acids. Due to their length and lack of homology across species to potentially induce cross-reactive T cells against most native human repeats, microconsensus repeat (RMC) sequences have been derived from a variety of human native repeat sequences. Briefly, after excluding low-quality human native repeat sequences, the remaining 34 human native repeat sequences were used to perform phylogenetic analysis (FIG. 2 and FIG. 3). The result showed that these sequences can be grouped into four groups of repeats that have more than 80% identity. Based on previous experience in developing a T cell-based vaccine, each microconsensus sequence should be derived from sequences that share more than 80% sequence identity with one another in order to detect cross-reactive T cell responses to native repeats used to generate this micro-consensus. As a result, four RMC sequences were generated.

[00269] Among those repeats that were not grouped into any of these four groups, only three other native repeats (R61-63) had less than 80% sequence identity with all four generated microconsensus repeat (RMC) sequences (Table 1). Table 1 shows the percent sequence identity of all ungrouped native MUC16 individual repeat sequences (R14, R1, R55, R56, R21, R58, R22, R59, R60, R61, R63 and R62) with RMC1-4. Bold indicates that the remaining ungrouped native repeat sequences have greater than 80% identity with at least one RMC, except for R61, R62 and R63 (GenBank: AAL65133.2).

Table 1

R14 R1 R55 R56 R21 R58 R22 R59 R60 R61 R63 R62 RMC No. 1 87.2 79.4 85.2 80.6 81.8 80.6 83.1 83.9 78.7 74.7 48.4 45.5 R.M.C. No. 2 85.3 80.6 87.7 83.9 83.8 80.6 83.1 83.9 80.0 76 fifty 45.5 RMC No. 3 77.6 70.3 75.5 75.5 76.6 76.8 79.9 79.4 75.5 70.8 54.8 47 RMC No. 4 77.6 74.8 83.2 79.4 82.5 80.0 83.1 82.6 78.7 77.9 51.6 47.7

[00270] To potentially increase the breadth of T cell responses elicited by the vaccine, these three repeats were included in the MUC16 tandem repeat central domain construct as separate elements. The remaining native repeats (R1, R14, R55, R56, R21, R58, R22, R59 and R60) share more than 80% sequence identity with at least one RMC and must be covered by at least one of the four RMC sequences.

[00271] In summary, the synthetic consensus approach was not used for the central domain of the tandem repeat. Instead, 4 RMC sequences derived from multiple human native repeat sequences were generated to potentially induce cross-reactive T cells against most human native repeats. Each RMC was derived from repeat sequences that share more than 80% sequence identity with one another. Native repeats R61, R62 and R63, all of which share less than 80% sequence identity with all 4 RMC sequences, were also included in the MUC16 tandem repeat central domain construct as separate elements to potentially increase the breadth of vaccine-induced T cell responses.

Example 2: MUC16 Carboxy-Terminal Domain Design Strategy

[00272] The carboxy-terminal domain of native human MUC16 is 284 amino acids long and consists of an extracellular ectodomain, a transmembrane domain, and a short cytoplasmic tail. In contrast to the central domain of the tandem repeat, the carboxy-terminal domain is well conserved across many species. However, the cytoplasmic tail of MUC16 contains several potential S/T/Y phosphorylation sites, suggesting that MUC16 is involved in the signal transduction pathway(s). (FIG. 4). FIG. 4 squares indicate potential S/T/Y phosphorylation sites. The N- and O-glycosylation sites are designated X and O'Brien, T. J. et al., respectively. Tumor biology: Journal of the International Society for Oncodevelopmental Biology and Medicine 22, 348-366 (2001). To prevent unwanted triggering of signaling downstream of translation, the cytoplasmic tail (amino acids 254-284 of the native carboxy-terminal domain) was not included in any of the MUC16 immunogens.

Generation of ectodomain and transmembrane domain of consensus MUC16

[00273] The human MUC16 consensus carboxy-terminal domain was constructed without the cytoplasmic tail using the MUC16 ectodomain and transmembrane domain 21 sequences from GenBank (www.ncbi.nlm.nih.gov/genbank/). The GenBank access numbers of these sequences are: AAL65133.2, XP_004059993.1, XP_014197952.1, XP_0039l4869.1, XP_007993338. l, XP_0l 1739103.1, XP_011932759.1, XP_011810358.1, XP_015296314.1, XP_010387164.1, XP_0089854l7.l, XP_0l 0347904.1, XP_012291241.1, XP_011287799.1, CR_003354138.3, XP_014410271.1, XP_0l 5096370.1, XP_011373099.1 , XP_011226599.1, XP_008703704. l, and XP_012496675.1.

[00274] The consensus sequence was generated using the DNASTAR® Lasergene software package (version 13.0.0.357). The MUC16 ectodomain and transmembrane domain sequences were imported into MegAlign and aligned using the ClustalW multiple sequence alignment program. The resulting MUC16 ectodomain and transmembrane domain sequence was determined to have 96.0%-96.4% identity with native human MUC16 ectodomain and transmembrane domain.

Rationale for introducing mutations in the carboxy-terminal domain to abolish the biological function of MUC16

[00275] Following the generation of the ectodomain and transmembrane domain of the MUC16 consensus, three asparagine (N) to alanine (A) (N -> A) mutations were introduced to abolish the biological function of the resulting ectodomain protein and the transmembrane domain of the consensus MUC16. These three mutations were introduced because the carboxy-terminal ectodomain of MUC16 is involved in tumorigenesis, tumor invasion, and metastasis. Rao, T. D. et al. PloS one 10 (2015). Rao et al. showed that the introduction of these three N->A mutations in the MUC16 ectodomain to abolish N-glycosylation dramatically reduced ERK1/2 and AKT phosphorylation and tumor growth in vivo in athymic nude mice transplanted with transfected 3T3 fibroblast cells. Reversal of biological function following the introduction of 3 N->A mutations into the synthetic MUC16 consensus construct has not been evaluated in vitro or in vivo.

Brief description of the design of the carboxy-terminal domain of MUC16

[00276] The ectodomain and transmembrane domain of consensus MUC16 were created and three mutations (N->A) were introduced to abolish N-glycosylation (FIG. 5). The modified ectodomain and transmembrane domain of consensus MUC16 have 95.3% identity with the ectodomain and transmembrane domain of native human MUC16. Table 2 shows the percent sequence homology of the generated ectodomain and transmembrane domain of MUC16 with the ectodomain and transmembrane domain of native human MUC16.

table 2

Identity percentage Divergence one 2 one \ 95.3 one 2 4.9 \ 2 one 2

1=Ectodomain and transmembrane domain of consensus MUC16

2=Ectodomain and transmembrane domain of human native MUC16

Example 3 Assembly Strategy for RMC, Native Repeats and Ectodomain and Transmembrane Domain (CTD) Synthetic Consensus MUC16 to Reduce the Likelihood of Introducing Off-target Epitopes

[00277] The elements to be included in the MUC16 design included four RMCs, three native repeats, and the ectodomain and transmembrane domain (CTD) of the synthetic MUC16 consensus. The initial design strategy was to generate the MUC16 immunogen (pre-synthetic MUC16 immunogen) by incorporating elements as shown in FIG. 6A-6B. However, since in this immunogen the fusions RMC1-RMC2, RMC2-RMC3, RMC3-RMC4, RMC4-R61, and the R63 ectodomain and transmembrane domain of the synthetic consensus MUC16 do not occur in the native MUC16 sequence, potential introduction of irrelevant, non-targeted epitopes is likely if if these fusions do not have high sequence identity with at least one native MUC16 repeat fusion. To determine the sequence identity of the synthetic immunogen fusions with all fusions of native MUCl6 repeats, the last 11 amino acids and 11 amino acids of the beginning of all adjacent native repeats were compared with the last 11 amino acids and 11 amino acids of the beginning of all adjacent synthetic immunogenic fusions (RMC1-RMC2, RMC2-RMC3, RMC3 -RMC4, RMC4-R61 and R63 Synthetic Consensus CTD). The results of the analysis showed that all synthetic immunogen fusions had greater than 95.2% sequence identity with at least one native repeat fusion, with the exception of the RMC4-R61 synthetic immunogen fusion, which had only up to 76.2% sequence identity with all native repeat fusions. repeats (FIG. 6A).

[00278] To reduce the likelihood of introducing off-target epitopes, a native repeat MUC16 59 (R59) was inserted in the RMC4-R61 fusion sequence between RMC4 and R61 (synthetic consensus IRC+R59 immunogen) (FIG. 6B). Sequence analysis showed that the RMC4-R59 and R59-R61 fusions had a higher identity with native repeat fusions (85.7% and 90.9%, respectively) compared to the RMC4-R61 fusion. Moreover, class I MHC epitope prediction using the Immune Epitope Database Resource Analysis Resource (//tools. iedb.org/mhci/) predicted the absence of strong binders in the HLA-A*02:0l haplotype at RMC4-R59 and R59-R61 fusions. .

[00279] Additional strategies have also been used to avoid introducing the RMC4-R61 fusion, including the following: using two plasmids (one to express the RMC1-RMC4 immunogen and the other to express the R61, R62, R63 immunogen and CTD synthetic consensus MUC16), adding furin cleavage site between RMC4 and native R61 of the presynthetic immunogen, and using an expression vector with two promoters, one promoter (hCMV) driving the expression of the RMC1-RMC4 immunogen and the other promoter (sCMV) driving the expression of R61, R62, R63 and CTD of the synthetic consensus immunogen MUC16. As a result, five different synthetic MUC16 consensus DNA plasmids were generated, as shown in Table 3. Schematic representations of the synthetic MUC16 consensus immunogens are shown in FIG. 7.

Table 3

Plasmid ID skeleton Name of the immunogen(s) Definitions pGX1435 pGX0001 IRC+R59 synthetic consensus MUC16 IRC+R59 : Integrated R epeats and Synthetic Consensus C arboxy Terminal Domain (integrated repeats and synthetic consensus carboxy-terminal domain + Native R epeat (native repeat) 59 pGX1436 pGX0001 RMC Synthetic Consensus MUC16 RMC: R epeat M micro- c onsensus
(re-micro-consensus pGX1437 pGX0001 NRC synthetic consensus MUC16 NRC: Native Repeats (native repeats) and Synthetic Consensus C arboxy Terminal Domain ( synthetic consensus carboxy-terminal domain) pGX1438 pGX0001 IRC synthetic consensus MUC16 IRC : Integrated Repeats and Synthetic Consensus C arboxy Terminal Domain pGX1439 pGX0003 (double promoter) RMC Synthetic Consensus MUC16+NRC Synthetic Consensus MUC16 See above

[00280] To have a higher level of expression, Kozak sequence and IgE were added to the N-terminus of all four synthetic consensus MUC16 immunogens. Yang, J. S. et al., The Journal of infectious diseases 184, 809-816 (2001). In addition, the codon usage of the DNA sequences encoding these immunogens has been adapted to the codon bias of Homo sapiens genes. Andre, S. et al. Journal of virology 72, 1497-1503 (1998); Deml, L. et al. Journal of virology 75, 10991-11001 (2001).

[00281] In addition, RNA optimization was also performed: regions with very high (> 80%) or very low (<30%) GC content, and cis-acting sequence motifs were excluded, such as TATA inner boxes, chi- sites and ribosomal entry sites. Schneider, R., et al, Journal of virology 71, 4892-4903 (1997); Muthumani, K. et al. Virology 314, 134-146 (2003). The synthesized synthetic MUC16 consensus immunogens were digested with BamHI and XhoI (pGXl435-pGXl438) or PmeI and XhoI or SalI and MluI (pGXl439) and cloned into the Inovio pGX0001 expression vector (pGXl435-38) or the Inovio pGXl493 dual promoter expression vector (pGXl493 ). All synthetic MUC16 consensus plasmids were subjected to full length sequencing and then analyzed and validated by two analysts for validation.

[00282] A representative comparative model of the central region of the tandem repeat of synthetic MUC16 consensus immunogens based on the synthetic MUC16 IRC consensus sequence is shown in FIG. 8A-8C. FIG. 8A is a schematic diagram of native MUC16 (based on GenBank AAL65133.2) and IRC synthetic consensus MUC16. There are clear size differences between the two molecules, and the native sequence contains additional domains not found in the synthetic consensus version. Both native MUC16 and the synthetic consensus MUC16 IRC contain a number of sea urchin sperm protein repeats, enterokinase and agrino(SEA)-like repeats, as well as a transmembrane domain. In the synthetic MUC16 consensus IRC, the repeat sequences are based on the microconsensus sequence protocol described in the Tandem Repeat Central Domain Design Strategy section. As shown in FIG. 8B, Comparative modeling illustrates size differences between native MUC16 and the synthetic consensus MUC16 IRC design. SEA-like repeat modules were modeled for both native MUC16 and IRC synthetic consensus MUC16. Due to the size of the native sequence, the simulation was only performed at low resolution and provided relative size information. FIG. 8C shows a comparative model of the synthetic consensus MUC16 IRC repeat region shown in cpk format. The large N-terminal region of MUC16 as well as the C-terminal region do not contain sequences with reliable parental templates and are therefore not amenable to modeling.

[00283] The nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) for the IRC+R59 synthetic consensus MUC16 are shown in Table 21 and Table 22, respectively. Nucleotide sequence (SEQ ID NO: 3) and the amino acid sequence (SEQ ID NO: 4) for RMC synthetic consensus MUC16 is shown in Table 23 and Table 24, respectively. The nucleotide sequence (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO: 6) for NRC synthetic consensus MUC16 are shown , respectively, in Table 25 and Table 26. The nucleotide sequence (SEQ ID NO: 7) and amino acid sequence (SEQ ID NO: 8) for IRC synthetic consensus MUC16 are shown in Table 27 and Table 28, respectively. : 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 and the respective amino acid positions are shown in Table 4.

Table 4

SEQ ID NO: 2 Abstract (IRC+R59 synthetic consensus MUC16) Description Amino acid position IgE leader sequence 1-18 Re-Micro Consensus 1 19-174 Re-Micro Consensus 2 175-330 Re-Micro Consensus 3 331-486 Re-Micro Consensus 4 487-642 Native repeat 59 643-798 Native repeat 61 799-954 Native repeat 62 955-1110 Native repeat 63 1111-1237 Mutations in the carboxy-terminal domain of synthetic MUC16 consensus to abolish N-glycosylation 1238-1490
N1408A
N1431A
N1437A SEQ ID NO: 4 Abstract (RMC Synthetic Consensus MUC16) Description Amino acid position IgE leader sequence 1-18 Re-Micro Consensus 1 19-174 Re-Micro Consensus 2 175-330 Re-Micro Consensus 3 331-486 Re-Micro Consensus 4 487-642 SEQ ID NO: 6 Abstract (NRC synthetic consensus MUC16) IgE leader sequence 1-18 Native repeat 61 19-174 Native repeat 62 175-330 Native repeat 63 331-457 Mutations in the carboxy-terminal domain of synthetic MUC16 consensus to abolish N-glycosylation 458-710
N628A
N651A
N657A SEQ ID NO: 8 Abstract (IRC synthetic consensus MUC16) IgE leader sequence 1-18 Re-Micro Consensus 1 19-174 Re-Micro Consensus 2 175-330 Re-Micro Consensus 3 331-486 Re-Micro Consensus 4 487-642 Furin cleavage site 643-649 Native repeat 61 650-805 Native repeat 62 806-961 Native repeat 63 962-1088 Mutations in the carboxy-terminal domain of synthetic MUC16 consensus to abolish N-glycosylation 1098-1341
N1259A
N1282A
N1288A

[00284] Differences between synthetic MUC16 consensus immunogens and native sequences are summarized in Table 5. Since the size of the central domain of the tandem repeat domain varies among species (human: 63 repeats, rhesus monkey: 4 repeats; mouse: 15 repeats), as well as the sizes of individual repeats vary greatly between species, so it is difficult to accurately align full-length synthetic MUC16 immunogenic sequences with species-specific native sequences. Therefore, for the central domain of the tandem repeat, only domain regions of the synthetic sea urchin sperm protein construct, enterokinase and agrin (SEA) domains were compared with the native human, mouse, and rhesus monkey SEA domain sequences, since this region is the most conserved region of the repeat sequence.

Table 5

Characteristics IRC+R59 synthetic consensus MUC16 RMC
synthetic consensus
MUC16 NRC
synthetic consensus MUC16 IRC
synthetic consensus MUC16 Synthetic consensus
MUC16-
SEA domains
RMC1-4 Synthetic consensus
MUC16-
SEA domains
R61-R63 Synthetic consensus
MUC16-
carboxy-terminal
domain Identity to native human
MUC16 / / / / 51.5% - 100% 100% 95.3% Identity to native
MUC16 rhesus monkey / / / / from 20.0% to
89.7% from 17.4% to
93.9% 95.7% Identity to native mouse
MUC16 / / / / from 21.6% to
71.1% from 15.1% to
71.3% 75.1% The number of amino acid mutations (compared to native human / / / / 0 12 Number of inserted mutations (not derived from consensus) 3 0 3 3 0 0 3 Molecular weight 1492 amino acids
(164 kDa) 644 amino acids
(71 kDa) 712 amino acids
(78 kDa) 1343 amino acids
(148 kDa) / / / Coding sequence length (bp) 4476 1932 2136 4029 / / /

Example 4: Construction and structure of a plasmid

pGX0001

[00285] The pGX0001 vector backbone is a modified pVAXl expression vector from 2998 bp. under the control of the human cytomegalovirus immediate early promoter (hCMV promoter). The original pVAXl was obtained from Thermo Fisher Scientific. The backbone of pGX0001 includes a kanamycin resistance gene (KanR) and a plasmid source of replication (pUC ori) for production purposes. These elements do not function in eukaryotic cells. A map and description of the modified expression vector pVAXl (pGX0001) are shown, respectively, in FIG. 9 and in table 6.

Table 6

Elements: Base pairs: CMV promoter: 137-724 T7 promoter/priming site 664-683 Multiple cloning site 696-811 Bovine growth hormone (GH) polyadenylation signal 829-1053 Kanamycin resistance gene 1226-2020 pUC origin 3219-2992

[00286] Modifications were made to pVAXl to create pGX0001. These modifications are listed in Table 7 and no problems were found regarding plasmid amplification and transcription and translation of the antigen. No further changes were observed in the sequence of pGX0001 in any of the plasmid products on the platform using pGX0001 as a backbone. Base pairs 2, 3 and 4 change from ACT to CTG in the backbone upstream of the CMV promoter.

Table 7

Modification base pair Description C>G 241 in the CMV promoter C>T 1158 backbone, downstream of the bovine growth hormone polyadenylation signal (bGH polyA) A>- 2092 backbone, downstream of the kanamycin resistance gene C>T 2493 at the origin of replication (pUC ori) G>C 2969 at the very end of the pUC Ori upstream of the RNASeH site

pGX0003

[00287] The pGX0003 vector backbone is a 4596 bp eukaryotic expression plasmid. with two multiple cloning sites for inserts under the control of two promoters: the hCMV promoter with SV40 poly A signal and the sCMV promoter with bGH poly A signal. This backbone also includes KanR and pUC ori. The map and description of pGX0003 are shown in FIG. 10 and in table 8.

Table 8

Elements: Base pairs: hCMV promoter: 3548-4373 Simian virus polyadenylation signal 40 4391-4596 Scmv promoter 2558-3060 Bovine growth hormone (GH) polyadenylation signal 2290-2528 Kanamycin resistance gene 298-1059 pUC origin 1227-1900

pGX1435

[00288] pGXl435 was generated by cloning the synthetic MUC16 consensus (IRC+R59 synthetic MUC16 consensus) DNA sequence into pGX0001 at the BamHI and XhoI sites. The associated mRNA production is driven by the human CMV promoter (hCMV promoter) and terminated by the bovine growth hormone 3' end polyadenylation signal (bGH poly A). The backbone of pGX0001 includes a kanamycin resistance gene (KanR) and a plasmid source of replication (pUC ori) for production purposes. These elements do not function in eukaryotic cells. A schematic diagram of pGXl435 and IRC+R59 synthetic consensus MUC16 is shown in FIG. 11 and plasmid elements and corresponding base pairs are shown in Table 9.

Table 9

Elements: Base pairs: hCMV promoter: 137-724 IRC+R59 coding sequence for synthetic consensus MUC16: 742-5217 bGH Poly A: 5261-5485 Kanamycin resistance (KanR): 5658-6452 pUC Ori: 6751-7424

pGX1436

[00289] pGXl436 was generated by cloning the synthetic microconsensus MUC16 (RMC synthetic consensus MUC16) DNA sequence into pGX0001 at the BamHI and XhoI sites. The associated mRNA production is driven by the human CMV promoter (hCMV promoter) and terminated by the bovine growth hormone 3' end polyadenylation signal (bGH poly A). The backbone of pGX0001 includes a kanamycin resistance gene (KanR) and a plasmid source of replication (pUC ori) for production purposes. These elements do not function in eukaryotic cells. A schematic diagram of pGXl436 and RMC synthetic consensus MUC16 is shown in FIG. 12 and plasmid elements and corresponding base pairs are shown in Table 10.

Table 10

Elements: Base pairs: hCMV promoter: 137-724 Synthetic MUC16 consensus RMC coding sequence: 742-2673 bGH Poly A: 2717-2941 Kanamycin resistance (KanR): 3114-3908 pUC Ori: 4207-4880

pGX1437

[00290] pGXl437 was generated by cloning the synthetic MUC16 consensus DNA sequence (Synthetic MUC16 consensus NRC) into pGX0001 at the BamHI and XhoI sites. The associated mRNA production is driven by the human CMV promoter (hCMV promoter) and terminated by the bovine growth hormone 3' end polyadenylation signal (bGH poly A). The backbone of pGX0001 includes a kanamycin resistance gene (KanR) and a plasmid source of replication (pUC ori) for production purposes. These elements do not function in eukaryotic cells. A schematic diagram of pGXl437 and NRC synthetic consensus MUC16 is shown in FIG. 13 and plasmid elements and corresponding base pairs are shown in Table 11.

Table 11

Elements: Base pairs: hCMV promoter: 137-724 Synthetic consensus MUC16 NRC coding sequence: 742-2877 bGH Poly A: 2921-3145 Kanamycin resistance (KanR): 3318-4112 pUC Ori: 4411-5084

pGX1438

[00291] pGXl438 was obtained by cloning the DNA sequence of synthetic consensus MUC16 (IRC synthetic consensus MUC16) in pGX0001 at the BamHI and XhoI sites. The associated mRNA production is driven by the human CMV promoter (hCMV promoter) and terminated by the bovine growth hormone 3' end polyadenylation signal (bGH poly A). The backbone of pGX0001 includes a kanamycin resistance gene (KanR) and a plasmid source of replication (pUC ori) for production purposes. These elements do not function in eukaryotic cells. A schematic diagram of pGXl438 and IRC synthetic consensus MUC16 is shown in FIG. 14 and plasmid elements and corresponding base pairs are shown in Table 12.

Table 12

Elements: Base pairs: hCMV promoter: 137-724 Synthetic MUC16 consensus IRC coding sequence: 742-4770 bGH Poly A: 4814-5038 Kanamycin resistance (KanR): 5211-6005 pUC Ori: 6304-6977

pGX1439

[00292] pGXl439 was generated by cloning the synthetic MUC16 consensus RMC DNA sequence in pGX0003 at the SalI and MluI sites and the synthetic MUC16 consensus NRC DNA sequence at the PmeI and XhoI sites.

[00293] pGXl439 is a dual-promoter DNA plasmid in which the mRNA-producing synthetic MUC16 consensus RMC is driven by the human CMV promoter (hCMV promoter) and terminated by the simian virus 40 polyadenylation signal (SV40 poly A), and in which the synthetic consensus NRC The mRNA-producing MUC16 is driven by the monkey CMV promoter (sCMV Promoter) and terminated by the bovine growth hormone polyadenylation signal (bGH poly A). The backbone of pGX0003 includes a kanamycin resistance gene (KanR) and a plasmid source of replication (pUC ori) for production purposes. These elements do not function in eukaryotic cells.

[00294] A schematic diagram of pGXl439 and RMC synthetic MUC16 consensus and NRC synthetic MUC16 consensus is shown in FIG. 15 and plasmid elements and corresponding base pairs are shown in Table 13.

Table 13

Elements: Base pairs: hCMV promoter: 5682-6506 Synthetic MUC16 consensus RMC coding sequence: 6524-8455 SV40 Poly A: 8457-8662 Kanamycin resistance (KanR): 298-1059 pUC Ori: 1227-1900 sCMV promoter 4703-5193 Synthetic consensus MUC16 NRC coding sequence: 2557-4692 bGH Poly A 2290-2528

Example 5 In Vitro Antigen Expression

[00295] Protein expression of pGXl435, pGXl436, pGXl437, pGXl438 and pGXl439 antigen was confirmed by Western blotting. Human rhabdomyosarcoma (RD) cells (ATCC, CCL-136) maintained in DMEM with 10% FBS (ThermoFisher) were transfected with pGXl435, pGXl436, pGXl437, pGXl438, pGXl439 or pGX0001 (6 μg/10 cm ² /dish) with using Turbofectin 8 (Origene). Forty-eight hours after transfection, the cells were lysed using RIPA cell lysis buffer (ThermoFisher) and the cell lysate was collected. After BCA analysis (ThermoFisher) to determine the concentration of total protein, 15 μg of cell lysate was subjected to electrophoresis in 4-12% SDS-PAGE gel (ThermoFisher) and detected using an anti-MUC16 monoclonal antibody (Abeam, clone EPSISR23-96, abl68360), then visualized with horseradish peroxidase (HRP) conjugated anti-rabbit IgG (Santa Cruz Biotech #sc-2004) using the ECL western blot system (GE Amersham). As a loading control, blots were re-examined for actin expression using an anti-β-actin monoclonal antibody (Santa Cruz Biotech, clone, C4).

162,5 кДа), а также белковые полосы для Участка 1 (

68,2 кДа) и Участка 2 (

77,2 кДа) (ФИГ. 16). В дорожке pGX0001 не были обнаружены белковые полосы, что указывает на то, что белковые полосы специфичны для синтетического консенсусного Mucl6. Анти-β-актиновые полосы были обнаружены с одинаковой интенсивностью, что указывает на то, что в каждой дорожке были загружены равные количества белка. Было обнаружено, что pGXl435 и pGXl438 экспрессируют соответствующие антигенные белки, а pGXl436, pGXl437 и pGXl439 экспрессируют свои соответствующие антигенные белковые участки.[00296] A protein band of expected molecular weight was found for synthetic consensus MUC16 constructs (

162.5 kDa), as well as protein bands for Region 1 (

68.2 kDa) and Site 2 (

77.2 kDa) (FIG. 16). No protein bands were found in the pGX0001 lane, indicating that the protein bands are specific to the synthetic Mucl6 consensus. Anti-β-actin bands were detected with the same intensity, indicating that equal amounts of protein were loaded in each lane. pGXl435 and pGXl438 were found to express their respective antigenic proteins, and pGXl436, pGXl437 and pGXl439 were found to express their respective antigenic protein regions.

Example 6 Immunogenicity of Mesothelin Synthetic Consensus Vaccine Constructs

Animals and immunizations

[00297] Female 8-week-old CB6F1 mice were purchased from Jackson Laboratories. All animals were housed in a temperature controlled, light cycling facility at BTS Research (San Diego, CA). Animal care was provided in accordance with National Institutes of Health guidelines and Animal Care and Use Proposal (ACUP) (BTS ACUP #15-091). The mice were divided into sixteen groups as shown in Table 14.

Table 14

Group n Design Construct Dose (mcg) Injection volume (µl) one eight pGX0001 thirty thirty 2 eight pGX1435 ten thirty 3 eight pGX1435 thirty thirty four eight pGX1435 fifty thirty 5 eight pGX1436 ten thirty 6 eight pGX1436 thirty thirty 7 eight pGX1436 fifty thirty eight eight pGX1437 ten thirty 9 eight pGX1437 thirty thirty ten eight pGX1437 fifty thirty eleven eight pGX1438 ten thirty 12 eight pGX1438 thirty thirty 13 eight pGX1438 fifty thirty fourteen eight pGX1439 ten thirty fifteen eight pGX1439 thirty thirty 16 eight pGX1439 fifty thirty

[00298] Mice in the immunization groups were vaccinated with the indicated doses of pGX0001 or pGXl435, pGXl436, pGXl437, pGXl438, pGXl439 in accordance with SOP R20-003147 CELLECTRA® 3P Mouse Treatment. Briefly, plasmids were prepared in sterile water for injection (VetOne) such that the indicated dose was delivered by intramuscular injection into the tibialis anterior muscle in an injection volume of 30 μl. Each intramuscular injection was immediately followed by electroporation (EP) using the adaptive DC electroporation device CELLECTRA® 2000 with a 3P matrix (Inovio Pharmaceuticals). The device was set to deliver two 0.1 A pulses with a pulse width of 52 ms, separated by 1 second. Due to the large number of mice and groups, half of the mice in each group received 3 immunizations 3 weeks apart. These mice were sacrificed one week after the last immunization and spleens were harvested for cellular immunity measurements. The other half of the mice in each group received 3 immunizations 3 weeks apart. Mice were sacrificed one week after the last immunization, and spleens were collected to obtain indicators of cellular immunity. No other tissue was collected.

Isolation of spleen lymphocytes

[00299] Splenocytes were isolated aseptically and placed in 5 ml of R10 medium (Rosewell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic, antimycotic agent). Splenocytes were isolated by mechanical disruption of the spleen using a Stomacher apparatus (Seward Laboratory Systems Inc.), the resulting product was filtered using a 40 μm cell sieve (BD Falcon). The resulting preperet was centrifuged and the pellet was treated for 5 minutes with ACK lysis buffer (Lonza) to lyse the erythrocytes. Splenocytes were then centrifuged, washed in PBS and then resuspended in R10 medium and immediately used for further analysis.

Determination of IFNγ by enzyme immunoassay (ELISpot)

[00300] An ELISpot analysis of mouse IFNγ to evaluate antigen-specific cellular responses was performed using a kit from MabTech (MabTech, # 3321-4APW-10). Ninety-six well plates pre-coated with anti-mouse IFNγ antibody (mAb AN18) were washed in PBS and blocked for 2 hours at room temperature with R10 medium and then added (in triplicate) at a cell count of 2 x 10 input ⁵ cells per well. A set of peptides (GenScript) was synthesized, each containing 15 amino acid residues with an overlap of 11 amino acids, representing the complete protein sequence of the synthetic MUC16 consensus for each construct. These sets of peptides were resuspended in DMSO (Sigma) and pooled at ~2 μg/mL peptide concentration into six peptide pools. These peptide pools contained peptides corresponding to the synthetic MUC16 consensus protein antigen as indicated in the scheme in FIG. 17. Concavalin A (Sigma) at a concentration of 5 μg/ml was used as a positive control, and complete culture medium was used as a negative control. Tablets were incubated for 18 hours at 37°C in an incubator in an atmosphere with 5% CO ₂ . Biotinylated anti-mouse IFNγ antibody (MabTech, mAb R4-6A2) was then added and the plates were incubated for 2 hours at room temperature. The plates were washed and anti-streptavidin-ALP antibody (MabTech) was added and the plates were incubated for 1 hour at room temperature. Spot detection was performed using BCIP/NBT substrate according to kit manufacturer's (MabTech) instructions. The spots on the plates were counted using an ELISPOT automatic reader (Cellular Technology). To display the data, the mean number of spot forming units (SFU) was adjusted to 1×10 ⁶ splenocytes.

[00301] Antigen-specific responses in the IFNγ ELISpot, expressed as the number of IFNγ spotting units (SFUs) per 1 x 10 ⁶ splenocytes, exceed the number of SFUs in the control when incubated with medium alone.

[00302] The immunogenicity of the synthetic MUC16 consensus construct was assessed at three doses (10 μg, 30 μg, and 50 μg) by ELISpot IFNγ determination and flow cytometry (n=8/group). Mice were immunized with an empty backbone plasmid (pGX0001) as a negative control (n=4/group). Vaccination with the full-length synthetic MUC16 consensus constructs (pGXl435, pGXl438 and pGXl439) elicited exceptionally robust cellular immune responses compared to negative control vaccinated mice.

[00303] The intensity of IFNγ production specific to the synthetic consensus MUC16 induced by pGXl435 was dose-dependent by ELISpot analysis (FIG. 18A). Specifically, the number of IFNγ SFUs in response to pGX1435 synthetic consensus MUC16 was 1564 ± 661, 3858 ± 2767 and 5407 ± 1959 at dose amounts of 10 μg, 30 μg and 50 μg, respectively. Synthetic consensus MUC16 IFNγ responses induced by pGXl435 were significantly higher compared to non-immunized mice at pGXl435 doses of 10 μg (p=0.002), 30 μg (p=0.028), and 50 μg (p=0.001). Detectable responses to the unique R59 epitope (peptide pool 6) induced by pGXl435 are also shown in FIG. 18D.

[00304] The intensity of IFNγ production specific to the synthetic consensus MUC16 induced by construct pGXl438 was dose-independent in ELISpot analysis, with lower doses eliciting a more robust response (FIG. 18B). Specifically, the number of IFNγ SFUs in response to pGXl438 synthetic consensus MUC16 was 5628 ± 3144, 7668 ± 3371 and 6005 ± 3472 at dose amounts of 10 μg, 30 μg and 50 μg, respectively. Synthetic consensus MUC16 IFNγ responses induced by pGXl438 were significantly higher compared to non-immunized mice at pGXl435 doses of 10 μg (p=0.008), 30 μg (p=0.002) and 50 μg (p=0.009).

[00305] The intensity of IFNγ production specific to the synthetic consensus MUC16 induced by construct pGXl439 was also dose-independent in ELISpot analysis, with lower doses producing a more robust response (FIG. 18C). Specifically, the number of IFNγ SFUs in response to pGX1439 synthetic consensus MUC16 was 3180 ± 1377, 6138 ± 2696 and 4862 ± 2069 at dose amounts of 10 μg, 30 μg and 50 μg, respectively. Synthetic consensus MUC16 IFNγ responses induced by pGXl439 were significantly higher compared to non-immunized mice at pGXl435 doses of 10 μg (p=0.002), 30 μg (p=0.002) and 50 μg (p=0.002).

[00306] IFNγ responses for full length constructs are summarized in Table 15.

Table 15

IFNγ responses in ELISpot to synthetic consensus MUC16 (pGX1435) fusion Drug code Group Dose amount Mean SFUs ± SDE p value pGX0001 Non-immunized 30 mcg 7 ± 8 n/a pGX1435 Immunized 10 mcg 1564 ± 661 0.002 30 mcg 3858 ± 2767 0.028 50 mcg 5407 ± 1959 0.001 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001). IFNγ ELISpot Responses to Furin Cleavage of Synthetic Consensus MUC16 (pGX1438) Drug code Group Dose amount Mean SFUs ± SDE p value pGX0001 Non-immunized 30 mcg 7 ± 8 n/a pGX1438 Immunized 10 mcg 5628 ± 3144 0.008 30 mcg 7668 ± 3371 0.002 50 mcg 6005 ± 3472 0.009 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001). IFNγ responses in ELISpot to the synthetic consensus MUC16 dual promoter (pGX1439) Drug code Group Dose amount Mean SFUs ± SDE p value pGX0001 Non-immunized 30 mcg 7 ± 8 n/a pGX1439 Immunized 10 mcg 3180 ± 1377 0.002 30 mcg 6138 ± 2696 0.002 50 mcg 4862 ± 2069 0.002 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001).

[00307] Vaccination with synthetic MUC16 consensus partial length constructs (pGXl436 and pGXl437) also elicited exceptionally robust cellular immune responses compared to vaccinated negative control mice.

[00308] The intensity of IFNγ production specific to the Site 1 synthetic consensus MUC16 consensus induced by construct pGXl436 was dose-dependent by ELISpot analysis (FIG. 19A). Specifically, the number of IFNγ SFUs in response to pGXl436 synthetic consensus MUC16 was 3609 ± 1377, 4711 ± 2365 and 5565 ± 3496 at dose amounts of 10 μg, 30 μg and 50 μg, respectively. Synthetic consensus MUC16 IFNγ responses induced by pGXl436 were significantly higher compared to non-immunized mice at pGXl436 doses of 10 μg (p=0.001), 30 μg (p=0.004) and 50 μg (p=0.014). When comparing the IFNγ responses to Region 1 induced by all MUC16 constructs, the responses induced by pGXl435 were significantly lower compared to pGXl438 (FIG. 19B).

[00309] The intensity of production of IFNγ, specific to the synthetic consensus MUC16 in relation to Region 2, induced by construct pGXl437, according to ELISpot analysis, was also dose-dependent (FIG. 19C). Specifically, the number of IFNγ SFUs in response to pGXl437 synthetic consensus MUC16 was 835 ± 428, 1893 ± 1291 and 2399 ± 1245 at dose amounts of 10 μg, 30 μg and 50 μg, respectively. Synthetic consensus MUC16 IFNγ responses induced by pGXl437 were significantly higher compared to non-immunized mice at pGXl437 doses of 10 μg (p=0.005), 30 μg (p=0.022) and 50 μg (p=0.005). There was a trend towards more stable production of IFNγ in relation to Section 2 with pGXl438 (FIG. 19D).

[00310] IFNγ responses to partial length constructs are summarized in Table 16.

Table 16

IFNγ responses in ELISpot to "Plot 1" synthetic consensus MUC16 (pGX1436) Drug code Group Dose amount Mean SFUs ± SDE p value pGX0001 Non-immunized 30 mcg 3±5 n/a pGX1436 Immunized 10 mcg 3609 ± 1377 0.001 30 mcg 4711 ± 2365 0.004 50 mcg 5565 ± 3496 0.014 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001). IFNγ responses in ELISpot to "Plot 2" of the synthetic consensus MUC16 (pGX1437) Drug code Group Dose amount Mean SFUs ± SDE p value pGX0001 Non-immunized 30 mcg 3±5 n/a pGX1437 Immunized 10 mcg 835 ± 428 0.005 30 mcg 1893±1291 0.022 50 mcg 2399 ± 1245 0.005 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001).

flow cytometry

[00311] Cellular immune responses induced by synthetic MUC16 consensus were further characterized by flow cytometry. 2 x 10 ⁶ splenocytes from vaccinated and unvaccinated mice were stimulated immediately after isolation with synthetic consensus peptide MUC16 for 6 hours in the presence of brefeldin A (BD Biosciences), monensin (BD Biosciences), and FITC-labeled mouse anti-CD107a antibody (BD Biosciences ). After stimulation with peptides, splenocytes were centrifuged and resuspended in 20 μl per well in BD Fc Block (BD Biosciences) mouse anti-mouse Fc blocking solution. The Fc Block solution was used with an initial dilution of 1:40 in PBS and incubation at 4°C for 5 minutes.

[00312] After incubation, the remaining extracellular antibodies (in PBS) were added in an amount of 30 μl per well and incubated at 4°C for 30 minutes. After addition of extracellular dye, the final volume in each well is 50 µl, including the Fc Block solution at a final dilution of 1:100 and extracellular antibodies at the appropriate working dilutions. Cells were then stained with viability dye (Vivid®, Thermo-Fisher) and the following extracellular antibodies: anti-mouse CD4 PerCP-Cy5.5 (BD Biosciences, clone RM4-5) and anti-mouse CD8a APC (BD Biosciences, clone 63-6, 7). Intracellular cytokines were then stained with the following antibodies: BV605 anti-mouse IFNγ antibody, APC-R700 anti-mouse IL-2 antibody, and PE anti-mouse TNF-α antibody (BD Biosciences). Cells were fixed and permeabilized (BD Biosciences, # 554714) for 20 minutes at 4°C. Intracellular staining was then stopped with the following antibodies: APC-Cy7 against mouse CD3e (BD Biosciences, clone 145-2C11), BV605 against mouse IFNγ (BD Biosciences, clone XMG1.2), APC-R700 against mouse IL-2 (BD Biosciences, clone JEs6-5H4) and anti-mouse TNF-α PE (BD Biosciences, clone MP6-XT22).

[00313] Intracellular staining data was obtained on 10-color FACS CANTO (BD Biosciences) and analysis was completed using FlowJo software. The flow cytometry gate strategy is shown below in FIG. 20. For a cell to be called antigen-specific by flow cytometry, the frequency of the reported parameter must be greater than the frequency of the media alone control. In order for a cell to be identified as producing antigen-specific CD107a, the cell must also be identified as positive for antigen-specific production of IFNγ and/or IL-2 and/or TNFα as determined by Boolean gated identification.

[00314] Full-length synthetic MUC16 consensus constructs elicited strong responses in the CD4 ⁺ T cell compartment, with pGXl438 eliciting the most robust response (FIGS. 21A-21C). Synthetic consensus MUC16 induced frequencies of antigen-specific CD4 ⁺ T cell responses that were significantly more efficient than non-immunized mice (0.29% ± 0.23%) at the 10 µg, 30 µg and 50 µg dose level groups for pGX1435, pGX1438 and pGXl439 (FIGS. 21A-21C).

[00315] CD4 ⁺ T cell intensity specific to the synthetic MUC16 consensus induced by the pGXl435 construct was dose-dependent (FIG. 21A). Specifically, CD4 ⁺ T cells when injected with the pGXl435 synthetic consensus MUC16 construct were 1.34% ± 0.45%, 2.27% ± 1.38%, and 3.12% ± 1.45% at dose amounts, respectively, 10 mcg, 30 mcg and 50 mcg. The number of CD4 ⁺ T cells when the synthetic consensus MUC16 construct pGXl435 was administered was significantly higher compared to non-immunized mice at pGXl435 dose amounts of 10 μg (p<0.001), 30 μg (p=0.020), and 50 μg (p=0.004). Specific CD4 ⁺ T cell responses to the pGXl435 synthetic consensus MUC16 construct consisted mainly of CD4 ⁺ T cells producing IFNγ ⁺ IL-2 ⁺ TNFα ⁺ , IFNγ ⁺ IL-2 ^- TNFα ⁺ or IFNγ ⁺ IL- 2 ^{- TNFα-} .

[00316] CD4 ⁺ T cell intensity specific to the synthetic MUC16 consensus induced by the pGXl438 construct was also dose-dependent (FIG. 21B). Specifically, CD4 ⁺ T cells when injected with the pGXl438 synthetic consensus MUC16 construct were 3.12% ± 1.19%, 3.73% ± 1.48%, and 4.06% ± 1.87% at dose amounts, respectively, 10 mcg, 30 mcg and 50 mcg. The number of CD4 ⁺ T cells when the synthetic consensus MUC16 construct pGXl438 was administered was significantly higher compared to non-immunized mice at pGXl438 dose amounts of 10 μg (p=0.001), 30 μg (p=0.001), and 50 μg (p=0.003). The specific CD4 ⁺ T cell responses to the pGXl438 synthetic consensus MUC16 construct consisted mainly of CD4 ⁺ T cells producing IFNγ ⁺ IL-2 ⁺ TNFα ⁺ , IFNγ ⁺ IL-2-TNFα ⁺ , or IFNγ ⁺ IL- 2 ^{- TNFα-} .

[00317] CD4 ⁺ T cell intensity specific to the synthetic MUC16 consensus induced by the pGXl439 construct was dose-dependent (FIG. 21C). Specifically, CD4 ⁺ T cells when injected with the pGXl439 synthetic consensus MUC16 construct were 1.99%±0.99%, 2.71%±0.51%, and 3.25%±1.29% at dose amounts, respectively, 10 mcg, 30 mcg and 50 mcg. The number of CD4 ⁺ T cells when the synthetic consensus MUC16 construct pGXl439 was administered was significantly higher compared to non-immunized mice at pGXl439 dose amounts of 10 μg (p=0.007), 30 μg (p<0.001), and 50 μg (p=0.001). Specific CD4 ⁺ T cell responses to the pGXl439 synthetic consensus MUC16 construct consisted primarily of CD4 ⁺ T cells producing IFNγ ⁺ IL-2 ⁺ TNFα ⁺ , IFNγ ⁺ IL-2-TNFα ⁺ , or IFNγ ⁺ IL- 2 ^{- TNFα-} . The frequency of antigen-specific CD4 ⁺ T cells is further detailed in Table 17.

[00318] All dose levels of full-length synthetic consensus MUC16 constructs induced higher CD4 ⁺ CD107a ⁺ T cell intensity compared to non-immunized mice (0.08% ± 0.04%), however, only the pGX1438 construct was significantly more stable at all doses. .

[00319] Specifically, the frequency of antigen-specific CD4 ⁺ CD107a ⁺ T cells upon administration of the pGXl435 construct was 0.27% ± 0.14%, 0.32% ± 0.37%, and 0.64% ± 0.34% at amounts doses, respectively, of 10 μg (p=0.011), 30 μg (p=0.297) and 50 μg (p=0.008) (FIG. 21A). The cytokine profile of CD4 ⁺ CD107a ⁺ T cells upon administration of the pGXl435 construct was similar across dose groups and consisted mainly of IFNγ ⁺ IL-2 ⁺ TNFα ⁺ and IFNγ ⁺ IL-2 ^- TNFα ⁺ cells (FIG. 22A).

[00320] The frequency of antigen-specific CD4 ⁺ CD107a ⁺ T cells when the pGXl438 construct was administered was 0.40% ± 0.18%, 0.54% ± 0.36%, and 0.53% ± 0.25% at dose amounts respectively, 10 μg (p=0.004), 30 μg (p=0.029) and 50 μg (p=0.004) (FIG. 21B). The cytokine profile of CD4 ⁺ CD107a ⁺ T cells when administered with the pGXl438 construct was similar across dose groups and consisted mainly of IFNγ ⁺ IL-2 ⁺ TNFα ⁺ and IFNγ ⁺ IL-2 ^- TNFα ⁺ cells (FIG. 22B)

[00321] The frequency of antigen-specific CD4 ⁺ CD107a ⁺ T cells when the pGXl439 construct was administered was 0.26% ± 0.18%, 0.36% ± 0.18%, and 0.50% ± 0.25% at dose amounts respectively, 10 μg (p=0.069), 30 μg (p=0.008) and 50 μg (p=0.006) (FIG. 22C). The cytokine profile of CD4 ⁺ CD107a ⁺ T cells when administered with the pGXl439 construct was similar across dose groups and consisted mainly of IFNγ ⁺ IL-2 ⁺ TNFα ⁺ and IFNγ ⁺ IL-2 ^- TNFα ⁺ cells (FIG. 22C). The frequency of antigen-specific CD4 ⁺ T cells with cytolytic potential is further shown in detail in Table 17.

Table 17

CD4 ⁺ and CD4 ⁺ CD107a ⁺ T cell responses to synthetic consensus MUC16 (pGX1435) fusion Drug code Group Dose amount % CD4 ⁺
±St.off. p value
% CD4 ⁺ % CD4 ⁺ CD107a ⁺
±St.off. p value
% CD4 ⁺ CD107a ⁺ pGX0001 Non-immunized 30 mcg 0.29 ± 0.23 n/a 0.08±0.04 n/a pGX1435 Immunized 10 mcg 1.34±0.45 <0.001 0.27±0.14 0.011 30 mcg 2.27 ± 1.38 0.020 0.32±0.37 0.297 50 mcg 3.12 ± 1.45 0.004 0.64±0.34 0.008 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001). CD4 ⁺ and CD4 ⁺ CD107a ⁺ T cell responses to furin cleavage of synthetic consensus MUC16 (pGX1438) Drug code Group Dose amount % CD4 ⁺
±St.off. p value
% CD4 ⁺ % CD4 ⁺ CD107a ⁺
±St.off. p value
% CD4 ⁺ CD107a ⁺ pGX0001 Non-immunized 30 mcg 0.29 ± 0.23 n/a 0.08±0.04 n/a pGX1438 Immunized 10 mcg 3.12 ± 1.19 0.001 0.40±0.18 0.004 30 mcg 3.73 ± 1.48 0.001 0.54±0.36 0.029 50 mcg 4.06 ± 1.87 0.003 0.53±0.25 0.004 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001). CD4 ⁺ and CD4 ⁺ CD107a ⁺ T cell responses to the dual promoter of synthetic consensus MUC16 (pGX1439) Drug code Group Dose amount % CD4 ⁺
±St.off. p value
% CD4 ⁺ % CD4 ⁺ CD107a ⁺
±St.off. p value
% CD4 ⁺ CD107a ⁺ pGX0001 Non-immunized 30 mcg 0.29 ± 0.23 n/a 0.08±0.04 n/a pGX1439 Immunized 10 mcg 1.99±0.99 0.007 0.26±0.18 0.069 30 mcg 2.71 ± 0.51 <0.001 0.36±0.18 0.008 50 mcg 3.25 ± 1.29 0.001 0.50±0.25 0.006 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001).

[00322] The frequency of antigen-specific CD8 ⁺ T cell responses induced by full length synthetic MUC16 consensus constructs was increased compared to control (0.13% ± 0.15%) across all dose groups for all constructs (FIGS. 23A-23C). Specifically, the frequency of antigen-specific responses of CD8 ⁺ T cells in all groups immunized with 10 μg (0.65% ± 0.28%, p=0.007), 30 μg (0.82% ± 0.46%, p=0.022) and 50 µg (1.60 ± 1.08%, p=0.034) pGXl435 was significantly more resistant compared to non-immunized mice. CD8 ⁺ T cell responses to pGXl435 synthetic consensus MUC16 were dose-dependent and consisted mainly of CD8 ⁺ T cells producing IFNγ ⁺ IL-2 ^- TNFα ^- and IFNγ ⁺ IL-2 ^- TNFα ⁺ (FIG. 23A) .

[00323] The frequency of antigen-specific responses of CD8 ⁺ T cells in groups immunized with 10 μg (1.30% ± 0.79%, p=0.024), 30 μg (1.33% ± 0.87%, p=0.034) and 50 µg (1.36%±0.71%, p=0.010) pGXl438 was also significantly more resistant compared to non-immunized mice. CD8 ⁺ T cell responses to pGX1438 synthetic consensus MUC16 were dose-independent, with very little difference between doses, and consisted primarily of CD8 ⁺ T cells producing IFNγ ⁺ IL-2 ^{- TNFα-} and IFNγ ⁺ IL-2 ^- TNFα ⁺ (FIG. 23B).

[00324] The frequency of antigen-specific responses of CD8 ⁺ T cells in groups immunized with 10 μg (1.36% ± 0.62%, p=0.004), 30 μg (2.07% ± 1.77%, p=0.089) and 50 μg (1.19% ± 0.69%, p=0.020) pGXl439 was also significantly more resistant compared to non-immunized mice. CD8 ⁺ T cell responses to pGX1439 synthetic consensus MUC16 were dose-independent and consisted mainly of CD8 ⁺ T cells producing IFNγ ⁺ IL-2 ^- TNFα ^- and IFNγ ⁺ IL-2 ^- TNFα ⁺ (FIG. 23C) . The frequency of antigen-specific CD8 ⁺ T cells is further detailed in Table 18.

[00325] Similar to the pattern with CD8 ⁺ antigen-specific T cells, full-length synthetic MUC16 consensus constructs induced a significant change in the frequency of CD8 ⁺ CD107a ⁺ T cells in all groups compared to non-immunized mice (0.05% ± 0.04%) over except for pGXl439 at a dose amount of 30 μg (FIGS. 24A-24C). Specifically, the frequency of CD8 ⁺ CD107a ⁺ antigen-specific T cells was 0.47±0.23%, 0.59±0.34%, and 1.19±0.90%, respectively, in the pGXl435 10 µg dose amount groups (p =0.005), 30 μg (p=0.013) and 50 μg (p=0.043) (FIG. 24A). The cytokine profile of CD8 ⁺ CD107a ⁺ T cells upon administration of the pGXl435 construct was similar across dose groups and consisted mainly of IFNγ ⁺ IL-2 ^- TNFα ^- and IFNγ ⁺ IL-2 ^- TNFα ⁺ cells (FIG. 23A).

[00326] The frequency of antigen-specific CD8 ⁺ CD107a ⁺ T cells was 0.80% ± 0.48%, 1.05% ± 0.77% and 1.05% ± 0.65%, respectively, in the pGXl438 dose amount groups 10 μg (p=0.016), 30 μg (p=0.039) and 50 μg (p=0.016) (FIG. 23B). The cytokine profile of CD8 ⁺ CD107a ⁺ T cells when administered with the pGXl438 construct was similar across dose groups and consisted mainly of IFNγ ⁺ IL-2 ^- TNFα ^- and IFNγ ⁺ IL-2 ^- TNFα ⁺ cells (FIG. 24B).

[00327] The frequency of antigen-specific CD8 ⁺ CD107a ⁺ T cells was 1.04% ± 0.65%, 1.68% ± 1.54% and 0.93% ± 0.68%, respectively, in the pGXl439 dose amount groups 10 μg (p=0.018), 30 μg (p=0.094) and 50 μg (p=0.038) (FIG. 23C). The cytokine profile of CD8 ⁺ CD107a ⁺ T cells when administered with the pGXl439 construct was similar across dose groups and consisted mainly of IFNγ ⁺ IL-2 ^- TNFα ^- and IFNγ ⁺ IL-2 ^- TNFα ⁺ cells (FIG. 24C). The frequency of antigen-specific CD8 ⁺ T cells with cytolytic potential is further detailed in Table 18.

Table 18

CD8 ⁺ and CD8 ⁺ CD107a ⁺ T cell responses to synthetic consensus MUC16 (pGX1435) fusion Drug code Group Dose amount %CD8 ⁺
±St.off. p value
%CD8 ⁺ %CD8 ⁺ CD107a ⁺
±St.off. p value
%CD8 ⁺ CD107a ⁺ pGX0001 Non-immunized 30 mcg 0.13±0.15 n/a 0.05±0.04 n/a pGX1435 Immunized 10 mcg 0.65 ± 0.28 0.007 0.47±0.23 0.005 30 mcg 0.82 ± 0.46 0.022 0.59±0.34 0.013 50 mcg 1.60 ± 1.08 0.034 1.19±0.90 0.043 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001). CD8 ⁺ and CD8 ⁺ CD107a ⁺ T cell responses to furin cleavage of synthetic consensus MUC16 (pGX1438) Drug code Group Dose amount %CD8 ⁺
±St.off. p value
%CD8 ⁺ %CD8 ⁺ CD107a ⁺
±St.off. p value
%CD8 ⁺ CD107a ⁺ pGX0001 Non-immunized 30 mcg 0.13±0.15 n/a 0.05±0.04 n/a pGX1438 Immunized 10 mcg 1.30±0.79 0.024 0.80±0.48 0.016 30 mcg 1.33±0.87 0.034 1.05±0.77 0.039 50 mcg 1.36±0.71 0.010 1.05±0.65 0.016 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001). CD8 ⁺ and CD8 ⁺ CD107a ⁺ T cell responses to the synthetic consensus MUC16 dual promoter (pGX1439) Drug code Group Dose quantity %CD8 ⁺
±St.off. p value
%CD8 ⁺ %CD8 ⁺ CD107a ⁺
±St.off. p value
%CD8 ⁺ CD107a ⁺ pGX0001 Non-immunized 30 mcg 0.13±0.15 n/a 0.05±0.04 n/a pGX1439 Immunized 10 mcg 1.36±0.62 0.004 1.04±0.65 0.018 30 mcg 2.07 ± 1.77 0.089 1.68±1.54 0.094 50 mcg 1.19 ± 0.69 0.020 0.93±0.68 0.038 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001).

[00328] Partial length synthetic MUC16 consensus constructs elicited strong responses in the CD4 ⁺ T cell compartment (FIGS. 25A-25B and FIGS. 26A-26B). CD4 ⁺ T cell intensity specific to the synthetic consensus MUC16 induced by the pGXl436 construct was dose-independent (FIG. 25A). Specifically, CD4 ⁺ T cells when injected with the pGXl436 synthetic consensus MUC16 construct were 1.74% ± 0.79%, 2.18% ± 1.15%, and 1.32% ± 1.74% at dose amounts, respectively, 10 mcg, 30 mcg and 50 mcg. The number of CD4 ⁺ T cells treated with the pGXl436 synthetic consensus MUC16 construct was significantly higher compared to non-immunized mice (0.17% ± 0.10%) at pGXl436 dose amounts of 10 μg (p=0.003) and 30 μg (p= 0.007) but not 50 µg (p=0.359). Specific CD4 ⁺ T cell responses to the pGXl436 synthetic consensus MUC16 construct consisted mainly of CD4 ⁺ T cells producing IFNγ ⁺ IL-2 ⁺ TNFα ⁺ , IFNγ ⁺ IL-2 ^- TNFα ⁺ or IFNγ ⁺ IL- 2 ^{- TNFα-} . The frequency of antigen-specific CD4 ⁺ CD107a ⁺ T cells was 0.45% ± 0.28%, 0.31% ± 0.26%, and 0.23% ± 0.30%, respectively, in the pGXl436 10 µg dose amount groups ( p=0.020), 30 μg (p=0.080), and 50 μg (p=0.369) (FIG. 25B). The cytokine profile of CD4 ⁺ CD107a ⁺ T cells when administered with the pGXl436 construct was similar across dose groups and consisted mainly of IFNγ ⁺ IL-2 ⁺ TNFα ⁺ and IFNγ ⁺ IL-2 ^- TNFα ⁺ cells (FIG. 25B). The frequency of antigen-specific CD4 ⁺ T cells with cytolytic potential is further detailed in Table 19.

[00329] CD4 ⁺ T cell intensity specific to the synthetic MUC16 consensus induced by the pGXl437 construct was dose-dependent (FIG. 26A). Specifically, CD4 ⁺ T cells when administered with the pGXl437 synthetic consensus MUC16 construct were 0.56% ± 0.38%, 1.14% ± 0.36%, and 1.87% ± 1.05% at dose amounts, respectively, 10 mcg, 30 mcg and 50 mcg. The number of CD4 ⁺ T cells treated with the pGXl437 synthetic consensus MUC16 construct was significantly higher compared to non-immunized mice (0.21% ± 0.14%) at pGXl437 dose amounts of 30 μg (p<0.001) and 50 μg (p= 0.011) but not 10 µg (p=0.063). Specific CD4 ⁺ T cell responses to the pGXl437 synthetic consensus MUC16 construct consisted mainly of CD4 ⁺ T cells producing IFNγ ⁺ IL-2 ⁺ TNFα ⁺ , IFNγ ⁺ IL-2 ^- TNFα ⁺ , or IFNγ ^- IL- 2 ^{- TNFα-} . The frequency of antigen-specific CD4 ⁺ CD107a ⁺ T cells was 0.09% ± 0.09%, 0.21% ± 0.05%, and 0.36% ± 0.21%, respectively, in the pGXl437 10 µg dose amount groups ( p=0.335), 30 μg (p<0.001) and 50 μg (p=0.016) (FIG. 26B). The cytokine profile of CD4 ⁺ CD107a ⁺ T cells when administered with the pGXl437 construct was similar across dose groups and consisted mainly of IFNγ ⁺ IL-2 ⁺ TNFα ⁺ and IFNγ ⁺ IL-2 ^- TNFα ⁺ cells (FIG. 26B). The frequency of antigen-specific CD4 ⁺ T cells with cytolytic potential is further detailed in Table 19.

Table 19

CD4 ⁺ and CD4 ⁺ CD107a ⁺ T cell responses to "Site 1" of the synthetic consensus MUC16 (pGX1436) Drug code Group Dose quantity % CD4 ⁺
±St.off. p value
% CD4 ⁺ % CD4 ⁺ CD107a ⁺
±St.off. p value
% CD4 ⁺ CD107a ⁺ pGX0001 Non-immunized 30 mcg 0.17 ± 0.10 n/a 0.04±0.03 n/a pGX1436 Immunized 10 mcg 1.74 ± 0.79 0.003 0.45±0.28 0.020 30 mcg 2.18 ± 1.15 0.007 0.31±0.26 0.080 50 mcg 1.32 ± 1.74 0.359 0.23±0.30 0.369 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001). CD4 ⁺ and CD4 ⁺ CD107a ⁺ T cell responses to "Plot 2" of synthetic consensus MUC16 (pGX1437) Drug code Group Dose amount % CD4 ⁺
±St.off. p value
% CD4 ⁺ % CD4 ⁺ CD107a ⁺
±St.off. p value
% CD4 ⁺ CD107a ⁺ pGX0001 Non-immunized 30 mcg 0.21±0.14 n/a 0.02±0.02 n/a pGX1437 Immunized 10 mcg 0.56 ± 0.38 0.063 0.09±0.09 0.335 30 mcg 1.14 ± 0.36 <0.001 0.21±0.05 <0.001 50 mcg 1.87 ± 1.05 0.011 0.36±0.21 0.016 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001).

[00330] Partial length synthetic MUC16 consensus constructs elicited strong responses in the CD8 ⁺ T cell compartment (FIGS. 27A-27B and FIGS. 28A-28B). The intensity of CD8 ⁺ T cells specific to the synthetic consensus MUC16 induced by the pGXl436 construct was dose-independent (FIG. 27A). Specifically, CD8 ⁺ T cells when administered with the pGXl436 synthetic consensus MUC16 construct were 1.76%±0.73%, 2.17%±0.81%, and 1.92%±2.13% at dose amounts, respectively, 10 mcg, 30 mcg and 50 mcg. The number of CD8 ⁺ T cells when injected with the synthetic consensus MUC16 construct pGXl436 was significantly higher compared to non-immunized mice (0.06% ± 0.08%) at pGXl436 dose amounts of 10 μg (p=0.002) and 30 μg (p= 0.001) but not 50 µg (p=0.192). The specific CD8 ⁺ T cell responses to the pGXl436 synthetic consensus MUC16 construct consisted primarily of CD8 ⁺ T cells producing IFNγ ⁺ IL-2 ^{- TNFα-} or IFNγ ⁺ IL-2 ^- TNFα ⁺ . The frequency of antigen-specific CD8 ⁺ CD107a ⁺ T cells was 1.55% ± 0.64%, 1.86% ± 0.73% and 1.72% ± 1.95%, respectively, in the pGXl436 10 μg dose amount groups (p =0.001), 30 μg (p=0.001) and 50 μg (p=0.193) (FIG. 26B). The cytokine profile of CD8 ⁺ CD107a ⁺ T cells when administered with the pGXl436 construct was similar across dose groups and consisted primarily of IFNγ ⁺ IL-2 ^- TNFα ^- or IFNγ ⁺ IL-2 ^- TNFα ⁺ cells (FIG. 27B). Overall, CD8 ⁺ T cell responses to Region 1 induced by pGXl435 are significantly lower compared to pGXl436 (FIG. 29A). The frequency of antigen-specific CD8 ⁺ T cells with cytolytic potential is further detailed in Table 20.

[00331] CD8 ⁺ T cell intensity specific to the synthetic MUC16 consensus induced by the pGXl437 construct was dose-dependent (FIG. 28A). Specifically, CD8 ⁺ T cells when injected with the pGXl437 synthetic consensus MUC16 construct were 0.26%±0.14%, 0.77%±0.47%, and 0.89%±0.37% at dose amounts, respectively, 10 mcg, 30 mcg and 50 mcg. The number of CD8 ⁺ T cells treated with the pGXl437 synthetic consensus MUC16 construct was significantly higher compared to non-immunized mice (0.07% ± 0.07%) at pGXl437 dose amounts of 30 μg (p=0.025) and 50 μg (p= 0.001) but not 10 µg (p=0.069). The specific responses of CD8 ⁺ T cells to the introduction of the pGXl437 synthetic consensus MUC16 construct consisted mainly of CD8 ⁺ T cells producing IFNγ ⁺ IL-2 ^- TNFα ⁺ or IFNγ ⁺ IL-2 ^- TNFα ^- . The frequency of antigen-specific CD8 ⁺ CD107a ⁺ T cells was 0.16% ± 0.10%, 0.58% ± 0.40%, and 0.89% ± 0.37%, respectively, in the pGXl437 10 µg dose amount groups ( p=0.022), 30 μg (p=0.030) and 50 μg (p=0.002) (Figure 28B). The cytokine profile of CD8 ⁺ CD107a ⁺ T cells when administered with the pGXl437 construct was similar across dose groups and consisted mainly of IFNγ ⁺ IL-2 ^- TNFα ^- and IFNγ ⁺ IL-2 ^- TNFα ⁺ cells (FIG. 28B). Overall, CD8 ⁺ T cell responses to Region 2 induced by pGXl435 and pGXl438 are significantly lower compared to pGXl437 (FIG. 29B). The frequency of antigen-specific CD8 ⁺ T cells with cytolytic potential is further detailed in Table 20.

Table 20

CD8 ⁺ and CD8 ⁺ CD107a ⁺ T cell responses to "Plot 1" of the synthetic consensus MUC16 (pGX1436) Drug code Group Dose amount %CD8 ⁺
±St.off. p value
%CD8 ⁺ %CD8 ⁺ CD107a ⁺
±St.off. p value
%CD8 ⁺ CD107a ⁺ pGX0001 Non-immunized 30 mcg 0.06 ± 0.08 n/a 0.03±0.03 n/a pGX1436 Immunized 10 mcg 1.76±0.73 0.002 1.55±0.64 0.001 30 mcg 2.17 ± 0.81 0.001 1.86±0.73 0.001 50 mcg 1.92 ± 2.13 0.192 1.72±1.95 0.193 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001). CD8 ⁺ and CD8 ⁺ CD107a ⁺ T cell responses to "Plot 2" of the synthetic consensus MUC16 (pGX1437) Drug code Group Dose amount %CD8 ⁺
±St.off. p value
%CD8 ⁺ %CD8 ⁺ CD107a ⁺
±St.off. p value
%CD8 ⁺ CD107a ⁺ pGX0001 Non-immunized 30 mcg 0.07 ± 0.07 n/a 0.03±0.02 n/a pGX1437 Immunized 10 mcg 0.26 ± 0.14 0.069 0.16±0.10 0.022 30 mcg 0.77 ± 0.47 0.025 0.58±0.40 0.030 50 mcg 0.89±0.37 0.001 0.89±0.37 0.002 Statistical significance was assigned at p≤0.05. P values are given in relation to non-immunized mice (immunized with pGX0001).

Statistical analysis

[00332] Statistical analysis was performed using IBM SPSS Statistics 22 (IBM Corporation). Analysis of differences between groups was performed using ANOVA using Tukey's Honest Significant Difference (HSD) post hoc test in order to adjust for multiple comparisons. Homogeneity of variance was confirmed using the F-statistic before multiple comparisons. For the entire statistical analysis, a p value of 0.050 was considered significant.

Conclusion

[00333] In general, full-length pGXl438 (furin cleavage) and pGXl439 (dual promoter) MUC16 constructs elicited more robust immune responses compared to pGXl435 (fusion) (FIGS. 30A-C). Compared to pGXl435, pGXl438 induced significantly more robust responses as measured by IFNγ in the ELISpot at dose amounts of 10 μg and 30 μg. Also, in the CD4 ⁺ T cell compartment, pGXl438 elicited significantly more robust immune responses compared to pGXl435 at a dose of 10 μg. There were no other significant differences in response intensity induced by the three full length MUC16 constructs, however there was a trend in favor of more robust CD8 ⁺ T cell responses induced by pGX1438 and pGX1439.

[00334] In addition, pGXl438 and pGXl439 induced pGXl436 equivalent Region 1 responses and pGXl437 equivalent Region 2 responses as measured by IFNγ detection in the CD4 ⁺ T cell compartment ELISpot. Responses equivalent to pGXl437 in the CD8 ⁺ T cell compartment were induced only by pGXl439.

[00335] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and should not be construed as limiting the scope of the invention, which is defined solely by the appended claims and their equivalents.

[00336] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications to the disclosed embodiments, including, without limitation, changes relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, compositions, or uses of the invention, may be made without departing from its spirit and scope.

Table 21: DNA coding sequence of IRC+R59 synthetic consensus MUC16

SEQ
ID
NO. SUBSEQUENCE one atggactgga cctggattct gttcctggtg gcagcagcaa cccgcgtgca ctccacagca gcaggacctc tgctggtgcc attcaccctg aactttacca tcacaaatct gcagtacgag gaggatatgc accacccagg cagcagaaag ttcaacacca cagagagggt gctgcagggc ctgctgggac caatgtttaa gaataccagc gtgggcctgc tgtattccgg atgcaggctg acactgctgc gctccgagaa ggacggagca gcaaccggcg tggatgccat ctgtacacac aggctggacc ccaagagccc tggcctggat cgggagcagc tgtactggga gctgtcccag ctgaccaacg gcatcaagga gctgggcccc tacacactgg accgcaacag cctgtatgtg aatggcttta cccaccggag ctccgtgcca aataccagca cacccggcac ctccacagtg gatctgggca cctccggcac accatctagc ctgccttctc caaccgcagc aggaccactg ctggtgcctt tcacactgaa ctttaccatt accaatctgc agtatgaaga ggacatgagg catcctggca gcagaaagtt caacacgaca gagagagtgc tgcaaggcct gctgaagcca ctgtttaaga atacctctgt gggccccctg tatagtggct gtagactgac actgctgcgc cccgaaaaag atggcgccgc cactggagtc gacgccatct gtacacacag actggacccc aagtctcccg gcctgaacag agaacagctg tattgggaac tgagcaagct gaccaatggc atcacagagc tgggccctta caccctggac agaaactctc tgtatgtgaa tggcttcacc cacaggacat ctgtgcctac cacaagcacc ccaggcacct ccaccgtcga tctgggcacc agcggcacac cttttagcct gccatcccct accaccgccg gaccactgct ggtgcccttc accctgaact ttaccataac caatctgcag tatgaggagg acatgcaccg gcccggcagc agaaagttca acactacaga gagagtgctg cagggcctgt tatcccctat ctttaagaat tcctctgtgg gcccactgta cagcggatgc aggctgacct ctctgcggcc cgaaaaagac ggagcagcaa caggaatgga tgccgtgtgc ctgtaccacc caaaccccaa gaggccaggc ctggatcgcg agcagctgta ttgggagctg agccagctga cccacaatat cacagagctg ggcccctact ctctggacag ggatagcctg tatgtgaacg gcttcaccca ccagaattccgtgcccacca catctacacc tggcaccagc acagtgtact gggccaccac aggcacccct agctcctttc caggacacac agcacctgga ccactgctga tcccattcac cctgaacttt accattacaa atctgcacta tgaggagaac atgcagcacc ccggcagccg caagttcaat accacagagc gggtgctgca aggcctgctg aagccgctgt ttaagaacac cagcgtggga cctctgtact ctggctgtcg cctgacactg ctgcggcccg agaagcatgg cgcagcaacc ggcgtggacg ctatttgcac ccacagactg gaccctaagg gcccaggcct ggatagggag cgcctgtatt gggaactgtc tcagctgacc aattctatca cagagctggg accatacacc ctggacaggg attctctgta cgtgaacggc ttcaatccac ggtctagcgt gcccaccaca agcacccctg gcacctccac agtgcacctg gccacctctg gcacaccctc ctctctgcct ggacacaccg caagccccct gctggtgctg ttcacaatca actttaccat tactaatctg agatacgagg agaacatgca ccaccctggc tccagaaagt tcaatactac cgaaagggtg ctgcaaggcc tgctgcgccc agtgtttaag aacacttccg tgggccccct gtatagcggc tgtaggctga cactgctgcg ccccaagaag gatggcgcag caaccaaggt cgacgctatc tgtacataca ggcccgaccc taaatcccca ggcctggacc gggagcagct gtattgggag ctgtctcagc tgacccactc cattaccgaa ctgggaccct acaccctgga cagagattcc ctgtacgtga atggcttcac acagaggagc tccgtgccta ccacatccat cccaggaacc cctacagtgg acctgggcac ctccggaacc ccagtgtcta agccaggacc atctgccgca agccacctgc tgatcctgtt caccctgaac tttaccataa cgaatctgcg gtacgaggag aacatgtggc caggctcccg gaagttcaat actaccgaga gagtgctgca aggcctgctg aggcctctgt ttaagaacac tagcgtgggc ccactgtatt ccggctctcg gctgaccctg ctgcggcccg aaaaagatgg agaggccaca ggcgtggatg ccatctgcac ccacagacct gacccaacag gacctggcct ggacagggag cagctgtacc tggagctgtc acagctgact cactccatta ccgagctggg cccctatac t ctggatagag actccctgta cgtcaacggc ttcacccacc ggtctagcgt gccaaccaca tctacaggcg tggtgagcga ggaacccttc accctgaact tcaccatcaa caatctgagg tacatggcag acatgggaca gccaggctct ctgaagttca acatcaccga taatgtgatg aagcacctgc tgagccctct gtttcagcgg agcagcctgg gagcaaggta caccggatgc cgcgtgatcg ccctgcggtc cgtgaagaac ggagcagaga cacgggtgga cctgctgtgc acatatctgc agcctctgag cggaccaggc ctgcccatca agcaggtgtt ccacgagctg tctcagcaga cccacggaat cacaaggctg ggaccctact ccctggacaa ggattctctg tacctgaacg gctataatga gcctggcctg gacgagcccc ctaccacacc caagcctgcc accacatttc tgccacccct gagcgaggca accacagcaa tgggatacca cctgaagacc ctgacactga acttcaccat cagcaatctg cagtattccc ccgatatggg caagggctct gccaccttta acagcacaga gggcgtgctg cagcacctgc tgcggcccct gttccagaag agctccatgg gcccttttta cctgggctgc cagctgatct ccctgaggcc tgaaaaagat ggagcagcaa ccggagtgga taccacatgt acataccacc cagaccccgt gggaccaggc ctggatatcc agcagctgta ctgggaactg tcccagctga ctcacggcgt gacacagctg ggcttctacg tgctggaccg cgattccctg tttatcaacg gctacgcccc tcagaatctg tctatccg gg gcgagtatca gatcaacttc cacatcgtga actggaatct gagcaatcct gacccaacct ctagcgagta catcgccctg ctgcgcgaca tccaggataa ggtgaccaca ctgtataagg gctcccagct gcacgacacc ttccggtttt gcctggtgac caacctgaca atggattcta tgctggtgac agtgaaggcc ctgttctcct ctaacctgga ccccagcctg gtggagcagg tgtttctgga taagaccctg aatgccagct cccactggct gggctccacc taccagctgg tggacatcca cgtgacagag atggagccaa gcgtgtacca gcccacctct agctcctcta cacagcactt ctacctgaac tttaccataa ctaatctgcc ctatagccag gatatcgccc agcctggcac cacaaactac cagcggaaca agagaaatat cgaggacgcc ctgaaccagc tgttcagaaa tagctccatc aagtcctatt tctctgattg ccaggtgagc acctttaggt ccgtgcctaa ttctcaccac acaggcgtgg actccctgtg cgccttttct ccactggcaa ggagagtgga tagggtggca atctacgagg agttcctgag gatgacccgc gccggaacac agctgcaggc ctttaccctg gacagatcta gcgtgctggt ggatggctat agccctaaca ggaatgagcc actgaccggc aactccgacc tgcccttctg ggccatcatc ctgatctgtc tggcaggcct gctgggcctg atcacctgcc tgatctgtgg ctttctggtg tgataa

Table 22: Protein sequence of the IRC+R59 synthetic consensus MUC 16

SEQ
ID
NO. SUBSEQUENCE 2 MDWTWILFLVAAATRVHSTAAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGLDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHRSSVPNTSTPGTSTVDLGTSGTPSSLPSPTAAGPLLVPFTLNFTITNLQYEEDMRHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKDGAATGVDAICTHRLDPKSPGLNREQLYWELSKLTNGITELGPYTLDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNTTERVLQGLLSPIFKNSSVGPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPYSLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTAPGPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKHGAATGVDAICTHRLDPKGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPRSSVPTTSTPGTSTVHLATSGTPSSLPGHTASPLLVLFTINFTITNLRYEENMHHPGSRKFNTTERVLQGLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELSQLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPSAASHLLILFTLNFTITNLRYEENMWPGSRKFNTTERVLQGLLRPLFKNTSVGPLYSGSRLTLLRPEKDGEATGVDAICTHRPDPTGPGLDREQLYLELSQLTHSITELGPYTLDRDSLYVNGFTHRSSVPTTSTGVVSEEPFTLNFTINNLRYMADMGQPGSLKFNITDNVMKHLLSPLFQRSSLGARYTGCRVIALRSVKN GAETRVDLLCTYLQPLSGPGLPIKQVFHELSQQTHGITRLGPYSLDKDSLYLNGYNEPGLDEPPTTPKPATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGVLQHLLRPLFQKSSMGPFYLGCQLISLRPEKDGAATGVDTTCTYHPDPVGPGLDIQQLYWELSQLTHGVTQLGFYVLDRDSLFINGYAPQNLSIRGEYQINFHIVNWNLSNPDPTSSEYIALLRDIQDKVTTLYKGSQLHDTFRFCLVTNLTMDSMLVTVKALFSSNLDPSLVEQVFLDKTLNASSHWLGSTYQLVDIHVTEMEPSVYQPTSSSSTQHFYLNFTITNLPYSQDIAQPGTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSVPNSHHTGVDSLCAFSPLARRVDRVAIYEEFLRMTRAGTQLQAFTLDRSSVLVDGYSPNRNEPLTGNSDLPFWAIILICLAG LLGLITCLICGFLV

Table 23: Synthetic consensus MUC16 RMC DNA coding sequence

SEQ
ID
NO. SUBSEQUENCE 3 atggattgga cttggattct gttcctggtc gccgccgcaa ctcgggtgca ttctactgct gctgggccac tgctggtgcc ttttacactg aacttcacca tcacaaatct gcagtacgag gaggacatgc accaccctgg ctctcggaag ttcaacacca cagagagagt gctgcagggc ctgctgggcc caatgtttaa gaataccagc gtgggcctgc tgtattccgg atgccggctg acactgctga gatccgagaa ggacggagca gcaaccggag tggatgccat ctgtacacac aggctggacc caaagtcccc aggcctggat agagagcagc tgtactggga gctgtctcag ctgaccaacg gcatcaagga gctgggcccc tacacactgg accggaacag cctgtatgtg aatggcttta cccacagaag ctccgtgcca aataccagca cacccggcac ctccacagtg gatctgggca cctctggcac accttctagc ctgccaagcc ctaccgcagc aggaccactg ctggtgccct tcacactgaa ctttaccatt accaatctgc agtatgaaga ggacatgagg cacccaggct cccgcaagtt caacactacc gagcgggtgc tgcaaggcct gctgaagcct ctgtttaaga atacctctgt gggcccactg tatagtggct gccggctgac actgctgcgg cccgaaaaag acggagcagc aaccggcgtg gatgctattt gcacccacag gctggacccc aagagcccag gcctgaaccg cgaacagctg tattgggagc tgtccaagct gaccaatggc atcacagagc tgggccccta caccctggac agaaattccc tgtacgtgaa tggcttcacc caccgcacat ctgtgcctac cacaagcacc ccaggcacct ccacagtgga tctgggcacc tccggcacac ccttttccct gccatctcca accacagcag gacctctgct ggtgccattc accctgaact ttaccattac taatctgcag tatgaagaag acatgcacag gcctggctct cgcaagttca acactactga gagggtgctg cagggcctgt taagcccaat ctttaagaat tcctctgtgg gccctctgta ttccggatgc aggctgacct ctctgcgccc agaaaaagat ggagcagcaa caggaatgga tgccgtgtgc ctgtaccacc ctaacccaaa gcggcccggc ctggacaggg agcagctgta ttgggaactg agccagctga cccacaatat cacagagctg ggcccttact ctctggaccg cgatagcctg tatgtgaacg gcttcaccca ccagaattcc gtgcccacca catctacacc tggcaccagc acagtgtact gggccaccac aggcaccccc agctcctttc ctggacacac agcaccagga cctctgctga tccccttcac cctgaacttt accataacaa atctgcacta tgaggagaac atgcagcacc ctggcagcag gaagttcaat accacagagc gcgtgctgca aggcctgctg aagccgctgt ttaagaacac cagcgtggga ccactgtaca gcggctgcag gctgaccctg ctgcgccctg agaagcatgg cgccgccacc ggcgtggatg ctatctgcac acatagactg gaccccaagg gacctggcct ggatagggag agactgtact gggaactgtc ccagctgacc aactcaatta cagagctggg cccatacacc ctggaccggg attctctgt a cgtgaacggc ttcaatccaa gatctagcgt ccctaccaca tctacccctg ggacaagtac cgtgcatctg gctacaagcg gaactccttc aagtctgcct ggacactgat

Table 24: Synthetic consensus MUC16 RMC protein sequence

SEQ
ID
NO. SUBSEQUENCE four MDWTWILFLVAAATRVHSTAAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGLDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHRSSVPNTSTPGTSTVDLGTSGTPSSLPSPTAAGPLLVPFTLNFTITNLQYEEDMRHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKDGAATGVDAICTHRLDPKSPGLNREQLYWELSKLTNGITELGPYTLDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNTTERVLQGLLSPIFKNSSVGPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPYSLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTAPGPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKHGAATGVDAICTHRLDPKGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPRSSVPTTSTPGTSTVHLATSGTPSSLPGH

Table 25: NRC DNA Coding Sequence of Synthetic Consensus MUC16

SEQ
ID
NO. SUBSEQUENCE 5 atggactgga cttggattct gttcctggtc gctgccgcca cccgcgtgca tagtgccgca tctcacctgc tgattctgtt caccctgaac ttcaccatca caaatctgcg gtacgaggag aacatgtggc caggcagccg caagttcaat accacagagc gggtgctgca gggcctgctg cggcccctgt ttaagaacac ctccgtgggc cccctgtatt ctggcagcag gctgaccctg ctgcgcccag agaaggacgg agaggcaaca ggcgtggatg ccatctgcac ccacaggcct gacccaacag gaccaggcct ggatagggag cagctgtacc tggagctgag ccagctgacc cactccatca cagagctggg accatacacc ctggacaggg attccctgta tgtgaacggc tttacccaca gaagctccgt gcccaccaca tctacaggcg tggtgagcga ggagcccttc accctgaact tcaccatcaa caatctgagg tacatggcag acatgggaca gccaggcagc ctgaagttca acatcaccga taatgtgatg aagcacctgc tgtcccctct gtttcagcgg tctagcctgg gagcaaggta caccggctgc agagtgatcg ccctgaggtc cgtgaagaac ggagcagaga cacgggtgga cctgctgtgc acatatctgc agcctctgag cggaccaggc ctgcctatca agcaggtgtt ccacgagctg tctcagcaga cccacggaat cacacgcctg ggaccttact ccctggacaa ggattctctg tacctgaacg gctataatga gccaggcctg gacgagcccc ctaccacacc caagcctgcc accacatttc tgccacccct gagcgaggca accacagcaa tgggatacca cctgaagacc ctgacactga acttcaccat cagcaatctg cagtattccc ccgatatggg caagggctct gccaccttta atagcacaga gggcgtgctg cagcacctgc tgaggcctct gttccagaag tcctctatgg gccccttcta cctgggatgc cagctgatct ccctgcgccc tgagaaggac ggagcagcaa ccggagtgga taccacatgt acataccacc cagaccccgt gggaccaggc ctggatatcc agcagctgta ttgggaactg tcccagctga cccacggcgt gacacagctg ggcttctatg tgctggaccg ggattctctg tttatcaacg gctacgcccc tcagaatctg agcatcagag gcgagtatca gatcaacttc cacatcgtga actggaatct gtctaatcct gatccaacca gctccgagta catcgccctg ctgcgggaca tccaggataa ggtgaccaca ctgtataagg gcagccagct gcacgacacc ttcagatttt gtctggtgac caacctgaca atggattcca tgctggtgac agtgaaggcc ctgttctcta gcaacctgga cccttctctg gtggagcagg tgtttctgga taagaccctg aatgcctcct ctcactggct gggctctacc taccagctgg tggacatcca cgtgacagag atggagccaa gcgtgtatca gcccaccagc tcctctagca cacagcactt ctacctgaac tttaccatca caaatctgcc ctatagccag gatatcgccc agcctggcac cacaaactac cagaggaaca agcgcaatat cgaggacgcc ctgaaccagc tgttcaggaa ttcctctatc aagtcctatt tctctgatt g ccaggtgagc acctttcgct ccgtgccaaa ttctcaccac acaggcgtgg actccctgtg cgccttttct cccctggcac ggagagtgga tagggtggca atctacgagg agttcctgcg gatgaccaga gccggcacac agctgcaggc ctttaccctg gacaggagct ccgtgctggt ggatggctat agccctaacc gcaatgagcc actgacaggc aattccgacc tgcccttctg ggccatcatc ctgatttgcc tggctggact gctggggctg attacctgtc tgatttgtgg gttcctggtg tgataa

Table 26: NRC Protein Sequence of Synthetic Consensus MUC 16

SEQ
ID
NO. SUBSEQUENCE 6 MDWTWILFLVAAATRVHSTAAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGLDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHRSSVPNTSTPGTSTVDLGTSGTPSSLPSPTAAGPLLVPFTLNFTITNLQYEEDMRHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKDGAATGVDAICTHRLDPKSPGLNREQLYWELSKLTNGITELGPYTLDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNTTERVLQGLLSPIFKNSSVGPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPYSLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTAPGPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKHGAATGVDAICTHRLDPKGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPRSSVPTTSTPGTSTVHLATSGTPSSLPGH

Table 27: Synthetic MUC16 consensus IRC DNA coding sequence

SEQ
ID
NO. SUBSEQUENCE 7 atggactgga cctggattct gttcctggtg gcagcagcaa ccagggtgca ctccacagca gcaggacctc tgctggtgcc attcaccctg aactttacca tcacaaatct gcagtacgag gaggatatgc accaccccgg cagccgcaag ttcaacacca cagagcgggt gctgcagggc ctgctgggac ctatgtttaa gaataccagc gtgggcctgc tgtattccgg atgcaggctg acactgctgc gctccgagaa ggacggagca gcaaccggcg tggatgccat ctgtacacac aggctggacc caaagtctcc cggcctggat cgcgagcagc tgtactggga gctgagccag ctgaccaacg gcatcaagga gctgggcccc tacacactgg accggaacag cctgtatgtg aatggcttca cccacagaag ctccgtgcca aatacctcca cacccggcac ctctacagtg gatctgggca cctctggcac accctctagc ctgcctagcc caaccgcagc aggaccactg ctggtgcctt tcacactgaa ctttaccatt accaatctgc agtatgaaga ggacatgcgg caccctggca gcagaaagtt caacactacc gagcgcgtgc tgcaaggcct gctgaagcca ctgtttaaga atacctctgt gggccccctg tatagtggct gtagactgac actgctgcgc cctgaaaaag atggcgccgc cactggagtc gacgctattt gcacccacag gctggacccc aagtccccag gcctgaacag agaacagctg tattgggagc tgtctaagct gaccaatggc atcacagagc tgggcccata caccctggac aggaactctc tgtacgtcaa tggcttcacc caccgcacaa gcgtgcctac cacatccacc ccaggcacct ctaccgtcga tctgggcacc agcggcacac cattttccct gccatctcct accaccgccg gaccactgct ggtgcccttc accctgaact ttaccataac caatctgcag tatgaggagg acatgcaccg gcccggctct agaaagttca acactactga acgggtgctg caaggcctgt taagccctat ctttaagaat tcctctgtgg gcccactgta cagcggatgc aggctgacct ctctgcggcc cgaaaaagac ggagcagcaa caggaatgga tgccgtgtgc ctgtaccacc caaaccccaa gaggcctggc ctggacagag agcagctgta ttgggaactg tcccagctga cccacaatat cacagagctg ggcccctaca gcctggacag agattccctg tatgtgaacg gcttcaccca ccagaattct gtgcccacca caagcacacc tggcacctcc acagtgtact gggccaccac aggcacccct agctcctttc caggacacac agcacctgga ccactgctga tccctttcac cctgaacttt accattacaa atctgcacta tgaggagaac atgcagcacc caggcagcag aaagttcaat accacagaga gggtgctgca aggcctgctg aagccgctgt ttaagaacac cagcgtggga cctctgtact ctggctgtcg cctgacactg ctgcggcccg agaagcatgg cgcagcaacc ggcgtggacg ctatttgcac tcatagactg gaccccaagg gacctggcct ggatagggag agactgtact gggaactgtc tcagctgacc aattccatta cagagctggg cccttacacc ctggaccggg acagcctgt a cgtcaacggc ttcaatccaa gatctagcgt gcccaccaca tccacccctg gcacctctac agtgcacctg gccaccagcg gaacaccctc ctctctgcct ggacacaggg gaaggaagcg gagaagcgcc gcatcccacc tgctgatcct gttcaccctg aactttacca taacgaatct gagatacgag gagaacatgt ggcctggctc ccgcaagttc aatactaccg aacgggtgct gcagggcctg ctgcggcccc tgtttaagaa cacttccgtg ggccccctgt attctggcag caggctgacc ctgctgcgcc cagagaagga cggagaggca acaggcgtgg atgccatctg cacccacagg cccgacccta caggaccagg cctggatagg gagcagctgt acctggagct gtcccagctg actcactcaa ttaccgaact gggaccttac accctggaca gggatagtct gtacgtgaat ggcttcaccc atcgcagctc cgtgccaacc acatctacag gcgtggtgag cgaggaaccc ttcaccctga acttcaccat caacaatctg aggtacatgg ccgacatggg ccagccaggc tccctgaagt tcaacatcac cgataatgtg atgaagcacc tgctgtctcc cctgtttcag aggtctagcc tgggagcaag gtacaccgga tgcagagtga tcgccctgag gtccgtgaag aacggagcag agacacgggt ggacctgctg tgcacatatc tgcagcctct gagcggacca ggcctgccca tcaagcaggt gttccacgag ctgtcccagc agacccacgg aatcacaagg ctgggacctt actccctgga caaggattct ctgtacctga acggctataa tgagccag gc ctggacgagc cccctaccac accaaagccc gccaccacat ttctgccacc cctgagcgag gcaaccacag caatgggata ccacctgaag accctgacac tgaacttcac catcagcaat ctgcagtatt cccccgatat gggcaagggc tctgccacct ttaacagcac agagggcgtg ctgcagcacc tgctgcggcc tctgttccag aagtcctcta tgggcccctt ctacctggga tgccagctga tctccctgcg gcccgaaaag gatggagcag caaccggagt ggataccaca tgtacatacc accctgaccc agtgggacca ggcctggata tccagcaatt atattgggaa ctgagtcagc tgacccacgg cgtgacacag ctgggcttct atgtgctgga cagggatagc ctgtttatca acggctacgc cccacagaat ctgtccatcc gcggcgagta tcagatcaac ttccacatcg tgaactggaa tctgagcaat cccgacccta ccagctccga gtacatcgcc ctgctgaggg acatccagga taaggtgacc acactgtata agggctccca gctgcacgac accttccgct tttgcctggt gaccaacctg acaatggatt ctatgctggt gacagtgaag gccctgttct ctagcaacct ggaccccagc ctggtggagc aggtgtttct ggataagacc ctgaatgcct cctctcactg gctgggcagc acctaccagc tggtggacat ccacgtgaca gagatggagc catccgtgta tcagcccacc agctcctcta gcacacagca cttctacctg aactttacca taactaatct gccctatagc caggatatcg cccagcctgg caccacaaac taccagc gga acaagagaaa tatcgaggac gccctgaacc agctgttccg gaattcctct atcaagtctt atttcagcga ttgccaggtg tccaccttta gatctgtgcc aaatagccac cacacaggcg tggactccct gtgcgccttt tctcccctgg caaggagggt ggatagggtg gcaatctacg aggagttcct gaggatgacc cgcgccggaa cacagctgca ggcctttacc ctggaccgga gctccgtgct ggtggatggc tattccccta acagaaatga gccactgaca ggcaactctg acctgccctt ctgggccatc atcctgatct gtctggcagg cctgctgggc ctgatcacct gcctgatctg tggctttctg gtgtgataa

Table 28: Synthetic consensus MUC16 IRC protein sequence

SEQ
ID
NO. SUBSEQUENCE eight MDWTWILFLVAAATRVHSTAAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGLDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHRSSVPNTSTPGTSTVDLGTSGTPSSLPSPTAAGPLLVPFTLNFTITNLQYEEDMRHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKDGAATGVDAICTHRLDPKSPGLNREQLYWELSKLTNGITELGPYTLDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNTTERVLQGLLSPIFKNSSVGPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPYSLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTAPGPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKHGAATGVDAICTHRLDPKGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPRSSVPTTSTPGTSTVHLATSGTPSSLPGHRGRKRRSAASHLLILFTLNFTITNLRYEENMWPGSRKFNTTERVLQGLLRPLFKNTSVGPLYSGSRLTLLRPEKDGEATGVDAICTHRPDPTGPGLDREQLYLELSQLTHSITELGPYTLDRDSLYVNGFTHRSSVPTTSTGVVSEEPFTLNFTINNLRYMADMGQPGSLKFNITDNVMKHLLSPLFQRSSLGARYTGCRVIALRSVKNGAETRVDLLCTYLQPLSGPGLPIKQVFHELSQQTHGITRLGPYSLDKDSLYLNGYNEPGLDEPPTTPKPATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGVLQHLLRPLFQKSSMGPFYLGCQLISLRPEK DGAATGVDTTCTYHPDPVGPGLDIQQLYWELSQLTHGVTQLGFYVLDRDSLFINGYAPQNLSIRGEYQINFHIVNWNLSNPDPTSSEYIALLRDIQDKVTTLYKGSQLHDTFRFCLVTNLTMDSMLVTVKALFSSNLDPSLVEQVFLDKTLNASSHWLGSTYQLVDIHVTEMEPSVYQPTSSSSTQHFYLNFTITNLPYSQDIAQPGTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSVPNSHHTGVDSLCAFSPLARRVDRVAIYEEFLRMTRAGTQLQAFTLDRSSVLVDGYSPNRNEPLTGNSDLPFWAIILICLAGLLGLITCLICGFLV

--->

SEQUENCE LIST

<110> INOVIO PHARMACEUTICALS, INC.

YAN, Jian

SLAGER, Anna

GARMAN, Bradley

Cook, Neil

<120> MUC16 ANTI-CANCER VACCINES AND THEIR USE

<130> I1005 US PRO

<160> 8

<170> Patent Version 3.5

<210> 1

<211> 4476

<212> DNA

<213> artificial sequence

<220>

<223> Microsensory repeat MUC16 + repeats 59, 61, 62, 63

+ synthetic terminal truncated carboxy terminal

domain of the amino acid sequence encoding the pGX1435 protein

<400> 1

atggactgga cctggattct gttcctggtg gcagcagcaa cccgcgtgca ctccacagca 60

gcaggacctc tgctggtgcc attcaccctg aactttacca tcacaaatct gcagtacgag 120

gaggatatgc accacccagg cagcagaaag ttcaacacca cagagagggt gctgcagggc 180

ctgctgggac caatgtttaa gaataccagc gtgggcctgc tgtattccgg atgcaggctg 240

acactgctgc gctccgagaa ggacggagca gcaaccggcg tggatgccat ctgtacacac 300

aggctggacc ccaagagccc tggcctggat cgggagcagc tgtactggga gctgtcccag 360

ctgaccaacg gcatcaagga gctgggcccc tacacactgg accgcaacag cctgtatgtg 420

aatggcttta cccaccggag ctccgtgcca aataccagca cacccggcac ctccacagtg 480

gatctgggca ccctccggcac accatctagc ctgccttctc caaccgcagc aggaccactg 540

ctggtgcctt tcacactgaa ctttaccatt accaatctgc agtatgaaga ggacatgagg 600

catcctggca gcagaaagtt caacacgaca gagagagtgc tgcaaggcct gctgaagcca 660

ctgtttaaga atacctctgt gggccccctg tatagtggct gtagactgac actgctgcgc 720

cccgaaaaag atggcgccgc cactggagtc gacgccatct gtacacacag actggacccc 780

aagtctcccg gcctgaacag agaacagctg tattgggaac tgagcaagct gaccaatggc 840

atcacagagc tgggccctta caccctggac agaaactctc tgtatgtgaa tggcttcacc 900

cacaggacat ctgtgcctac cacaagcacc ccaggcacct ccaccgtcga tctgggcacc 960

agcggcacac cttttagcct gccatcccct accaccgccg gaccactgct ggtgcccttc 1020

accctgaact ttaccataac caatctgcag tatgaggagg acatgcaccg gcccggcagc 1080

agaaagttca acactacaga gagagtgctg cagggcctgt tatcccctat ctttaagaat 1140

tcctctgtgg gcccactgta cagcggatgc aggctgacct ctctgcggcc cgaaaaagac 1200

ggagcagcaa caggaatgga tgccgtgtgc ctgtaccacc caaaccccaa gaggccaggc 1260

ctggatcgcg agcagctgta ttgggagctg agccagctga cccacaatat cacagagctg 1320

ggcccctact ctctggacag ggatagcctg tatgtgaacg gcttcaccca ccagaattcc 1380

gtgcccacca catctacacc tggcaccagc acagtgtact gggccaccac aggcacccct 1440

agctcctttc caggacacac agcacctgga ccactgctga tcccattcac cctgaacttt 1500

accattacaa atctgcacta tgaggagaac atgcagcacc ccggcagccg caagttcaat 1560

accacagagc gggtgctgca aggcctgctg aagccgctgt ttaagaacac cagcgtggga 1620

cctctgtact ctggctgtcg cctgacactg ctgcggcccg agaagcatgg cgcagcaacc 1680

ggcgtggacg ctatttgcac ccacagactg gaccctaagg gcccaggcct ggatagggag 1740

cgcctgtatt gggaactgtc tcagctgacc aattctatca cagagctggg accatacacc 1800

ctggacaggg attctctgta cgtgaacggc ttcaatccac ggtctagcgt gcccaccaca 1860

agcacccctg gcacctccac agtgcacctg gccacctctg gcacaccctc ctctctgcct 1920

ggacacaccg caagccccct gctggtgctg ttcacaatca actttaccat tactaatctg 1980

agatacgagg agaacatgca ccaccctggc tccagaaagt tcaatactac cgaaagggtg 2040

ctgcaaggcc tgctgcgcc agtgtttaag aacacttccg tgggccccct gtatagcggc 2100

tgtaggctga cactgctgcg ccccaagaag gatggcgcag caaccaaggt cgacgctatc 2160

tgtacataca ggcccgaccc taaatcccca ggcctggacc gggagcagct gtattgggag 2220

ctgtctcagc tgacccactc cattaccgaa ctgggaccct acaccctggga cagagattcc 2280

ctgtacgtga atggcttcac acagaggagc tccgtgccta ccacatccat ccggaacc 2340

cctacagtgg acctgggcac ctcgggaacc ccagtgtcta agccaggacc atctgccgca 2400

agccacctgc tgatcctgtt caccctgaac tttaccataa cgaatctgcg gtacgaggag 2460

aacatgtggc caggctcccg gaagttcaat actaccgaga gagtgctgca aggcctgctg 2520

aggcctctgt ttaagaacac tagcgtgggc ccactgtatt ccggctctcg gctgaccctg 2580

ctgcggcccg aaaaagatgg agaggccaca ggcgtggatg ccatctgcac ccacagacct 2640

gacccaacag gacctggcct ggacagggag cagctgtacc tggagctgtc acagctgact 2700

cactccatta ccgagctggg cccctatact ctggatagag actccctgta cgtcaacggc 2760

ttcacccacc ggtctagcgt gccaaccaca tctacaggcg tggtgagcga ggaacccttc 2820

accctgaact tcaccatcaa caatctgagg tacatggcag acatgggaca gccaggctct 2880

ctgaagttca acatcaccga taatgtgatg aagcacctgc tgagccctct gtttcagcgg 2940

agcagcctgg gagcaaggta caccggatgc cgcgtgatcg ccctgcggtc cgtgaagaac 3000

ggagcagaga cacgggtgga cctgctgtgc acatatctgc agcctctgag cggaccaggc 3060

3120

ggaccctact ccctggacaa ggattctctg tacctgaacg gctataatga gcctggcctg 3180

gacgagcccc ctaccacacc caagcctgcc accacatttc tgccacccct gagcgaggca 3240

accacagcaa tgggatacca cctgaagacc ctgacactga acttcaccat cagcaatctg 3300

3360

cagcacctgc tgcggcccct gttccagaag agctccatgg gcccttttta cctgggctgc 3420

3480

acataccacc cagaccccgt gggaccaggc ctggatatcc agcagctgta ctgggaactg 3540

tcccagctga ctcacggcgt gacacagctg ggcttctacg tgctggaccg cgattccctg 3600

tttatcaacg gctacgcccc tcagaatctg tctatccggg gcgagtatca gatcaacttc 3660

cacatcgtga actggaatct gagcaatcct gacccaacct ctagcgagta catcgccctg 3720

ctgcgcgaca tccaggataa ggtgaccaca ctgtataagg gctcccagct gcacgacacc 3780

ttccggtttt gcctggtgac caacctgaca atggattcta tgctggtgac agtgaaggcc 3840

ctgttctcct ctaacctgga ccccagcctg gtggagcagg tgtttctgga taagaccctg 3900

aatgccagct cccactggct gggctccacc taccagctgg tggacatcca cgtgacagag 3960

atggagccaa gcgtgtacca gcccacctct agctcctcta cacagcactt ctacctgaac 4020

tttaccataa ctaatctgcc ctatagccag gatatcgccc agcctggcac cacaaactac 4080

cagcggaaca agagaaatat cgaggacgcc ctgaaccagc tgttcagaaa tagctccatc 4140

aagtcctatt tctctgattg ccaggtgagc acctttaggt ccgtgcctaa ttctcaccac 4200

acaggcgtgg actccctgtg cgccttttct ccactggcaa ggagagtgga tagggtggca 4260

atctacgagg agttcctgag gatgacccgc gccggaacac agctgcaggc ctttaccctg 4320

gacagatcta gcgtgctggt ggatggctat agccctaaca ggaatgagcc actgaccggc 4380

aactccgacc tgcccttctg ggccatcatc ctgatctgtc tggcaggcct gctgggcctg 4440

atcacctgcc tgatctgtgg ctttctggtg tgataa 4476

<210> 2

<211> 1490

<212> Protein

<213> artificial sequence

<220>

<223> Microsensory repeat MUC16 + repeats 59, 61, 62, 63

+ synthetic consensus carboxy-terminal domain

amino acid sequence encoding pGX1435 protein

<400> 2

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

5 10 15

His Ser Thr Ala Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe

20 25 30

Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asp Met His His Pro Gly Ser

35 40 45

Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Gly Pro

50 55 60

Met Phe Lys Asn Thr Ser Val Gly Leu Leu Tyr Ser Gly Cys Arg Leu

65 70 75 80

Thr Leu Leu Arg Ser Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala

85 90 95

Ile Cys Thr His Arg Leu Asp Pro Lys Ser Pro Gly Leu Asp Arg Glu

100 105 110

Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr Asn Gly Ile Lys Glu Leu

115 120 125

Gly Pro Tyr Thr Leu Asp Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr

130 135 140

His Arg Ser Ser Val Pro Asn Thr Ser Thr Pro Gly Thr Ser Thr Val

145 150 155 160

Asp Leu Gly Thr Ser Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr Ala

165 170 175

Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn

180 185 190

Leu Gln Tyr Glu Glu Asp Met Arg His Pro Gly Ser Arg Lys Phe Asn

195 200 205

Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Lys Pro Leu Phe Lys Asn

210 215 220

Thr Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg

225 230 235 240

Pro Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys Thr His

245 250 255

Arg Leu Asp Pro Lys Ser Pro Gly Leu Asn Arg Glu Gln Leu Tyr Trp

260 265 270

Glu Leu Ser Lys Leu Thr Asn Gly Ile Thr Glu Leu Gly Pro Tyr Thr

275 280 285

Leu Asp Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr His Arg Thr Ser

290 295 300

Val Pro Thr Thr Ser Thr Pro Gly Thr Ser Thr Val Asp Leu Gly Thr

305 310 315 320

Ser Gly Thr Pro Phe Ser Leu Pro Ser Pro Thr Thr Ala Gly Pro Leu

325 330 335

Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr Glu

340 345 350

Glu Asp Met His Arg Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg

355 360 365

Val Leu Gln Gly Leu Leu Ser Pro Ile Phe Lys Asn Ser Ser Val Gly

370 375 380

Pro Leu Tyr Ser Gly Cys Arg Leu Thr Ser Leu Arg Pro Glu Lys Asp

385 390 395 400

Gly Ala Ala Thr Gly Met Asp Ala Val Cys Leu Tyr His Pro Asn Pro

405 410 415

Lys Arg Pro Gly Leu Asp Arg Glu Gln Leu Tyr Trp Glu Leu Ser Gln

420 425 430

Leu Thr His Asn Ile Thr Glu Leu Gly Pro Tyr Ser Leu Asp Arg Asp

435 440 445

Ser Leu Tyr Val Asn Gly Phe Thr His Gln Asn Ser Val Pro Thr Thr

450 455 460

Ser Thr Pro Gly Thr Ser Thr Val Tyr Trp Ala Thr Thr Gly Thr Pro

465 470 475 480

Ser Ser Phe Pro Gly His Thr Ala Pro Gly Pro Leu Leu Ile Pro Phe

485 490 495

Thr Leu Asn Phe Thr Ile Thr Asn Leu His Tyr Glu Glu Asn Met Gln

500 505 510

His Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly

515 520 525

Leu Leu Lys Pro Leu Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser

530 535 540

Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys His Gly Ala Ala Thr

545 550 555 560

Gly Val Asp Ala Ile Cys Thr His Arg Leu Asp Pro Lys Gly Pro Gly

565 570 575

Leu Asp Arg Glu Arg Leu Tyr Trp Glu Leu Ser Gln Leu Thr Asn Ser

580 585 590

Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser Leu Tyr Val

595 600 605

Asn Gly Phe Asn Pro Arg Ser Ser Val Pro Thr Thr Ser Thr Pro Gly

610 615 620

Thr Ser Thr Val His Leu Ala Thr Ser Gly Thr Pro Ser Ser Leu Pro

625 630 635 640

Gly His Thr Ala Ser Pro Leu Leu Val Leu Phe Thr Ile Asn Phe Thr

645 650 655

Ile Thr Asn Leu Arg Tyr Glu Glu Asn Met His His Pro Gly Ser Arg

660 665 670

Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Arg Pro Val

675 680 685

Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr

690 695 700

Leu Leu Arg Pro Lys Lys Asp Gly Ala Ala Thr Lys Val Asp Ala Ile

705 710 715 720

Cys Thr Tyr Arg Pro Asp Pro Lys Ser Pro Gly Leu Asp Arg Glu Gln

725 730 735

Leu Tyr Trp Glu Leu Ser Gln Leu Thr His Ser Ile Thr Glu Leu Gly

740 745 750

Pro Tyr Thr Leu Asp Arg Asp Ser Leu Tyr Val Asn Gly Phe Thr Gln

755 760 765

Arg Ser Ser Val Pro Thr Thr Ser Ile Pro Gly Thr Pro Thr Val Asp

770 775 780

Leu Gly Thr Ser Gly Thr Pro Val Ser Lys Pro Gly Pro Ser Ala Ala

785 790 795 800

Ser His Leu Leu Ile Leu Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu

805 810 815

Arg Tyr Glu Glu Asn Met Trp Pro Gly Ser Arg Lys Phe Asn Thr Thr

820 825 830

Glu Arg Val Leu Gln Gly Leu Leu Arg Pro Leu Phe Lys Asn Thr Ser

835 840 845

Val Gly Pro Leu Tyr Ser Gly Ser Arg Leu Thr Leu Leu Arg Pro Glu

850 855 860

Lys Asp Gly Glu Ala Thr Gly Val Asp Ala Ile Cys Thr His Arg Pro

865 870 875 880

Asp Pro Thr Gly Pro Gly Leu Asp Arg Glu Gln Leu Tyr Leu Glu Leu

885 890 895

Ser Gln Leu Thr His Ser Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp

900 905 910

Arg Asp Ser Leu Tyr Val Asn Gly Phe Thr His Arg Ser Ser Val Pro

915 920 925

Thr Thr Ser Thr Gly Val Val Ser Glu Glu Pro Phe Thr Leu Asn Phe

930 935 940

Thr Ile Asn Asn Leu Arg Tyr Met Ala Asp Met Gly Gln Pro Gly Ser

945 950 955 960

Leu Lys Phe Asn Ile Thr Asp Asn Val Met Lys His Leu Leu Ser Pro

965 970 975

Leu Phe Gln Arg Ser Ser Leu Gly Ala Arg Tyr Thr Gly Cys Arg Val

980 985 990

Ile Ala Leu Arg Ser Val Lys Asn Gly Ala Glu Thr Arg Val Asp Leu

995 1000 1005

Leu Cys Thr Tyr Leu Gln Pro Leu Ser Gly Pro Gly Leu Pro Ile

1010 1015 1020

Lys Gln Val Phe His Glu Leu Ser Gln Gln Thr His Gly Ile Thr

1025 1030 1035

Arg Leu Gly Pro Tyr Ser Leu Asp Lys Asp Ser Leu Tyr Leu Asn

1040 1045 1050

Gly Tyr Asn Glu Pro Gly Leu Asp Glu Pro Pro Thr Thr Pro Lys

1055 1060 1065

Pro Ala Thr Thr Phe Leu Pro Pro Leu Ser Glu Ala Thr Thr Ala

1070 1075 1080

Met Gly Tyr His Leu Lys Thr Leu Thr Leu Asn Phe Thr Ile Ser

1085 1090 1095

Asn Leu Gln Tyr Ser Pro Asp Met Gly Lys Gly Ser Ala Thr Phe

1100 1105 1110

Asn Ser Thr Glu Gly Val Leu Gln His Leu Leu Arg Pro Leu Phe

1115 1120 1125

Gln Lys Ser Ser Met Gly Pro Phe Tyr Leu Gly Cys Gln Leu Ile

1130 1135 1140

Ser Leu Arg Pro Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Thr

1145 1150 1155

Thr Cys Thr Tyr His Pro Asp Pro Val Gly Pro Gly Leu Asp Ile

1160 1165 1170

Gln Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr His Gly Val Thr

1175 1180 1185

Gln Leu Gly Phe Tyr Val Leu Asp Arg Asp Ser Leu Phe Ile Asn

1190 1195 1200

Gly Tyr Ala Pro Gln Asn Leu Ser Ile Arg Gly Glu Tyr Gln Ile

1205 1210 1215

Asn Phe His Ile Val Asn Trp Asn Leu Ser Asn Pro Asp Pro Thr

1220 1225 1230

Ser Ser Glu Tyr Ile Ala Leu Leu Arg Asp Ile Gln Asp Lys Val

1235 1240 1245

Thr Thr Leu Tyr Lys Gly Ser Gln Leu His Asp Thr Phe Arg Phe

1250 1255 1260

Cys Leu Val Thr Asn Leu Thr Met Asp Ser Met Leu Val Thr Val

1265 1270 1275

Lys Ala Leu Phe Ser Ser Asn Leu Asp Pro Ser Leu Val Glu Gln

1280 1285 1290

Val Phe Leu Asp Lys Thr Leu Asn Ala Ser Ser His Trp Leu Gly

1295 1300 1305

Ser Thr Tyr Gln Leu Val Asp Ile His Val Thr Glu Met Glu Pro

1310 1315 1320

Ser Val Tyr Gln Pro Thr Ser Ser Ser Ser Thr Gln His Phe Tyr

1325 1330 1335

Leu Asn Phe Thr Ile Thr Asn Leu Pro Tyr Ser Gln Asp Ile Ala

1340 1345 1350

Gln Pro Gly Thr Thr Asn Tyr Gln Arg Asn Lys Arg Asn Ile Glu

1355 1360 1365

Asp Ala Leu Asn Gln Leu Phe Arg Asn Ser Ser Ile Lys Ser Tyr

1370 1375 1380

Phe Ser Asp Cys Gln Val Ser Thr Phe Arg Ser Val Pro Asn Ser

1385 1390 1395

His His Thr Gly Val Asp Ser Leu Cys Ala Phe Ser Pro Leu Ala

1400 1405 1410

Arg Arg Val Asp Arg Val Ala Ile Tyr Glu Glu Phe Leu Arg Met

1415 1420 1425

Thr Arg Ala Gly Thr Gln Leu Gln Ala Phe Thr Leu Asp Arg Ser

1430 1435 1440

Ser Val Leu Val Asp Gly Tyr Ser Pro Asn Arg Asn Glu Pro Leu

1445 1450 1455

Thr Gly Asn Ser Asp Leu Pro Phe Trp Ala Ile Ile Leu Ile Cys

1460 1465 1470

Leu Ala Gly Leu Leu Gly Leu Ile Thr Cys Leu Ile Cys Gly Phe

1475 1480 1485

Leu Val

1490

<210> 3

<211> 1932

<212> DNA

<213> artificial sequence

<220>

<223> MUC16 microconsensus amino acid sequence repeat,

encoding protein pGX1436

<400> 3

atggattgga cttggattct gttcctggtc gccgccgcaa ctcgggtgca ttctactgct 60

gctgggccac tgctggtgcc ttttacactg aacttcacca tcacaaatct gcagtacgag 120

gaggacatgc accaccctgg ctctcggaag ttcaacacca cagagagagt gctgcagggc 180

ctgctgggcc caatgtttaa gaataccagc gtgggcctgc tgtattccgg atgccggctg 240

acactgctga gatccgagaa ggacggagca gcaaccggag tggatgccat ctgtacacac 300

aggctggacc caaagtcccc aggcctggat agagagcagc tgtactggga gctgtctcag 360

ctgaccaacg gcatcaagga gctgggcccc tacacactgg accggaacag cctgtatgtg 420

aatggcttta cccacagaag ctccgtgcca aataccagca cacccggcac ctccacagtg 480

gatctgggca ccctctggcac accttctagc ctgccaagcc ctaccgcagc aggaccactg 540

ctggtgccct tcacactgaa ctttaccatt accaatctgc agtatgaaga ggacatgagg 600

cacccaggct cccgcaagtt caacactacc gagcgggtgc tgcaaggcct gctgaagcct 660

ctgtttaaga atacctctgt gggcccactg tatagtggct gccggctgac actgctgcgg 720

cccgaaaaag acggagcagc aaccggcgtg gatgctattt gcacccacag gctggacccc 780

aagagcccag gcctgaaccg cgaacagctg tattgggagc tgtccaagct gaccaatggc 840

atcacagagc tgggccccta caccctggac agaaattccc tgtacgtgaa tggcttcacc 900

caccgcacat ctgtgcctac cacaagcacc ccaggcacct ccacagtgga tctgggcacc 960

tccggcacac ccttttccct gccatctcca accacagcag gacctctgct ggtgccattc 1020

accctgaact ttaccattac taatctgcag tatgaagaag acatgcacag gcctggctct 1080

cgcaagttca acactactga gagggtgctg cagggcctgt taagcccaat ctttaagaat 1140

tcctctgtgg gccctctgta ttccggatgc aggctgacct ctctgcgccc agaaaaagat 1200

ggagcagcaa caggaatgga tgccgtgtgc ctgtaccacc ctaacccaaa gcggcccggc 1260

ctggacaggg agcagctgta ttgggaactg agccagctga cccacaatat cacagagctg 1320

ggcccttact ctctggaccg cgatagcctg tatgtgaacg gcttcaccca ccagaattcc 1380

gtgcccacca catctacacc tggcaccagc acagtgtact gggccaccac aggcaccccc 1440

agctcctttc ctggacacac agcaccagga cctctgctga tccccttcac cctgaacttt 1500

accataacaa atctgcacta tgaggagaac atgcagcacc ctggcagcag gaagttcaat 1560

accacagagc gcgtgctgca aggcctgctg aagccgctgt ttaagaacac cagcgtggga 1620

ccactgtaca gcggctgcag gctgaccctg ctgcgccctg agaagcatgg cgccgccacc 1680

ggcgtggatg ctatctgcac acatagactg gaccccaagg gacctggcct ggatagggag 1740

agactgtact gggaactgtc ccagctgacc aactcaatta cagagctggg cccatacacc 1800

ctggaccggg attctctgta cgtgaacggc ttcaatccaa gatctagcgt ccctaccaca 1860

tctacccctg ggacaagtac cgtgcatctg gctacaagcg gaactccttc aagtctgcct 1920

ggacactgat a.a. 1932

<210> 4

<211> 642

<212> Protein

<213> artificial sequence

<220>

<223> MUC16 amino acid sequence consensus repeat,

encoding protein pGX1436

<400> 4

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

5 10 15

His Ser Thr Ala Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe

20 25 30

Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asp Met His His Pro Gly Ser

35 40 45

Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Gly Pro

50 55 60

Met Phe Lys Asn Thr Ser Val Gly Leu Leu Tyr Ser Gly Cys Arg Leu

65 70 75 80

Thr Leu Leu Arg Ser Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala

85 90 95

Ile Cys Thr His Arg Leu Asp Pro Lys Ser Pro Gly Leu Asp Arg Glu

100 105 110

Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr Asn Gly Ile Lys Glu Leu

115 120 125

Gly Pro Tyr Thr Leu Asp Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr

130 135 140

His Arg Ser Ser Val Pro Asn Thr Ser Thr Pro Gly Thr Ser Thr Val

145 150 155 160

Asp Leu Gly Thr Ser Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr Ala

165 170 175

Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn

180 185 190

Leu Gln Tyr Glu Glu Asp Met Arg His Pro Gly Ser Arg Lys Phe Asn

195 200 205

Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Lys Pro Leu Phe Lys Asn

210 215 220

Thr Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg

225 230 235 240

Pro Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys Thr His

245 250 255

Arg Leu Asp Pro Lys Ser Pro Gly Leu Asn Arg Glu Gln Leu Tyr Trp

260 265 270

Glu Leu Ser Lys Leu Thr Asn Gly Ile Thr Glu Leu Gly Pro Tyr Thr

275 280 285

Leu Asp Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr His Arg Thr Ser

290 295 300

Val Pro Thr Thr Ser Thr Pro Gly Thr Ser Thr Val Asp Leu Gly Thr

305 310 315 320

Ser Gly Thr Pro Phe Ser Leu Pro Ser Pro Thr Thr Ala Gly Pro Leu

325 330 335

Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr Glu

340 345 350

Glu Asp Met His Arg Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg

355 360 365

Val Leu Gln Gly Leu Leu Ser Pro Ile Phe Lys Asn Ser Ser Val Gly

370 375 380

Pro Leu Tyr Ser Gly Cys Arg Leu Thr Ser Leu Arg Pro Glu Lys Asp

385 390 395 400

Gly Ala Ala Thr Gly Met Asp Ala Val Cys Leu Tyr His Pro Asn Pro

405 410 415

Lys Arg Pro Gly Leu Asp Arg Glu Gln Leu Tyr Trp Glu Leu Ser Gln

420 425 430

Leu Thr His Asn Ile Thr Glu Leu Gly Pro Tyr Ser Leu Asp Arg Asp

435 440 445

Ser Leu Tyr Val Asn Gly Phe Thr His Gln Asn Ser Val Pro Thr Thr

450 455 460

Ser Thr Pro Gly Thr Ser Thr Val Tyr Trp Ala Thr Thr Gly Thr Pro

465 470 475 480

Ser Ser Phe Pro Gly His Thr Ala Pro Gly Pro Leu Leu Ile Pro Phe

485 490 495

Thr Leu Asn Phe Thr Ile Thr Asn Leu His Tyr Glu Glu Asn Met Gln

500 505 510

His Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly

515 520 525

Leu Leu Lys Pro Leu Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser

530 535 540

Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys His Gly Ala Ala Thr

545 550 555 560

Gly Val Asp Ala Ile Cys Thr His Arg Leu Asp Pro Lys Gly Pro Gly

565 570 575

Leu Asp Arg Glu Arg Leu Tyr Trp Glu Leu Ser Gln Leu Thr Asn Ser

580 585 590

Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser Leu Tyr Val

595 600 605

Asn Gly Phe Asn Pro Arg Ser Ser Val Pro Thr Thr Ser Thr Pro Gly

610 615 620

Thr Ser Thr Val His Leu Ala Thr Ser Gly Thr Pro Ser Ser Leu Pro

625 630 635 640

Gly His

<210> 5

<211> 2136

<212> DNA

<213> artificial sequence

<220>

<223> MUC16 repeats 61, 62, 63 + synthetic consensus carboxy-terminal

domain of the amino acid sequence encoding the pGX 1437 protein

<400> 5

atggactgga cttggattct gttcctggtc gctgccgcca cccgcgtgca tagtgccgca 60

tctcacctgc tgattctgtt caccctgaac ttcaccatca caaatctgcg gtacgaggag 120

aacatgtggc caggcagccg caagttcaat accacagagc gggtgctgca gggcctgctg 180

cggcccctgt ttaagaacac ctccgtgggc cccctgtatt ctggcagcag gctgaccctg 240

ctgcgcccag agaaggacgg agaggcaaca ggcgtggatg ccatctgcac ccacaggcct 300

gacccaacag gaccaggcct ggatagggag cagctgtacc tggagctgag ccagctgacc 360

cactccatca cagagctggg accatacacc ctggacaggg attccctgta tgtgaacggc 420

tttacccaca gaagctccgt gcccaccaca tctacaggcg tggtgagcga ggagcccttc 480

accctgaact tcaccatcaa caatctgagg tacatggcag acatgggaca gccaggcagc 540

ctgaagttca acatcaccga taatgtgatg aagcacctgc tgtcccctct gtttcagcgg 600

tctagcctgg gagcaaggta caccggctgc agagtgatcg ccctgaggtc cgtgaagaac 660

ggagcagaga cacgggtgga cctgctgtgc acatatctgc agcctctgag cggaccaggc 720

ctgcctatca agcaggtgtt ccacgagctg tctcagcaga cccacggaat cacacgcctg 780

ggaccttact ccctggacaa ggattctctg tacctgaacg gctataatga gccaggcctg 840

gacgagcccc ctaccacacc caagcctgcc accacatttc tgccacccct gagcgaggca 900

accacagcaa tgggatacca cctgaagacc ctgacactga acttcaccat cagcaatctg 960

cagtattccc ccgatatggg caagggctct gccaccttta atagcacaga gggcgtgctg 1020

cagcacctgc tgaggcctct gttccagaag tcctctatgg gccccttcta cctgggatgc 1080

1140

acataccacc cagaccccgt gggaccaggc ctggatatcc agcagctgta ttgggaactg 1200

tcccagctga cccacggcgt gacacagctg ggcttctatg tgctggaccg ggattctctg 1260

tttatcaacg gctacgcccc tcagaatctg agcatcagag gcgagtatca gatcaacttc 1320

cacatcgtga actggaatct gtctaatcct gatccaacca gctccgagta catcgccctg 1380

ctgcgggaca tccaggataa ggtgaccaca ctgtataagg gcagccagct gcacgacacc 1440

ttcagatttt gtctggtgac caacctgaca atggattcca tgctggtgac agtgaaggcc 1500

ctgttctcta gcaacctgga cccttctctg gtggagcagg tgtttctgga taagaccctg 1560

aatgcctcct ctcactggct gggctctacc taccagctgg tggacatcca cgtgacagag 1620

atggagccaa gcgtgtatca gcccaccagc tcctctagca cacagcactt ctacctgaac 1680

tttaccatca caaatctgcc ctatagccag gatatcgccc agcctggcac cacaaactac 1740

cagaggaaca agcgcaatat cgaggacgcc ctgaaccagc tgttcaggaa ttcctctatc 1800

aagtcctatt tctctgattg ccaggtgagc acctttcgct ccgtgccaaa ttctcaccac 1860

acaggcgtgg actccctgtg cgccttttct cccctggcac ggagagtgga tagggtggca 1920

atctacgagg agttcctgcg gatgaccaga gccggcacac agctgcaggc ctttaccctg 1980

gacaggagct ccgtgctggt ggatggctat agccctaacc gcaatgagcc actgacaggc 2040

aattccgacc tgcccttctg ggccatcatc ctgatttgcc tggctggact gctggggctg 2100

attacctgtc tgatttgtgg gttcctggtg tgataa 2136

<210> 6

<211> 710

<212> Protein

<213> artificial sequence

<220>

<223> MUC16 repeats 61, 62, 63 + synthetic consensus truncated carboxy-terminal

domain of the amino acid sequence encoding the pGX 1437 protein

<400> 6

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

5 10 15

His Ser Ala Ala Ser His Leu Leu Ile Leu Phe Thr Leu Asn Phe Thr

20 25 30

Ile Thr Asn Leu Arg Tyr Glu Glu Asn Met Trp Pro Gly Ser Arg Lys

35 40 45

Phe Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Arg Pro Leu Phe

50 55 60

Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser Gly Ser Arg Leu Thr Leu

65 70 75 80

Leu Arg Pro Glu Lys Asp Gly Glu Ala Thr Gly Val Asp Ala Ile Cys

85 90 95

Thr His Arg Pro Asp Pro Thr Gly Pro Gly Leu Asp Arg Glu Gln Leu

100 105 110

Tyr Leu Glu Leu Ser Gln Leu Thr His Ser Ile Thr Glu Leu Gly Pro

115 120 125

Tyr Thr Leu Asp Arg Asp Ser Leu Tyr Val Asn Gly Phe Thr His Arg

130 135 140

Ser Ser Val Pro Thr Thr Ser Thr Gly Val Val Ser Glu Glu Pro Phe

145 150 155 160

Thr Leu Asn Phe Thr Ile Asn Asn Leu Arg Tyr Met Ala Asp Met Gly

165 170 175

Gln Pro Gly Ser Leu Lys Phe Asn Ile Thr Asp Asn Val Met Lys His

180 185 190

Leu Leu Ser Pro Leu Phe Gln Arg Ser Ser Leu Gly Ala Arg Tyr Thr

195 200 205

Gly Cys Arg Val Ile Ala Leu Arg Ser Val Lys Asn Gly Ala Glu Thr

210 215 220

Arg Val Asp Leu Leu Cys Thr Tyr Leu Gln Pro Leu Ser Gly Pro Gly

225 230 235 240

Leu Pro Ile Lys Gln Val Phe His Glu Leu Ser Gln Gln Thr His Gly

245 250 255

Ile Thr Arg Leu Gly Pro Tyr Ser Leu Asp Lys Asp Ser Leu Tyr Leu

260 265 270

Asn Gly Tyr Asn Glu Pro Gly Leu Asp Glu Pro Pro Thr Thr Pro Lys

275 280 285

Pro Ala Thr Thr Phe Leu Pro Pro Leu Ser Glu Ala Thr Thr Ala Met

290 295 300

Gly Tyr His Leu Lys Thr Leu Thr Leu Asn Phe Thr Ile Ser Asn Leu

305 310 315 320

Gln Tyr Ser Pro Asp Met Gly Lys Gly Ser Ala Thr Phe Asn Ser Thr

325 330 335

Glu Gly Val Leu Gln His Leu Leu Arg Pro Leu Phe Gln Lys Ser Ser

340 345 350

Met Gly Pro Phe Tyr Leu Gly Cys Gln Leu Ile Ser Leu Arg Pro Glu

355 360 365

Lys Asp Gly Ala Ala Thr Gly Val Asp Thr Thr Cys Thr Tyr His Pro

370 375 380

Asp Pro Val Gly Pro Gly Leu Asp Ile Gln Gln Leu Tyr Trp Glu Leu

385 390 395 400

Ser Gln Leu Thr His Gly Val Thr Gln Leu Gly Phe Tyr Val Leu Asp

405 410 415

Arg Asp Ser Leu Phe Ile Asn Gly Tyr Ala Pro Gln Asn Leu Ser Ile

420 425 430

Arg Gly Glu Tyr Gln Ile Asn Phe His Ile Val Asn Trp Asn Leu Ser

435 440 445

Asn Pro Asp Pro Thr Ser Glu Tyr Ile Ala Leu Leu Arg Asp Ile

450 455 460

Gln Asp Lys Val Thr Thr Leu Tyr Lys Gly Ser Gln Leu His Asp Thr

465 470 475 480

Phe Arg Phe Cys Leu Val Thr Asn Leu Thr Met Asp Ser Met Leu Val

485 490 495

Thr Val Lys Ala Leu Phe Ser Ser Asn Leu Asp Pro Ser Leu Val Glu

500 505 510

Gln Val Phe Leu Asp Lys Thr Leu Asn Ala Ser Ser His Trp Leu Gly

515 520 525

Ser Thr Tyr Gln Leu Val Asp Ile His Val Thr Glu Met Glu Pro Ser

530 535 540

Val Tyr Gln Pro Thr Ser Ser Ser Ser Thr Gln His Phe Tyr Leu Asn

545 550 555 560

Phe Thr Ile Thr Asn Leu Pro Tyr Ser Gln Asp Ile Ala Gln Pro Gly

565 570 575

Thr Thr Asn Tyr Gln Arg Asn Lys Arg Asn Ile Glu Asp Ala Leu Asn

580 585 590

Gln Leu Phe Arg Asn Ser Ser Ile Lys Ser Tyr Phe Ser Asp Cys Gln

595 600 605

Val Ser Thr Phe Arg Ser Val Pro Asn Ser His His Thr Gly Val Asp

610 615 620

Ser Leu Cys Ala Phe Ser Pro Leu Ala Arg Arg Val Asp Arg Val Ala

625 630 635 640

Ile Tyr Glu Glu Phe Leu Arg Met Thr Arg Ala Gly Thr Gln Leu Gln

645 650 655

Ala Phe Thr Leu Asp Arg Ser Ser Val Leu Val Asp Gly Tyr Ser Pro

660 665 670

Asn Arg Asn Glu Pro Leu Thr Gly Asn Ser Asp Leu Pro Phe Trp Ala

675 680 685

Ile Ile Leu Ile Cys Leu Ala Gly Leu Leu Gly Leu Ile Thr Cys Leu

690 695 700

Ile Cys Gly Phe Leu Val

705 710

<210> 7

<211> 4029

<212> DNA

<213> artificial sequence

<220>

<223> MUC16 microsensing repeats with cleavage site before the repeats

61, 62, 63 + synthetic consensus carboxy-terminal

domain of the amino acid sequence encoding the pGX1438 protein

<400> 7

atggactgga cctggattct gttcctggtg gcagcagcaa ccagggtgca ctccacagca 60

gcaggacctc tgctggtgcc attcaccctg aactttacca tcacaaatct gcagtacgag 120

gaggatatgc accaccccgg cagccgcaag ttcaacacca cagagcgggt gctgcagggc 180

ctgctgggac ctatgtttaa gaataccagc gtgggcctgc tgtattccgg atgcaggctg 240

acactgctgc gctccgagaa ggacggagca gcaaccggcg tggatgccat ctgtacacac 300

aggctggacc caaagtctcc cggcctggat cgcgagcagc tgtactggga gctgagccag 360

ctgaccaacg gcatcaagga gctgggcccc tacacactgg accggaacag cctgtatgtg 420

aatggcttca cccacagaag ctccgtgcca aatacctcca cacccggcac ctctacagtg 480

gatctgggca ccctctggcac accctctagc ctgcctagcc caaccgcagc aggaccactg 540

ctggtgcctt tcacactgaa ctttaccatt accaatctgc agtatgaaga ggacatgcgg 600

caccctggca gcagaaagtt caacactacc gagcgcgtgc tgcaaggcct gctgaagcca 660

ctgtttaaga atacctctgt gggccccctg tatagtggct gtagactgac actgctgcgc 720

cctgaaaaag atggcgccgc cactggagtc gacgctattt gcacccacag gctggacccc 780

aagtccccag gcctgaacag agaacagctg tattgggagc tgtctaagct gaccaatggc 840

atcacagagc tgggcccata caccctggac aggaactctc tgtacgtcaa tggcttcacc 900

caccgcacaa gcgtgcctac cacatccacc ccaggcacct ctaccgtcga tctgggcacc 960

agcggcacac cattttccct gccatctcct acccgccg gaccactgct ggtgcccttc 1020

accctgaact ttaccataac caatctgcag tatgaggagg acatgcaccg gcccggctct 1080

agaaagttca acactactga acgggtgctg caaggcctgt taagccctat ctttaagaat 1140

tcctctgtgg gcccactgta cagcggatgc aggctgacct ctctgcggcc cgaaaaagac 1200

ggagcagcaa caggaatgga tgccgtgtgc ctgtaccacc caaaccccaa gaggcctggc 1260

ctggacagag agcagctgta ttgggaactg tcccagctga cccacaatat cacagagctg 1320

ggcccctaca gcctggacag agattccctg tatgtgaacg gcttcaccca ccagaattct 1380

gtgcccacca caagcaccc tggcacctcc acagtgtact gggccaccac aggcacccct 1440

agctcctttc caggacacac agcacctgga ccactgctga tccctttcac cctgaacttt 1500

accattacaa atctgcacta tgaggagaac atgcagcacc caggcagcag aaagttcaat 1560

accacagaga gggtgctgca aggcctgctg aagccgctgt ttaagaacac cagcgtggga 1620

cctctgtact ctggctgtcg cctgacactg ctgcggcccg agaagcatgg cgcagcaacc 1680

ggcgtggacg ctatttgcac tcatagactg gaccccaagg gacctggcct ggatagggag 1740

agactgtact gggaactgtc tcagctgacc aattccatta cagagctggg cccttacacc 1800

ctggaccggg acagcctgta cgtcaacggc ttcaatccaa gatctagcgt gcccaccaca 1860

tccacccctg gcacctctac agtgcacctg gccaccagcg gaacaccctc ctctctgcct 1920

ggacacaggg gaaggaagcg gagaagcgcc gcatcccacc tgctgatcct gttcaccctg 1980

aactttacca taacgaatct gagatacgag gagaacatgt ggcctggctc ccgcaagttc 2040

aatactaccg aacgggtgct gcagggcctg ctgcggcccc tgtttaagaa cacttccgtg 2100

ggccccctgt attctggcag caggctgacc ctgctgcgcc cagagaagga cggagaggca 2160

acaggcgtgg atgccatctg cacccacagg cccgacccta caggaccagg cctggatagg 2220

gagcagctgt acctggagct gtcccagctg actcactcaa ttaccgaact gggaccttac 2280

accctggaca gggatagtct gtacgtgaat ggcttcaccc atcgcagctc cgtgccaacc 2340

acatctacag gcgtggtgag cgaggaaccc ttcaccctga acttcaccat caacaatctg 2400

aggtacatgg ccgacatggg ccagccaggc tccctgaagt tcaacatcac cgataatgtg 2460

atgaagcacc tgctgtctcc cctgtttcag aggtctagcc tgggagcaag gtacaccgga 2520

tgcagagtga tcgccctgag gtccgtgaag aacggagcag agacacgggt ggacctgctg 2580

tgcacatatc tgcagcctct gagcggacca ggcctgccca tcaagcaggt gttccacgag 2640

ctgtcccagc agacccgg aatcacaagg ctgggacctt actccctgga caaggattct 2700

ctgtacctga acggctataa tgagccaggc ctggacgagc cccctaccac acccaaagccc 2760

gccaccacat ttctgccacc cctgagcgag gcaaccacag caatgggata ccacctgaag 2820

accctgacac tgaacttcac catcagcaat ctgcagtatt cccccgatat gggcaagggc 2880

tctgccacct ttaacagcac agagggcgtg ctgcagcacc tgctgcggcc tctgttccag 2940

aagtcctcta tgggcccctt ctacctggga tgccagctga tctccctgcg gcccgaaaag 3000

gatggagcag caaccggagt ggataccaca tgtacatacc accctgaccc agtgggacca 3060

ggcctggata tccagcaatt atattgggaa ctgagtcagc tgacccacgg cgtgacacag 3120

ctgggcttct atgtgctgga cagggatagc ctgtttatca acggctacgc cccacagaat 3180

ctgtccatcc gcggcgagta tcagatcaac ttccacatcg tgaactggaa tctgagcaat 3240

ccgacccta ccagctccga gtacatcgcc ctgctgagg acatccagga taaggtgacc 3300

acactgtata agggctccca gctgcacgac accttccgct tttgcctggt gaccaacctg 3360

acaatggatt ctatgctggt gacagtgaag gccctgttct ctagcaacct ggaccccagc 3420

ctggtggagc aggtgtttct ggataagacc ctgaatgcct cctctcactg gctgggcagc 3480

acctaccagc tggtggacat ccacgtgaca gagatggagc catccgtgta tcagcccacc 3540

agctcctcta gcacacagca cttctacctg aactttacca taactaatct gccctatagc 3600

3660

gccctgaacc agctgttccg gaattcctct atcaagtctt atttcagcga ttgccaggtg 3720

tccaccttta gatctgtgcc aaatagccac cacacaggcg tggactccct gtgcgccttt 3780

tctcccctgg caaggagggt ggatagggtg gcaatctacg aggagttcct gaggatgacc 3840

cgcgccggaa cacagctgca ggcctttacc ctggaccggga gctccgtgct ggtggatggc 3900

tattccccta acagaaatga gccactgaca ggcaactctg acctgccctt ctgggccatc 3960

atcctgatct gtctggcagg ctgctgggc ctgatcacct gcctgatctg tggctttctg 4020

gtgtgataa 4029

<210> 8

<211> 1341

<212> Protein

<213> artificial sequence

<220>

<223> MUC16 microsensing repeats with cleavage site before the repeats

61, 62, 63 + synthetic consensus carboxy-terminal

domain of the amino acid sequence encoding the pGX1438 protein

<400> 8

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

5 10 15

His Ser Thr Ala Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe

20 25 30

Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asp Met His His Pro Gly Ser

35 40 45

Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Gly Pro

50 55 60

Met Phe Lys Asn Thr Ser Val Gly Leu Leu Tyr Ser Gly Cys Arg Leu

65 70 75 80

Thr Leu Leu Arg Ser Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala

85 90 95

Ile Cys Thr His Arg Leu Asp Pro Lys Ser Pro Gly Leu Asp Arg Glu

100 105 110

Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr Asn Gly Ile Lys Glu Leu

115 120 125

Gly Pro Tyr Thr Leu Asp Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr

130 135 140

His Arg Ser Ser Val Pro Asn Thr Ser Thr Pro Gly Thr Ser Thr Val

145 150 155 160

Asp Leu Gly Thr Ser Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr Ala

165 170 175

Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn

180 185 190

Leu Gln Tyr Glu Glu Asp Met Arg His Pro Gly Ser Arg Lys Phe Asn

195 200 205

Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Lys Pro Leu Phe Lys Asn

210 215 220

Thr Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg

225 230 235 240

Pro Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys Thr His

245 250 255

Arg Leu Asp Pro Lys Ser Pro Gly Leu Asn Arg Glu Gln Leu Tyr Trp

260 265 270

Glu Leu Ser Lys Leu Thr Asn Gly Ile Thr Glu Leu Gly Pro Tyr Thr

275 280 285

Leu Asp Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr His Arg Thr Ser

290 295 300

Val Pro Thr Thr Ser Thr Pro Gly Thr Ser Thr Val Asp Leu Gly Thr

305 310 315 320

Ser Gly Thr Pro Phe Ser Leu Pro Ser Pro Thr Thr Ala Gly Pro Leu

325 330 335

Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr Glu

340 345 350

Glu Asp Met His Arg Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg

355 360 365

Val Leu Gln Gly Leu Leu Ser Pro Ile Phe Lys Asn Ser Ser Val Gly

370 375 380

Pro Leu Tyr Ser Gly Cys Arg Leu Thr Ser Leu Arg Pro Glu Lys Asp

385 390 395 400

Gly Ala Ala Thr Gly Met Asp Ala Val Cys Leu Tyr His Pro Asn Pro

405 410 415

Lys Arg Pro Gly Leu Asp Arg Glu Gln Leu Tyr Trp Glu Leu Ser Gln

420 425 430

Leu Thr His Asn Ile Thr Glu Leu Gly Pro Tyr Ser Leu Asp Arg Asp

435 440 445

Ser Leu Tyr Val Asn Gly Phe Thr His Gln Asn Ser Val Pro Thr Thr

450 455 460

Ser Thr Pro Gly Thr Ser Thr Val Tyr Trp Ala Thr Thr Gly Thr Pro

465 470 475 480

Ser Ser Phe Pro Gly His Thr Ala Pro Gly Pro Leu Leu Ile Pro Phe

485 490 495

Thr Leu Asn Phe Thr Ile Thr Asn Leu His Tyr Glu Glu Asn Met Gln

500 505 510

His Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly

515 520 525

Leu Leu Lys Pro Leu Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser

530 535 540

Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys His Gly Ala Ala Thr

545 550 555 560

Gly Val Asp Ala Ile Cys Thr His Arg Leu Asp Pro Lys Gly Pro Gly

565 570 575

Leu Asp Arg Glu Arg Leu Tyr Trp Glu Leu Ser Gln Leu Thr Asn Ser

580 585 590

Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser Leu Tyr Val

595 600 605

Asn Gly Phe Asn Pro Arg Ser Ser Val Pro Thr Thr Ser Thr Pro Gly

610 615 620

Thr Ser Thr Val His Leu Ala Thr Ser Gly Thr Pro Ser Ser Leu Pro

625 630 635 640

Gly His Arg Gly Arg Lys Arg Arg Ser Ala Ala Ser His Leu Leu Ile

645 650 655

Leu Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Arg Tyr Glu Glu Asn

660 665 670

Met Trp Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln

675 680 685

Gly Leu Leu Arg Pro Leu Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr

690 695 700

Ser Gly Ser Arg Leu Thr Leu Leu Arg Pro Glu Lys Asp Gly Glu Ala

705 710 715 720

Thr Gly Val Asp Ala Ile Cys Thr His Arg Pro Asp Pro Thr Gly Pro

725 730 735

Gly Leu Asp Arg Glu Gln Leu Tyr Leu Glu Leu Ser Gln Leu Thr His

740 745 750

Ser Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser Leu Tyr

755 760 765

Val Asn Gly Phe Thr His Arg Ser Ser Val Pro Thr Thr Ser Thr Gly

770 775 780

Val Val Ser Glu Glu Pro Phe Thr Leu Asn Phe Thr Ile Asn Asn Leu

785 790 795 800

Arg Tyr Met Ala Asp Met Gly Gln Pro Gly Ser Leu Lys Phe Asn Ile

805 810 815

Thr Asp Asn Val Met Lys His Leu Leu Ser Pro Leu Phe Gln Arg Ser

820 825 830

Ser Leu Gly Ala Arg Tyr Thr Gly Cys Arg Val Ile Ala Leu Arg Ser

835 840 845

Val Lys Asn Gly Ala Glu Thr Arg Val Asp Leu Leu Cys Thr Tyr Leu

850 855 860

Gln Pro Leu Ser Gly Pro Gly Leu Pro Ile Lys Gln Val Phe His Glu

865 870 875 880

Leu Ser Gln Gln Thr His Gly Ile Thr Arg Leu Gly Pro Tyr Ser Leu

885 890 895

Asp Lys Asp Ser Leu Tyr Leu Asn Gly Tyr Asn Glu Pro Gly Leu Asp

900 905 910

Glu Pro Pro Thr Thr Pro Lys Pro Ala Thr Thr Phe Leu Pro Pro Leu

915 920 925

Ser Glu Ala Thr Thr Ala Met Gly Tyr His Leu Lys Thr Leu Thr Leu

930 935 940

Asn Phe Thr Ile Ser Asn Leu Gln Tyr Ser Pro Asp Met Gly Lys Gly

945 950 955 960

Ser Ala Thr Phe Asn Ser Thr Glu Gly Val Leu Gln His Leu Leu Arg

965 970 975

Pro Leu Phe Gln Lys Ser Ser Met Gly Pro Phe Tyr Leu Gly Cys Gln

980 985 990

Leu Ile Ser Leu Arg Pro Glu Lys Asp Gly Ala Ala Thr Gly Val Asp

995 1000 1005

Thr Thr Cys Thr Tyr His Pro Asp Pro Val Gly Pro Gly Leu Asp

1010 1015 1020

Ile Gln Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr His Gly Val

1025 1030 1035

Thr Gln Leu Gly Phe Tyr Val Leu Asp Arg Asp Ser Leu Phe Ile

1040 1045 1050

Asn Gly Tyr Ala Pro Gln Asn Leu Ser Ile Arg Gly Glu Tyr Gln

1055 1060 1065

Ile Asn Phe His Ile Val Asn Trp Asn Leu Ser Asn Pro Asp Pro

1070 1075 1080

Thr Ser Ser Glu Tyr Ile Ala Leu Leu Arg Asp Ile Gln Asp Lys

1085 1090 1095

Val Thr Thr Leu Tyr Lys Gly Ser Gln Leu His Asp Thr Phe Arg

1100 1105 1110

Phe Cys Leu Val Thr Asn Leu Thr Met Asp Ser Met Leu Val Thr

1115 1120 1125

Val Lys Ala Leu Phe Ser Ser Asn Leu Asp Pro Ser Leu Val Glu

1130 1135 1140

Gln Val Phe Leu Asp Lys Thr Leu Asn Ala Ser Ser His Trp Leu

1145 1150 1155

Gly Ser Thr Tyr Gln Leu Val Asp Ile His Val Thr Glu Met Glu

1160 1165 1170

Pro Ser Val Tyr Gln Pro Thr Ser Ser Ser Ser Thr Gln His Phe

1175 1180 1185

Tyr Leu Asn Phe Thr Ile Thr Asn Leu Pro Tyr Ser Gln Asp Ile

1190 1195 1200

Ala Gln Pro Gly Thr Thr Asn Tyr Gln Arg Asn Lys Arg Asn Ile

1205 1210 1215

Glu Asp Ala Leu Asn Gln Leu Phe Arg Asn Ser Ser Ile Lys Ser

1220 1225 1230

Tyr Phe Ser Asp Cys Gln Val Ser Thr Phe Arg Ser Val Pro Asn

1235 1240 1245

Ser His His Thr Gly Val Asp Ser Leu Cys Ala Phe Ser Pro Leu

1250 1255 1260

Ala Arg Arg Val Asp Arg Val Ala Ile Tyr Glu Glu Phe Leu Arg

1265 1270 1275

Met Thr Arg Ala Gly Thr Gln Leu Gln Ala Phe Thr Leu Asp Arg

1280 1285 1290

Ser Ser Val Leu Val Asp Gly Tyr Ser Pro Asn Arg Asn Glu Pro

1295 1300 1305

Leu Thr Gly Asn Ser Asp Leu Pro Phe Trp Ala Ile Ile Leu Ile

1310 1315 1320

Cys Leu Ala Gly Leu Leu Gly Leu Ile Thr Cys Leu Ile Cys Gly

1325 1330 1335

Phe Leu Val

1340

<---

Claims (67)

1. A combination for the treatment or prevention of MUC16-expressing cancer, comprising a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, wherein said nucleic acid molecule contains: (a) a nucleic acid sequence encoding amino acids 19-1490 of SEQ ID NO: 2; or (b) a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to amino acids 19-1490 of SEQ ID NO: 2, wherein said amino acid sequence contains alanine at amino acid position 1408, alanine at amino acid position 1431, and alanine at amino acid position 1437 with respect to SEQ ID NO: 2. 2. A combination for the treatment or prevention of MUC16-expressing cancer, comprising a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, wherein said nucleic acid molecule contains: (a) nucleotides 55-4470 of SEQ ID NO: 1; or (b) a nucleic acid sequence that is at least 95% identical to nucleotides 55-4470 of SEQ ID NO: 1, wherein said nucleic acid sequence encodes alanine at amino acid position 1408, alanine at amino acid position 1431, and alanine at amino acid position 1437 relative to SEQ ID NO: 2. 3. A combination for the treatment or prevention of MUC16-expressing cancer, comprising a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, wherein said nucleic acid molecule contains: (a) a nucleic acid sequence encoding SEQ ID NO: 2; or (b) a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to SEQ ID NO: 2, wherein said amino acid sequence contains alanine at amino acid position 1408, alanine at amino acid position 1431, and alanine at amino acid position 1437 relative to SEQ ID NO: 2. 4. A combination for the treatment or prevention of MUC16-expressing cancer, comprising a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, wherein said nucleic acid molecule contains: (a) SEQ ID NO: 1; or (b) a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 1, wherein said nucleic acid sequence encodes alanine at amino acid position 1408, alanine at amino acid position 1431, and alanine at amino acid position 1437 relative to SEQ ID NO: 2 . 5. A combination for the treatment or prevention of MUC16-expressing cancer, comprising a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, wherein said nucleic acid molecule contains: (a) a nucleic acid sequence encoding amino acids 19-642 of SEQ ID NO: 4; or (b) a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to amino acids 19-642 of SEQ ID NO: 4. 6. A combination for the treatment or prevention of MUC16-expressing cancer, comprising a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, wherein said nucleic acid molecule contains: (a) nucleotides 55-1926 of SEQ ID NO: 3; or (b) a nucleic acid sequence that is at least 95% identical to nucleotides 55-1926 of SEQ ID NO: 3. 7. A combination for the treatment or prevention of MUC16-expressing cancer, comprising a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, wherein said nucleic acid molecule contains: (a) a nucleic acid sequence encoding SEQ ID NO: 4; or (b) a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to SEQ ID NO: 4. 8. A combination for the treatment or prevention of MUC16-expressing cancer, comprising a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, wherein said nucleic acid molecule contains: (a) SEQ ID NO: 3; or (b) a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 3. 9. A combination for the treatment or prevention of MUC16-expressing cancer, comprising a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, wherein said nucleic acid molecule contains: (a) a nucleic acid sequence encoding amino acids 19-710 of SEQ ID NO: 6; or (b) a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to amino acids 19-710 of SEQ ID NO: 6, wherein said amino acid sequence contains alanine at amino acid position 628, alanine at amino acid position 651, and alanine at amino acid position 657 with respect to SEQ ID NO: 6. 10. A combination for the treatment or prevention of MUC16-expressing cancer, comprising a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, wherein said nucleic acid molecule contains: (a) nucleotides 55-2130 of SEQ ID NO: 5; or (b) a nucleic acid sequence that is at least 95% identical to nucleotides 55-2130 of SEQ ID NO: 5, wherein said nucleic acid sequence encodes alanine at amino acid position 628, alanine at amino acid position 651, and alanine at amino acid position 657 relative to SEQ ID NO: 6. 11. A combination for the treatment or prevention of MUC16-expressing cancer, comprising a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, wherein said nucleic acid molecule contains: (a) a nucleic acid sequence encoding SEQ ID NO: 6; or (b) a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to SEQ ID NO: 6, wherein said amino acid sequence contains alanine at amino acid position 628, alanine at amino acid position 651, and alanine at amino acid position 657 relative to SEQ ID NO:6. 12. A combination for the treatment or prevention of MUC16-expressing cancer, containing a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, where said nucleic acid molecule contains: (a) SEQ ID NO: 5; or (b) a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 5, wherein said nucleic acid sequence encodes alanine at amino acid position 628, alanine at amino acid position 651, and alanine at amino acid position 657 relative to SEQ ID NO: 6 . 13. Combination for the treatment or prevention of MUC16-expressing cancer, containing a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, where said nucleic acid molecule contains: (a) a nucleic acid sequence encoding amino acids 19-642 and 650-1341 of SEQ ID NO: 8; or (b) a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to amino acids 19-642 and 650-1341 of SEQ ID NO: 8, wherein said amino acid sequence contains alanine at amino acid position 1259, alanine at amino acid position 1282, and alanine at amino acid position 1288 relative to SEQ ID NO: 8. 14. A combination for the treatment or prevention of MUC16-expressing cancer, containing a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, where said nucleic acid molecule contains: (a) nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7; or (b) a nucleic acid sequence that is at least 95% identical to nucleotides 55-1926 and 1948-4023 of SEQ ID NO: 7, wherein said nucleic acid sequence encodes alanine at amino acid position 1259, alanine at amino acid position 1282, and alanine at amino acid position 1288 with respect to SEQ ID NO: 8. 15. A combination for the treatment or prevention of MUC16-expressing cancer, comprising a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, wherein said nucleic acid molecule contains: (a) a nucleic acid sequence encoding amino acids 19-1341 of SEQ ID NO: 8; or (b) a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to amino acids 19-1341 of SEQ ID NO: 8, wherein said amino acid sequence contains alanine at amino acid position 1259, alanine at amino acid position 1282, and alanine at amino acid position 1288 with respect to SEQ ID NO: 8. 16. A combination for the treatment or prevention of MUC16-expressing cancer, containing a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, where said nucleic acid molecule contains: (a) nucleotides 55-4023 of SEQ ID NO: 7; or (b) a nucleic acid sequence that is at least 95% identical to nucleotides 55-4023 of SEQ ID NO: 7, wherein said nucleic acid sequence encodes alanine at amino acid position 1259, alanine at amino acid position 1282, and alanine at amino acid position 1288 relative to SEQ ID NO: 8. 17. Combination for the treatment or prevention of MUC16-expressing cancer, containing a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, where said nucleic acid molecule contains: (a) a nucleic acid sequence encoding SEQ ID NO: 8; (b) a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to SEQ ID NO: 8, wherein said amino acid sequence contains alanine at amino acid position 1259, alanine at amino acid position 1282, and alanine at amino acid position 1288 relative to SEQ ID NO: 8. 18. A combination for the treatment or prevention of MUC16-expressing cancer, containing a nucleic acid molecule encoding the MUC16 antigen and an immune checkpoint inhibitor, where said nucleic acid molecule contains: (a) SEQ ID NO: 7; or (b) a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 7, wherein said nucleic acid sequence encodes alanine at amino acid position 1259, alanine at amino acid position 1282, and alanine at amino acid position 1288 relative to SEQ ID NO: 8 . 19. The combination according to any one of paragraphs. 1-18, wherein said immune checkpoint inhibitor is programmed cell death-1 (PD-l), programmed cell death ligand-1 (PD-L1), or a combination thereof. 20. The combination according to any one of paragraphs. 1-18, wherein said immune checkpoint inhibitor is an antibody to programmed cell death-1 (PD-1) protein or an antigen-binding fragment thereof. 21. The combination of claim 20, wherein said anti-PD-1 antibody is nivolumab. 22. The combination according to any one of paragraphs. 1-18, wherein said immune checkpoint inhibitor is an antibody to programmed cell death ligand-1 (PD-L1) or an antigen-binding fragment thereof. 23. The combination according to any one of paragraphs. 1-22, wherein said nucleic acid molecule encoding the MUC16 antigen is an expression vector. 24. The combination of claim 23, wherein said nucleic acid molecule is operably linked to a regulatory element selected from a polyadenylation promoter or signal. 25. The combination of claim 24, wherein said promoter is the human cytomegalovirus immediate-early response promoter (hCMV promoter). 26. The combination of claim 24, wherein said polyadenylation signal is a bovine growth hormone (bGH poly-A) polyadenylation signal sequence. 27. The combination according to any one of paragraphs. 23-26, where said vector is a plasmid. 28. An immunogenic composition containing an effective amount of the combination according to any one of paragraphs. 1-27 and a pharmaceutically acceptable carrier. 29. Vaccine for the treatment or prevention of MUC16-expressing cancer, containing a combination according to any one of paragraphs. 1-27 and a pharmaceutically acceptable excipient, and optionally wherein said vaccine further comprises an adjuvant, preferably wherein said adjuvant is IL-12, IL-15, IL-28, or RANTES. 30. A method of treating a subject with a cancer cell expressing MUC16, comprising the introduction of a therapeutically effective amount of the vaccine according to claim 29; optionally where said drug is formulated to be administered by electroporation; and optionally where said drug is formulated to be administered at one or more sites on the subject. 31. A method of vaccinating a subject against a cancer cell expressing MUC16, comprising administering an amount of the vaccine according to claim 29 effective to induce a humoral immune response, optionally wherein said drug is prepared for administration by electroporation and optionally where said drug is prepared for administration in one or more places on the subject.

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