SEQUENCE LISTING

Incorporated by reference in its entirety herein is a nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 14,638 Byte ASCII (Text) file named “740352_ST25.TXT,” created on Aug. 21, 2018.

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 is an amino acid sequence of a human Brachyury protein.

SEQ ID NO: 2 is a nucleic acid sequence encoding a human Brachyury protein.

SEQ ID NO: 3 is an amino acid sequence of a murine Brachyury protein.

SEQ ID NO: 4 is a nucleic acid sequence encoding a murine Brachyury protein.

SEQ ID NO: 5 is a Brachyury class IIA epitope.

SEQ ID NO: 6 is a Brachyury class IIB epitope.

FIELD

This application relates to the field of cancer therapeutics, specifically to the use of a Brachyury protein and non-poxvirus, non-yeast vectors encoding a Brachyury protein for the treatment of cancer.

BACKGROUND

The Brachyury gene was initially cloned from mouse developmental mutants characterized by an arrest in mesoderm formation (Hermann et al,Nature1990; 343:617-22) has been recognized as gene that is important in mesoderm development during gastrulation. Brachyury is a member of a family of transcription factors, designated T-box transcription factors, these factors are characterized by a conserved DNA-binding domain (Papaioannou et al.,Bioessays1998; 20:9-19). These transcription factors play an essential role in the formation and organization of mesoderm in vertebrates (see, for example, Edwards et al.,Genome Res1996; 6:226-33). In addition to the important role of the T-box proteins in the control of developmental processes, several members of this family are deregulated in cancer. For example, the human Tbx2 gene has been reported to be amplified in pancreatic cancer cell lines (Mahlamaki et al.,Genes Chromosomes Cancer2002, 35:353-8) and is overexpressed in BRCA-1- and BRCA-2-mutated breast tumors (Sinclair et al.,Cancer Res2002; 62:3587-91). In addition, Tbx3 expression has been shown to be augmented in certain human breast cancer cell lines (Fan et al.,Cancer Res2004; 64:5132-9). Expression of Brachyury has also been documented in human teratocarcinoma lines: a subset of germ cell tumors, teratocarcinomas are embryonal carcinoma cells with competence for mesoderm differentiation (Gokhale et al.,Cell Growth Differ2000; 11:157-62) and in chordomas (see, for example, Vojovic et al.,J Pathol2006; 209:157-65).

Immunotherapeutic interventions against cancer depend on the identification of tumor antigens able to elicit a host immune response against the tumor cells. Good targets are molecules that are selectively expressed by malignant cells and that are also essential for malignant transformation and/or tumor progression. The epithelial-mesenchymal transition (EMT) has been recognized as a key step during the progression of primary tumors into metastases (Thiery et al.,Nat Rev Cancer2002; 2:442-54). Several molecules have been identified that play a key role in EMT during tumor progression (Huber et al.,Curr Opin Cell Biol2005; 17:548-58), among them the transcription factors Twist, Snail, and Slug (Yang et al.,Cell2004; 117:927-39; Cano et al.,Nat Cell Biol2000; 2: 76-83). Molecules that trigger EMT could function to prevent tumor invasion and metastasis. However, a need remains for reagents that induce an effective immune response to cancer, including a CD4 and a CD8 T cell response.

SUMMARY

It is disclosed herein that Brachyury protein or a Brachyury polypeptide can be used to induce Brachyury-specific CD4+ T cells in vivo and ex vivo. It is also disclosed that Brachyury protein and Brachyury polypeptides can be used to stimulate the production of both Brachyury-specific CD4+ T cells and Brachyury-specific CD8+ T cells. Brachyury is expressed in numerous human cancers, such as in cancer of the small intestine, stomach, kidney bladder, uterus, ovary, testes, lung, colon, prostate, bronchial tube, chronic lymphocytic leukemia (CLL), other B cell-based malignancies, and breast cancer, such as infiltrating ductal carcinomas of the breast. Thus, Brachyury protein, Brachyury polypeptides, and nucleic acids encoding Brachyury protein and/or polypeptides, can be used to produce Brachyury specific CD4+ T cells, and CD8+ T cells, that can be used for the treatment or prevention of cancer.

In some embodiments, methods are disclosed for inducing CD4+ Brachyury-specific T cells and/or CD8+ Brachyury specific T cells. The methods include the use of a Brachyury protein, a Brachyury polypeptide, nucleic acids encoding the Brachyury protein and/or Brachyury polypeptides, or host cells expressing the Brachyury protein or polypeptide, such as such as aSalmonellaorListeriahost cells. These agents can be administered either alone or in conjunction with another agent, such as a cytokine and/or another cancer therapy. In some embodiments, methods are disclosed for treating a subject with a cancer, such as a breast cancer, cancer of the small intestine, stomach, kidney, bladder, uterus, ovaries, testes lung, colon or prostate, or a tumor of B cell origin, or for preventing these cancers in a subject. In some embodiments, the methods include measuring Brachyury-specific CD4+ T cells. In further embodiments, the methods also induce CD8+ Brachyury specific T cells.

Non-pox non-yeast vectors encoding a Brachyury protein are disclosed that can be used to induce CD4+ Brachyury-specific T cells and/or CD8+ Brachyury-specific T cells. In some non-limiting examples, the vector is an alphavirus, a lentivirus, an adenovirus, a measles virus or a poliovirus vector. In additional embodiments, host cells transformed with these vectors, such asSalmonellaandListeriahost cells are provided.

In additional embodiments, methods are provided for inhibiting the growth of a cancer cell in a subject. These methods include contacting a dendritic cell with a protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth as SEQ ID NO: 1, a polypeptide comprising at least 15 consecutive amino acids of the amino acid sequence set forth at SEQ ID NO: 1 that specifically binds a Major Histocompatibility Class (MHC class II) molecule, or aListeriaorSalmonellahost cell expressing the protein or the polypeptide thereby preparing a specific antigen presenting cell. These methods also include administering the antigen presenting cell to the subject, thereby inducing an immune response and inhibiting the growth of the cancer cell.

The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

