JP2018527286A - Identification of immunogenic MHC class II peptides for immune-based therapy - Google Patents

Abstract

The present invention provides compositions, methods and vaccines that can stimulate the immune system and can be used to treat malignant tumors associated with overexpression of HER-3 protein. Such a composition comprises an epitope of the HER-3 protein. [Selection] Figure 1

Description

(Cross-reference of related applications)
This application is a continuation of application number PCT / US15 / 41034, filed July 17, 2015. The application claims priority to US Provisional Patent Application No. 62 / 076,789 filed on November 7, 2014 and US Provisional Patent Application No. 62 / 025,681 filed July 17, 2014. . The entire contents of each patent application are incorporated herein by reference.

  In 25-30% of breast cancers, amplification and overexpression of the growth factor receptor gene HER2 (also known as human epidermal growth factor receptor-2, neu / erbB2) results in enhanced tumor invasiveness and recurrence and Associated with a high risk of death (Slamon, D. et al., 1987, Science 235: 177; Yarden, Y., 2001, Oncology 1: 1). This oncogene encodes a 185 kilodalton (kDa) transmembrane receptor tyrosine kinase. HER2 stands out from the others in several ways as one of the four members of the human epidermal growth factor receptor (EGFR) family. First, HER2 is an orphan receptor. No high affinity ligand has been identified. Second, HER2 becomes a preferred partner for other EGFR family members (HER1 / EGFR, HER3 and HER4), forming heterodimers, which have high ligand affinity and excellent Signaling activity. Third, full-length HER2 undergoes proteolytic cleavage and releases a soluble extracellular domain (ECD). ECD shedding has been shown to be an alternative activation mechanism for full-length HER2, both in vitro and in vivo, because a membrane anchor fragment with kinase activity remains. The central role of HER2 in EGFR family signaling correlates with its involvement in the development of several types of cancer, including breast cancer, ovarian cancer, colon cancer and gastric cancer, regardless of its expression level (Slamon, D Et al., 1989, Science 244: 707; Hynes, N. et al., 1994, Biochem. Biophys. Acta. 1198: 165). HER2 may also confer resistance to certain chemotherapy on tumor cells (Pegram, M. et al., 1997, Oncogene 15: 537). HER2 is an important target for cancer therapeutics given its very important role in tumorigenesis.

  The human EGF receptor (HER) family of receptor tyrosine kinases regulate a great variety of biological processes, including cell proliferation, migration, invasion and survival. This family consists of four members: EGFR (HER1), HER2 (neu or ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Currently, 11 including epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor alpha (TGFα), amphiregulin (AR), epiregulin, betacellulin and heregulin The ligands have been reported. These ligands bind directly to their cognate receptors, which leads to the formation of receptor homodimers or heterodimers that cause activation of multiple signaling pathways. Dysregulation of HER-family members, either through activation of mutations, receptor overexpression or abnormal ligand release, leads to diverse human tumor development. HER3 is overexpressed in breast, ovarian and lung cancers and this genetic feature correlates with poor prognosis. HER3, when activated by heregulin, dimerizes with HER2 and EGFR to form a potent oncogenic receptor heterodimer. Within this complex, HER3 preferentially recruits PI3 kinase to its cytoplasmic docking site, thereby regulating cell growth and survival. To date, HER3 is inactive as a kinase due to the apparently aberrant sequence characteristics in its kinase domain, and the HER-family kinase activity is impaired to initiate a signaling event. It has been speculated that it requires heterodimerization with a non-member. Consistent with this, it was shown that HER2 requires HER3 to drive breast tumor cell proliferation. However, recent findings have shown that HER3 can also phosphorylate Pyk2 and consequently activate the MAPK pathway in human glioma cells. Furthermore, monoclonal antibodies specific for HER3 can also inhibit the growth and migration of cancerous cell lines. Interestingly, recently, cancer cells circumvent HER-family inhibitor therapy by upregulating HER3 signaling, and HER3 inhibition abrogates HER2-driven tamoxifen resistance in breast cancer cells Indicated. Furthermore, resistance to EGFR small molecule inhibitor gefitinib (Iressa) therapy has also been shown to be linked to signal activation of HER3.

  HER3 is a receptor protein and plays an important role in regulating normal cell growth. HER3 lacks its own kinase activity and transduces signals across the cell membrane depending on the presence of HER2. At the beginning of transcription, the HER3 mRNA precursor contains 28 exons and 27 introns. The fully spliced HER3 mRNA is composed of 28 exons after splicing introns.

  During the past decade, targeted therapies have emerged as the basis for cancer therapeutics. Members of the EGF receptor family, ie EGFR (or HER1) and ErbB2 (or HER2 / neu), are becoming particularly interesting targets, because these receptor tyrosine kinases ( This is because RTK) is deregulated in many cancers. The oncogenic function of ErbB3 or HER3, another member of the EGF receptor family, has only recently been examined due to mediating resistance to HER2 and its major role in therapies targeting the PI3K pathway It came to be. Activation of mutations in HER3 and / or its overexpression has been identified in several different tumor types, including breast cancer, gastric cancer, colon cancer, bladder cancer and melanoma, and worse overall in these tumors A prognostic prognosis.

Slamon, D.C. Et al., 1987, Science 235: 177. Yarden, Y.M. 2001, Oncology 1: 1. Slamon, D.C. Et al., 1989, Science 244: 707. Hynes, N .; Et al., 1994, Biochem. Biophys. Acta. 1198: 165 Pegram, M .; Et al., 1997, Oncogene 15: 537.

  Despite advances in the art, it remains uncertain whether effective immune responses can be generated in humans using cell or protein-based vaccine strategies that target HER-3. Accordingly, there is a need in the art for additional immunotherapy approaches to treat or prevent breast cancer and other malignancies associated with HER-3 protein overexpression. The present invention satisfies this need.

  The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

1 is a graph showing immunogenic peptides derived from HER-3 showing the ability to activate CD4 T cells across many patients (SEQ ID NOs: 1-3, respectively, in apparent order). FIG. 5 is a graph showing an overall screen of HER3 using a group of 10 peptide fragments. Also shown is a HER3 screen graph using a single peptide (SEQ ID NOs: 4-7, respectively, in order of appearance). FIG. 5 is a graph showing an overall screen of HER3 using a group of 10 peptide fragments. Also shown is a HER3 screen graph using a single peptide (SEQ ID NOs: 4 and 7, respectively, in apparent order). FIG. 5 is a graph showing an overall screen of HER3 using a group of 10 peptide fragments. Also shown is a HER3 screen graph using a single peptide (SEQ ID NOS: 1-3, in order of appearance, respectively). FIG. 2 is a graph showing the production of IFN-γ from different HER3 peptides. FIG. 2 is a graph showing the production of IFN-γ from different HER3 peptides. FIG. 6 is a graph showing the generation of IFN-γ from a “reverse” screen that has been previously identified and then sensitized to the peptide and the HER3 extracellular domain. FIG. 6 is a graph showing the generation of IFN-γ from a “reverse” screen that has been previously identified and then sensitized to the peptide and the HER3 extracellular domain. FIG. 6 is a graph showing the production of IFN-γ from a “reverse” screen starting with a peptide and sensitizing to the peptide and HER3 extracellular domain in a patient who has not previously been sensitized to the HER extracellular domain. Graph showing IFN-γ production from a “reverse” screen starting with a full peptide library and sensitizing to peptides and HER3 extracellular domain in patients who have not previously sensitized to the HER extracellular domain It is. Graph showing IFN-γ production from a “reverse” screen starting with a full peptide library and sensitizing to peptides and HER3 extracellular domain in patients who have not previously sensitized to the HER extracellular domain It is. FIG. 6 is a graph showing the production of IFN-γ from a “reverse” screen starting with a peptide and sensitizing to the peptide and HER3 extracellular domain in a patient who has not previously been sensitized to the HER extracellular domain. FIG. 4 is a graph showing a continuous peptide screen in UPCC 15107-24 donor. FIG. 4 is a graph showing a continuous peptide screen in UPCC 15107-38 donor. FIG. 6 is a graph showing “reverse” sensitization in UPCC 15107-38 and UPCC 15107-24 donors. Immunogenicity in UPCC 15107-30 and UPCC 15107-32 donors (both patients are known not to show anti-HER3 reactivity to identified HER3 peptides and / or native HER3 ECD) FIG. 3 is a graph showing that DC1 pulsed with a HER3 epitope sensitized CD4 + Th1 and overcome anti-HER3 immune tolerance. It is a graph which shows that a CD4 + HER3 epitope shows MHC class II promiscuousness. Placing activated HER-3 CD4 + cells next to cells expressing HER-3 in the chamber causes the HER-3 CD4 + cells to cause apoptosis or death of breast cancer cells expressing HER-3 FIG. FIG. 6 shows a method for identifying immunogenic, class II promiscuous HER3 CD4 + peptides using HER3 ECD as a tumor antigen to generate anti-HER3 Th1 cellular immunity. FIG. 6 is a graph showing that the immunogenicity of the identified CD4 + HER3 ECD epitope was confirmed by “reverse” sensitization. A HER3 ECD screen was performed with the single peptide indicated. FIG. 6 is a graph showing additional results confirming the immunogenicity of identified CD4 + HER3 ECD epitopes by “reverse” sensitization. 3 shows photographs of immunohistochemical scoring of HER staining A and B are HER family overexpression in Barrett's esophagus with low grade dysplasia (LGD) or high grade dysplasia (HGD) (Figure 22A) and high grade dysplastic Barrett's lesion (with carcinoma) HGD). Without associated invasive cancer (HGD) (Figure 22B).

  The present invention provides isolated peptides of HER family proteins and other receptor tyrosine kinases. In one embodiment, the present invention provides isolated peptides from one or more of HER-1, HER-3 and c-MET proteins. In one embodiment, the peptide of the invention corresponds to an epitope of HER-1. In one embodiment, the peptide of the invention corresponds to an epitope of HER-3. In one embodiment, the peptide of the invention corresponds to an epitope of c-MET.

  In some embodiments, the corresponding HER family of proteins and other receptor tyrosine kinase epitopes are immunogenic. The present invention further provides compositions comprising one or more peptides of the present invention. In one embodiment, the invention provides a chimeric peptide, the chimeric peptide comprising one or more peptides of the invention.

  In one embodiment, the invention includes a composition comprising a multivalent peptide. A multivalent peptide includes two or more peptides of the invention.

  Further provided are methods of stimulating an immune response and methods of treating cancer in a subject. Also provided are vaccines for use in therapy and prevention. The peptides of the invention can elicit an immune response, either alone or in the context of a chimeric peptide, as described herein. In one embodiment, the immune response is a humoral response. In another embodiment, the immune response is a cell-mediated response. According to some embodiments, the peptides of the invention confer a protective effect.

  In another embodiment, HER3 expression can be used as a marker of tumor progression in precancerous lesions at the gastroesophageal junction.

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

  In general, both the nomenclature used herein and laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization are well known and commonly used in the art. .

  Standard techniques are used for nucleic acid and peptide synthesis. Techniques and procedures are generally described in the prior art in the art and various general references (eg, Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, Y As well as Ausubel et al., 2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout this description.

  The nomenclature used herein and the laboratory procedures used in analytical chemistry and organic synthesis described below are well known in the art and commonly used. Chemical synthesis and analysis are performed using standard techniques or modifications thereof.

  As used herein, each of the following terms has the meaning associated with it in this section.

  As used herein, the articles “a” and “an” are used to refer to one or more (ie, at least one) of the grammatical objects of the article. By way of example, “an element” means one element or more than one element.

  “About” when referring to a measurable value, for example, an amount, a time period, etc., as used herein, is ± 20% from a particular value, or ± 10%, or ± It is intended to encompass variations of 5%, or ± 1%, or ± 0.1%, and such variations are appropriate for performing the disclosed methods.

  When used in the context of an organism, tissue, cell or component thereof, the term “abnormal” refers to at least one observable or detectable characteristic (eg, age, treatment, time of day, etc.) An organism, tissue, cell or component thereof that differs from an organism, tissue, cell or component thereof that exhibits “normal” (expected) characteristics. The characteristics that are normal or expected for one cell or tissue type may be abnormal for different cell or tissue types.

  As used herein, “adjuvant therapy” for breast cancer refers to any treatment given after primary therapy (ie, surgery) to increase long-term survival opportunities. “Neoadjuvant therapy” is a treatment given before primary therapy.

  The term “antigen” or “ag” as used herein is defined as a molecule that causes an immune response. This immune response can be involved in either the production of antibodies or the activation of certain immunologically competent cells or both. One skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. In addition, antigens can be obtained from recombinant or genomic DNA. Those skilled in the art will understand that any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will thus encode the term “antigen” as used herein. Will. Furthermore, one skilled in the art will understand that the antigen need not be encoded solely by the full-length nucleotide sequence of the gene. The present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences can be easily placed in various combinations to elicit a desired immune response. Becomes clear. Furthermore, those skilled in the art will also appreciate that an antigen need not be encoded by a “gene” at all. It will be readily apparent that the antigen may be produced synthetically or obtained from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

  An “antigen-presenting cell” (APC) is a cell that is capable of activating T cells, including but not limited to mononuclear cells / macrophages, B cells and dendritic cells (DC).

  “APC loaded with antigen” or “APC pulsed with antigen” includes APC that has been exposed to and activated by an antigen. For example, APC can be Ag loaded during culturing in vitro, for example, in the presence of an antigen. Alternatively, APC can be loaded by exposing it to the antigen in vivo. “Antigen-loaded APC” has traditionally been made in two ways: (1) “pulsing” a small peptide fragment known as an antigenic peptide directly onto the exterior of the APC, or (2) APC Incubate with total protein or protein particles and then have APC ingest the total protein or protein particles. These proteins are digested by APC into small peptide fragments and finally transported to the APC surface where they are presented. Furthermore, antigen-loaded APCs can also be generated by introducing a polynucleotide encoding the antigen into the cell.

  The term “anti-tumor effect” as used herein refers to various physiological symptoms associated with decreased tumor volume, decreased number of tumor cells, decreased number of metastases, increased life expectancy, or cancerous condition. It refers to the biological effects that can be revealed by remission. In the first place, “anti-tumor activity” can also be revealed by the ability of the peptides, polynucleotides, cells and antibodies of the present invention in preventing tumor development.

  The term “autoimmune disease” as used herein is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self antigens. In particular, examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), nutrition Impaired epidermolysis bullosa, epididymis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis , Rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthritis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis.

  As used herein, the term “self” is intended to refer to any material that is obtained from an individual and that is subsequently being reintroduced into the same individual.

  The term “B cell” as used herein is defined as a cell derived from bone marrow and / or spleen. B cells can develop into plasma cells, which produce antibodies.

  The term “cancer”, as used herein, is defined as cell hyperproliferation, resulting in aberrant growth, lack of differentiation, local tissue invasion as a result of the abandonment of normal control, a unique trait of cancer. And / or metastasis occurs. Examples include but are not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain tumor, lymphoma, leukemia, lung cancer, germ cell tumor, etc. Can be mentioned.

  A “disease” is an animal health condition in which the animal's health condition continues to deteriorate if the animal is unable to maintain homeostasis and the disease does not ameliorate.

  A “disorder” in an animal refers to a case where the animal is capable of maintaining homeostasis, but the health status of the animal is less favorable than in the absence of the disorder. Disorders that are left untreated do not necessarily cause a further decline in the animal's health.

  A disease or disorder is “reduced” if the severity or frequency of at least one sign or symptom of the disease or disorder experienced by the patient is reduced.

  “Effective amount” or “therapeutically effective amount” is used interchangeably herein and, as described herein, is a compound, formulation, material or composition effective to achieve a particular biological result. Refers to the amount of a thing. Such results can include, but are not limited to, inhibition of viral infection as determined by any suitable means in the art.

  As used herein, “endogenous” refers to any material belonging to or produced within an organism, cell, tissue or system.

  As used herein, the term “exogenous” refers to any material that is introduced from or produced outside an organism, cell, tissue or system.

  “HER receptor” is a receptor protein tyrosine kinase belonging to the HER receptor family, EGFR (ErbB1, HER1) receptor, HER2 (ErbB2) receptor, HER3 (ErbB3) receptor and HER4 (ErbB4) receptor including. HER receptors generally bind to HER ligands and / or can dimerize with another HER receptor molecule; an extracellular domain; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain As well as a carboxyl-terminal signaling domain with several tyrosine residues that can be phosphorylated. The HER receptor may be a “native sequence” HER receptor or an “amino acid sequence variant” thereof. Preferably, the HER receptor is a native sequence human HER receptor.

  “HER pathway” refers to a signaling network mediated by the HER receptor family.

  “HER activation” refers to activation or phosphorylation of any one or more HER receptors. In general, activation of HER results in signal transduction (eg, signal transduction caused by the intracellular kinase domain of the HER receptor that phosphorylates tyrosine residues or substrate polypeptides in the HER receptor). HER activation can be mediated by binding of a HER ligand to a HER dimer containing the HER receptor of interest. Binding of a HER ligand to a HER dimer can activate the kinase domain of one or more HER receptors in the dimer, thereby in one or more HER receptors. Phosphorylation of tyrosine residues and / or phosphorylation of tyrosine residues in additional substrate polypeptides, eg, Akt intracellular kinase or MAPK intracellular kinase.

  “HER3” and “ErbB3” are disclosed, for example, in US Pat. Nos. 5,183,884 and 5,480,968, and Kraus et al., PNAS (USA) 86: 9193-9197 (1989). Refers to a receptor polypeptide.

  “HER3 extracellular domain” or “HER3ECD” refers to a domain of HER3 that is outside the cell and that is anchored or circulating in the cell membrane, including fragments thereof. In one embodiment, the extracellular domain of HER3 may comprise four domains: domain I, domain II, domain III and domain IV. In one embodiment, the HER3 ECD comprises amino acids 1-636 (numbering including the signal peptide). In one embodiment, HER3 domain III comprises amino acids 328-532 (numbering including signal peptide).

  “Homologous” as used herein is a subunit between two macromolecular molecules, eg, two nucleic acid molecules, eg, two DNA molecules or two RNA molecules, or two polypeptide molecules. Refers to sequence similarity. If the same monomer subunit occupies a position of a subunit in both two molecules, for example if adenine occupies a position in each of the two DNA molecules, these molecules Is completely homologous or 100% homologous. The percent homology between two sequences is a direct function of the number of matching or homologous positions, for example, in a sequence of two compounds, half of the positions (eg, 10 subunits high in length) If the 5 positions in the molecule are homologous, the two sequences are 50% identical, and if 90% of the positions, eg 9 out of 10 are matched or homologous, the two sequences The sequences share 90% homology. As an example, the DNA sequences 5'ATTGCC3 'and 5'TATGGC3' share 50% homology.

  Furthermore, when the term “homology” or “identity” is used herein to refer to nucleic acids and proteins, it is understood that it applies to homology or identity at both the nucleic acid and amino acid sequence levels. Should.

  The term “hyperproliferative disease” is defined as a disease that results from cellular hyperproliferation. Exemplary hyperproliferative diseases include, but are not limited to, cancer or autoimmune diseases. Other hyperproliferative diseases can include, for example, vascular occlusion, restenosis, atherosclerosis or inflammatory bowel disease.

  As used herein, “instruction material” includes publications, records, schematics or any other representational medium that can be used to convey the usefulness of the compositions and methods of the invention. Instructions for use of the kit of the present invention are attached to, for example, a container containing the nucleic acid, peptide and / or composition of the present invention, or shipped together with a container containing the nucleic acid, peptide and / or composition. be able to. Alternatively, the instructional material and the compound may be shipped separately from the container, with the intention that the instructional material and the compound be used together by the recipient.

  “Immune response” as used herein means the activation of a host immune system, eg, a mammalian immune system, in response to the introduction of an antigen. The immune response can take the form of a cellular response or a humoral response, or both.

  “Isolated” means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not in an “isolated state”, but the same nucleic acid or peptide that is partially or completely separated from coexisting material in its natural state is “isolated. It is. The isolated nucleic acid or protein may be present in substantially purified form, or may be present in an externally applied environment such as, for example, a host cell.

  By the term “modulating” as used herein, as compared to the level of response in a subject in the absence of treatment or compound, and / or otherwise identical, but not yet. By mediating a detectable increase or decrease in response level in a subject when compared to the response level in a treated subject. The term encompasses perturbing and / or affecting a native signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

  “Peptide”, “protein” or “polypeptide” as used herein can mean amino acid-linked sequences and can be natural, synthetic, or natural and synthetic variants or combinations. .

  As used herein, “population” includes reference to an isolated culture comprising a homogeneous, substantially homogeneous, or heterogeneous cell culture. In general, a “population” can also be considered an “isolated” cell culture.

  As used herein, a “recombinant cell” is a host cell that contains a recombinant polynucleotide.

  A “sample” or “biological sample” as used herein is obtained from a subject, including but not limited to organs, tissues, exosomes, blood, plasma, saliva, urine and other body fluids. Means biological material. The sample can be material from the subject of any source.

  “Signal 1” as used herein generally refers to the first biochemical signal passed from activated DCs to T cells. Signal 1 is provided by an antigen expressed on the surface of the DC and is sensed by the T cell through the T cell receptor.

  “Signal 2” as used herein generally refers to a second signal provided to the T cell by the DC. Signal 2 is provided by “costimulatory” molecules on activated DCs, usually CD80 and / or CD86 (but other costimulatory molecules are also known) and sensed by the T cell through the surface receptor CD28. Is done.

  “Signal 3” as used herein generally refers to a signal generated from a soluble protein (usually a cytokine) produced by activated DC. These are sensed through receptors on T lymphocytes. The third signal instructs the T cell as to what phenotypic or functional characteristics it should acquire in order to best cope with the current threat.

  By the term “specifically binds” as used herein, recognizes and binds to another molecule or feature in a sample, but substantially recognizes another molecule or feature. Means a molecule that does not bind to them, for example, an antibody.

  The terms “subject”, “patient”, “individual” and the like are used interchangeably herein, whether any animal or those suitable for the methods described herein, whether in vitro or in situ. Refers to cells. In certain non-limiting embodiments, the patient, subject or individual is a human.

  The term “T cell” as used herein is defined as a cell derived from the thymus that is involved in a variety of cell-mediated immune responses.

  The term “T-helper”, when used herein with reference to cells, includes a subgroup of lymphocytes (white blood cells or leukocyte types) that includes different cell types that can be identified by one skilled in the art. Indicates. In particular, T-helper cells according to the present disclosure include effector Th cells (eg, Th1, Th2 and Th17). These Th cells secrete cytokines, proteins or peptides that activate or interact with other leukocytes.

  “Th1 T cells”, as used herein, produce the cytokine IFN-γ at high levels and survive in host cells, against certain disease-causing microorganisms, and also against cancer. Refers to T cells that are believed to be extremely effective.

  “Th17 T cells” as used herein are considered highly effective against disease-causing microorganisms that produce cytokines IL-17 and IL-22 at high levels and survive on mucosal surfaces. Refers to the T cell being

  A “therapeutically effective amount” is the amount of a compound of the invention that, when administered to a patient, ameliorates the symptoms of the disease. The amount of a compound of the invention that constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and severity of the disease, the age of the patient to be treated, and the like. A therapeutically effective amount can be routinely determined by one of ordinary skill in the art in view of the skilled person's own knowledge and the present disclosure.

  The terms “treat”, “treating” and “treatment” refer to the therapeutic or prophylactic strategies described herein. The method of “treatment” exceeds the expected survival for prevention, cure, delay, reduced severity or remission of one or more symptoms of a disorder or relapse disorder, or when no such treatment is given, In order to prolong the survival of a subject, the composition of the present invention can be used to ultimately acquire a subject in need of such treatment, eg, a subject suffering from or suffering from a disease or disorder Take advantage of administering to subjects at risk.

  The term “vaccine” as used herein is defined as a material used to elicit an immune response after administration to an animal, preferably a mammal, more preferably a human. Once introduced into a subject, a vaccine can elicit an immune response including, but not limited to, the generation of antibodies, cytokines and / or other cellular responses.

  A “variant” with respect to a peptide or polypeptide retains at least one biological activity, although the amino acid sequence differs by amino acid insertion, deletion or conservative substitution. A variant may also mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of amino acids, ie, replacing amino acids with different amino acids with similar properties (eg, hydrophilicity, charged region extent and distribution) typically results in minor changes in the art. It is recognized that These minor changes can be identified, in part, by examining the hydrophobic hydrophilicity index of amino acids according to understanding in the art. Kyte et al. Mol. Biol. 157: 105-132 (1982). The hydrophobic hydrophilicity index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydrophobic hydrophilicity index can be substituted and still retain protein function. In one aspect, amino acids having a hydrophobic hydrophilicity index of ± 2 are substituted. The hydrophilicity of amino acids can also be used to indicate substitutions that would result in a protein that retains biological function. In the peptide context, examining the hydrophilicity of an amino acid makes it possible to calculate the largest local average hydrophilicity of the peptide, which can be well correlated with antigenicity and immunogenicity. It is a useful measure that has been reported. U.S. Pat. No. 4,554,101, which is incorporated herein by reference in its entirety. As understood in the art, substitution of amino acids with similar hydrophilicity values can yield peptides that retain biological activity, eg, immunogenicity. Substitutions can be performed with amino acids having hydrophilic values within ± 2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with such observations, amino acid substitutions corresponding to biological functions are related to the relative similarity of amino acids, particularly those amino acids, indicated by hydrophobicity, hydrophilicity, charge, size and other properties. It is understood that it depends on the side chain.

  Scope: Throughout this disclosure, various aspects of this invention can be presented as a range format. It should be understood that the description as a range format is merely for convenience and brevity and should not be construed as a firm limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges and individual numerical values within that range. For example, a description of a range, for example, 1-6 is a partial range, for example, 1-3, 1-4, 1-5, 2-4, 2-6, 3-6 etc., and within that range Individual numbers, such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6, should be considered specifically disclosed. This applies regardless of the range width.

DESCRIPTION The present invention provides immunological compositions comprising HER family proteins and other receptor tyrosine kinase (RTK) peptides. In one embodiment, the present invention provides isolated peptides from one or more of HER-1, HER-3 and c-MET proteins. In one embodiment, the peptides of the invention are useful for eliciting an immune response. Compositions comprising the peptides of the present invention are also useful as prophylactic therapeutic agents for initial protection and as therapeutic agents for the treatment of ongoing conditions.

  The present invention also provides a method for treating or preventing cancer. Such methods include the step of administering a peptide of the invention or a combination of peptides of the invention to a subject in need thereof. As a result of the administration of such peptides, anti-tumor immunity is induced. Accordingly, the present invention provides methods for inducing anti-tumor immunity in a subject, such methods comprising a peptide of the invention or a combination of peptides of the invention, and pharmaceutical and cell compositions derived therefrom. Administering to a subject.

  The present invention includes a method for inducing a T cell response in a mammal. The method includes administering an antigen presenting cell (APC) that specifically induces T cell proliferation. In one embodiment, the method comprises administering a dendritic cell vaccine pulsed with a peptide of the invention, thereby specifically inducing T cell proliferation against an antigen corresponding to the peptide.

  In one embodiment, APC pulsed with a peptide of the invention can be used to culture and expand T cells. Once APC is used to expand and proliferate the T cells to obtain a sufficient number of antigen-specific T cells, the antigen-specific T cells so obtained are administered to the mammal, whereby the antigen in the mammal Induces a specific T cell response.

  The present invention includes preparations of activated DC. In one embodiment, the DC preparation is greater than 90% pure. In another embodiment, the DC preparation is fully activated. For example, the DC is activated using a DC activation regimen that involves contacting the DC with a TLR agonist (eg, LPS). In another embodiment, the process of mobilizing calcium is used in conjunction with other DC activation regimens (eg, activators) that enhance cytokines of different third signals to activate DCs.

  The present invention includes mature, antigen-loaded DCs activated by any DC activation regimen. The DCs of the present invention produce desirable levels of cytokines and chemokines. In one embodiment, the present invention provides a method of pulsing and activating cells, whereby the cells remain active after cryopreservation. The benefits of the DC preparation of the invention are that from a single leukocyte apheresis (collection from a patient), the cells are efficient in multiple (eg, 10 or more) “booster” doses in addition to the initial vaccine. These frozen doses can be thawed as needed at a remote treatment site, without the need for any specialized cell processing facility or further quality control testing.

  The present invention also relates to cryopreservation of these activated DCs, which are cryopreserved to retain efficacy and function in antigen presentation and production of various cytokines and chemokines upon thawing, Thus, activated DCs that have been cryopreserved and subsequently thawed are clinically effective at the same level as freshly collected and activated DCs.

  The present invention contemplates that the present invention provides a method for generating and cryopreserving DCs that have superior function in generating stronger signals to T cells, and thus more A powerful, DC-based vaccine is obtained. By effectively cryopreserving such cells, samples can be stored, thawed and used later, thereby reducing the need to repeat the pheresis and erutation processes during vaccine production. Let It is an advantage that the DCs can be frozen and then later thawed and removed, which means that the vaccine produced in a single shot can be subdivided and frozen, and then for weeks This means that it can be administered to patients one at a time over a period of months or years, resulting in a “booster” vaccination that enhances immunity.

  This embodiment also includes the use of HER3 expression as a marker of tumor progression in a premalignant lesion of the gastroesophageal junction, also known as Barrett's esophagus. This marker has prognostic and therapeutic use in invasive esophageal gastric cancer.

Compositions The present invention provides isolated peptides of HER family proteins and other receptor tyrosine kinases. In one embodiment, the present invention provides isolated peptides from one or more of HER-1, HER-3 and c-MET proteins. In one embodiment, the peptides of the invention correspond to epitopes of the corresponding HER protein or c-MET protein. In some embodiments, the corresponding HER protein or c-MET protein epitope is immunogenic.

  The present invention provides a composition comprising one or more peptides of the present invention. The invention also provides a composition comprising one or more chimeric peptides. In one embodiment, the chimeric peptide further comprises one epitope of the corresponding HER protein or c-MET protein.

  Further provided are compositions having one or more multivalent peptides. These multivalent peptides contain more than one epitope of the invention.

  Methods of stimulating an immune response using the compositions of the present invention and methods of treating cancer in a subject are included in the present invention. Vaccines are also provided for use in therapy and prevention. The epitopes of the invention can elicit an immune response, either alone or in the context of a chimeric peptide, as described herein. In one embodiment, the immune response is a humoral response. In another embodiment, the immune response is a cell-mediated response. According to some embodiments, the epitope or peptide of the invention confers a protective effect.

In one embodiment, the epitope of HER-3 or other peptide of the invention is
p11-13 (peptides 51-75): KLYERCEVVMGNLEIVLTGHNADLFSFLQW (SEQ ID NO: 1);
p81-83 (peptides 401-425): SWPPHMMHNFSVSNLTTIGGRSLYN (SEQ ID NO: 2);
p84-86 (peptides 416-440): TTIGGRSLYNRGFSLLIMKNLNVTS (SEQ ID NO: 3);
p12 (peptides 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4);
p81 (peptides 401-415): SWPPHMMHNFSVFSNL (SEQ ID NO: 5);
p84 (peptides 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6);
p91 (peptides 451 to 465): AGRIYISANRQLCYH (SEQ ID NO: 7)
including.

  The HER-3 peptide or any peptide of the present invention may be cyclic or linear. If the epitope is circular, it can be circular in any suitable manner. For example, disulfide bonds can be formed between selected pairs of cysteines (Cys) to provide the desired conformation. It is believed that the formation of a cyclic epitope can result in a conformation that improves the humoral response and thus improves the protective effect.

  The HER-3 epitope identified by SEQ ID NO: 4 corresponds to positions 56-70 of the HER-3 protein. The HER-3 epitope identified by SEQ ID NO: 5 corresponds to positions 401-415 of the HER-3 protein. The HER-3 epitope identified by SEQ ID NO: 6 corresponds to positions 416-430 of the HER-3 protein. The HER-3 epitope identified by SEQ ID NO: 7 corresponds to positions 451-465 of the HER-3 protein.

  As described herein, an epitope of HER-3 of the present invention also encompasses peptides that are functional equivalents of the peptide identified by SEQ ID NO. Such functional equivalents have an altered sequence, in which case one or more amino acids in the sequence of the corresponding HER-3 epitope are substituted, or one or more amino acids are It has been deleted from or added to the corresponding reference sequence. For example, 1 to 3 amino acids can be added to the amino terminus, the carboxy terminus, or both. In some examples, the HER-3 epitope is glycosylated.

  In other examples, the HER-3 epitope can be a retro-inverso isomer of the HER-3 epitope. Retro-inverso modification involves the reversal of all amide bonds within the peptide backbone. This reversal can be achieved by reversing the direction of the sequence and reversing the asymmetry of each amino acid residue by using D-amino acids instead of L-amino acids. This retro-inverso isomer form can retain at least some peptide bond planarity and conformational constraints.

  Non-conservative amino acid substitutions and / or conservative substitutions can be made. A substitution is a conservative amino acid substitution if the substituted amino acid has structural or chemical properties similar to the corresponding amino acid in the reference sequence. By way of example, conservative amino acid substitutions include replacing one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine and isoleucine with another such amino acid; one hydroxyl-containing amino acid, such as serine and When replacing threonine with another such amino acid; when replacing one acidic residue such as glutamic acid or aspartic acid with another such residue; replacing one amide-containing residue such as asparagine and glutamine When exchanging with another such residue; when exchanging one aromatic residue such as phenylalanine and tyrosine with another such residue; one basic residue such as lysine, arginine and histidine In exchange for another such residue; and one small amino acid Acid, such as alanine, serine, threonine, methionine and glycine, which may be replaced with another such amino acids.

  In some examples, deletions and additions are located at the amino terminus, carboxy terminus, or both of one sequence of the peptides of the invention. For example, an equivalent of a HER-3 epitope is at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91%, at least 92%, at least with the corresponding HER-3 epitope sequence Have an amino acid sequence that is 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical. Sequences that are at least 90% identical have only one change, ie, any combination of deletions, additions or substitutions, per 10 amino acids of the reference sequence. Percent identity is determined by comparing the variant amino acid sequence to a reference sequence using programs known or developed in the art.

  In the case of a functional equivalent longer than the sequence of the corresponding HER-3 epitope, the functional equivalent is a sequence that is at least 90% identical to the sequence of the HER-3 epitope and the wild-type HER-3 protein It may have a sequence within which is adjacent to the sequence of the HER-3 epitope.

  By altering the sequence of the epitope and then assaying the resulting polypeptide for the ability to stimulate an immune response, eg, the production of antibodies, the functional equivalent of a HER-3 epitope can be identified . Such antibodies can be found in a variety of body fluids, including serum and ascites. Briefly, a body fluid sample is isolated from a warm-blooded animal, such as a human, for which it is desired to determine whether an antibody specific for a HER-3 polypeptide is present. Incubating the body fluid with the HER-3 polypeptide under conditions sufficient for and for a time sufficient to form an immune complex between the polypeptide and the antibody specific for the protein; Preferably, the assay is performed using an ELISA method.

  According to other embodiments of the invention, a chimeric peptide and a composition comprising one or more chimeric peptides are provided. According to various embodiments, the chimeric peptide comprises a HER-3 epitope, another epitope, and a linker that connects the HER-3 epitope to other epitopes. In one embodiment, other epitopes can include, but are not limited to, another HER-3 epitope, HER-1 epitope, HER-2 epitope, and c-MET epitope. It is further understood that any suitable linker may be used. For example, depending on the epitope used, the HER-3 epitope can be linked to either the amino terminus or the carboxy terminus of other epitopes. The location and selection of other epitopes depends on the structural features of the HER-3 epitope, ie whether it is an alpha helix or beta-turn or chain.

  In one embodiment, the linker can be a peptide about 2 to about 15 amino acids, about 2 to about 10 amino acids, or about 2 to about 6 amino acids in length. The chimeric peptide may be linear or cyclic. Furthermore, the HER-3 epitope, other epitopes and / or linkers may be in the form of retro-inverso. Thus, the HER-3 epitope alone may be in retro-inverso form. Alternatively, the HER-3 epitope and other epitopes may be in retro-inverso form. In another example, the HER-3 epitope, other epitopes and linkers may be in the form of retro-inverso.

  In another embodiment, the peptides of the present invention can be combined into a mixture instead of taking the form of a chimeric peptide. In any event, a composition of the invention comprising a peptide can be a useful agent for pulsing antigen presenting cells (eg, dendritic cells) to produce a cellular vaccine. In another embodiment, a composition of the invention comprising a peptide can be an immunogen useful for inducing the production of antibodies. The compositions of the invention can also be used to immunize a subject and delay or prevent tumor development. The compositions of the invention can be used in vaccines to provide a protective effect.

  According to a further embodiment of the invention, a composition comprising a mixture of two or more of the peptides or chimeric peptides of the invention is provided. In some examples, each of the two or more chimeric peptides has a different HER-3 epitope. In other examples, one of the HER-3 epitopes is selected from SEQ ID NOs: 1-7.

  The peptides of the invention, including chimeric peptides, can be prepared using well-known techniques. For example, peptides can be synthesized and prepared using either recombinant DNA technology or chemical synthesis. The peptides of the present invention may be synthesized individually or as longer polypeptides composed of two or more peptides. The peptides of the present invention are preferably isolated, ie, substantially free of other naturally occurring host cell proteins and fragments thereof.

  The peptide and chimeric peptide of the present invention can be synthesized using a commercially available peptide synthesizer. For example, Kaumaya et al., “De Novo” Engineering of Peptide Immunogenic and Antigenic Determinants as Biogenetics as Biogenetics, Biosciences, Peptides, Peptides, Peptides, Peptides, Peptides, Peptides. The chemical methods described in can be used. For example, a HER-3 epitope can be synthesized co-linearly with other epitopes to form a chimeric peptide. Peptide synthesis can be performed using Fmoc / t-But chemistry. Peptides and chimeric peptides can be made circular in any suitable manner. For example, using a method that differentially protects cysteine residues, performs iodine oxidation, adds water to facilitate removal of Acm groups, and forms disulfide bonds simultaneously, and / or a silyl chloride-sulfoxide method. Thus, a disulfide bond can be obtained.

  Peptides and chimeric peptides can also be generated using cell-free translation systems and RNA molecules derived from DNA constructs encoding epitopes or peptides. Instead, the epitope or chimeric peptide is generated by transfecting a host cell with an expression vector containing a DNA sequence encoding the respective epitope or chimeric peptide, and then inducing expression of the polypeptide in the host cell. You can also In the case of production of recombinants, recombinant constructs containing one or more of the sequences encoding epitopes, chimeric peptides or variants thereof can be obtained using conventional methods such as calcium phosphate transfection, DEAE-dextran mediated transfection. , Introduced into host cells by microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, or infection.

  The peptides of the present invention may contain modifications, such as glycosylation, side chain oxidation, or phosphorylation, but only if the modification does not destroy the biological activity of the peptide. . Other modifications include, for example, the incorporation of D-amino acids or other amino acid mimetics that can be used to increase the serum half-life of the peptide.

  The peptides of the invention can be prepared as a combination, the combination comprising two or more peptides of the invention for use as a vaccine for a disease, eg, cancer. The peptides may be in cocktails or conjugated to each other using standard techniques. For example, a peptide can be expressed as a single polypeptide sequence. The peptides in the combination may be the same or different.

  The present invention should also be construed to include “mutants”, “derivatives” and “variants” of the peptides of the present invention (or the DNA encoding said peptides). A variant is a peptide in which one or more amino acids are changed (or one or more base pairs are changed when referring to a nucleotide sequence encoding said amino acid) and thus obtained The resulting peptide (or DNA) is not identical to the sequences listed herein, but has the same biological properties as the peptides disclosed herein.

  The present invention also provides a polynucleotide encoding at least one peptide selected from peptides having any one or more of SEQ ID NOs: 1-7. Nucleic acid sequences include both DNA sequences that are transcribed into RNA and RNA sequences that are translated into peptides. According to another embodiment, the polynucleotide of the present invention is deduced from the amino acid sequence of the peptide of the present invention. As is known in the art, several alternative polynucleotides are possible due to overlapping codons, but these retain the biological activity of the translated peptide.

  In addition, the invention also encompasses isolated nucleic acids that encode peptides having substantial homology to the peptides disclosed herein. Preferably, the nucleotide sequence of the isolated nucleic acid encoding the peptide of the present invention is “substantially homologous”, ie about 60% homologous to the nucleotide sequence of the isolated nucleic acid encoding the peptide of the present invention, more preferably , About 70% homology, even more preferably about 80% homology, more preferably about 90% homology, even more preferably about 95% homology, even more preferably about 99% homology.

  The scope of the present invention includes homologues, analogs, variants, derivatives and salts, including shorter and longer peptides and polynucleotides; and analogs of peptides and polynucleotides having one or more amino acid or nucleic acid substitutions As well as amino acids or nucleic acid derivatives known in the art, non-natural amino acids or nucleic acids and synthetic amino acids or nucleic acids, provided that these modifications preserve the biological activity of the original molecule. I want you to clearly understand what must be done. Specifically, in accordance with the principles of the present invention, any active fragments of active peptides, as well as extensions, conjugates and mixtures are disclosed.

  The present invention includes any isolated nucleic acid that is homologous to the nucleic acid described or referred to herein, provided that these homologous DNAs are the biology of the peptides disclosed herein. Should be construed as having active activity.

  The nucleic acid of the present invention includes a sequence of RNA or DNA encoding the peptide of the present invention, which makes the nucleotide sequence more stable, whether with or without cells. Or, those skilled in the art will appreciate that any modified form thereof, including chemical modifications of RNA, is also encompassed. Nucleotide chemical modifications can also be used to enhance the efficiency with which a cell incorporates a nucleotide sequence or the expression of a nucleotide in a cell. The present invention contemplates any combination of nucleotide sequence modifications.

  Furthermore, mutants, derivatives of the proteins of the invention using methods of recombinant DNA well known in the art, such as those described in Sambrook and Russell, supra, and Ausubel et al., Supra. Alternatively, any number of procedures can be used to generate variant forms. Procedures for introducing amino acid changes into a peptide or polypeptide by altering the DNA sequence encoding the polypeptide are well known in the art and are also described in these and other articles. Yes.

  Nucleic acids encoding the peptides of the invention can be incorporated into suitable vectors, eg, retroviral vectors. These vectors are well known in the art. Nucleic acids, or vectors usefully containing nucleic acids, can be transferred into the desired cells, and such cells are preferably obtained from the patient. Advantageously, the present invention provides an off-the-shelf composition that allows rapid modification of a patient's own cells (or another mammal's own cells). Thus, it becomes possible to quickly and easily generate modified cells having excellent cancer cell killing properties.

In vectors and other related aspects, the invention includes an isolated nucleic acid encoding one or more of the peptides having a sequence selected from the group consisting of SEQ ID NOs: 1-7.

  In one embodiment, the present invention comprises a nucleic acid sequence encoding one or more peptides of the present invention, which sequence is operably linked to a nucleic acid comprising a promoter / regulatory sequence, and thus the latter The nucleic acid is preferably capable of directing the expression of the protein encoded by the former nucleic acid. Accordingly, the present invention provides an expression vector and method for introducing exogenous DNA into a cell and simultaneously expressing the exogenous DNA in the cell, such as Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold. Spring Harbor Laboratory, New York), and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York). Integration of the desired polynucleotide into the vector and selection of the vector is well known in the art, as described, for example, in Sambrook et al., Supra, and Ausubel et al., Supra.

  Polynucleotides can also be cloned into several types of vectors. However, this invention should not be construed as limited to any particular vector. Instead, the present invention should be construed to encompass a very wide range of vectors that are readily available and / or well known in the art. For example, the polynucleotides of the invention can be cloned into vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

  In certain embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector, and a mammalian cell vector. There are numerous expression vector systems that include at least some or all of the compositions discussed above. Systems based on prokaryotic and / or eukaryotic vectors can be utilized and used with the present invention to produce polynucleotides or their cognate polypeptides. Many such systems are commercially available and widely available.

  Furthermore, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012) and Ausubel et al. (1997) and other virology and molecular biology manuals. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, eg, WO 01/96584; WO 01/29058; and US Pat. No. 6,326,193.

  In order to express the desired nucleotide sequence of the present invention, at least one module in each promoter functions to position the start site for RNA synthesis. The best known example is the TATA box, but in some promoters lacking the TATA box, for example, the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 gene, a separate overlay that overlaps the start site. The elements themselves help to fix the starting location.

  Additional promoter elements, ie enhancers, regulate the frequency of transcription initiation. Typically these are located in the region 30-110 bp upstream from the start site, but recently several promoters have been shown to contain functional elements also downstream of the start site. ing. The spacing between promoter elements is often flexible, so that the function of the promoter is preserved even when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can work together or function independently to activate transcription.

  A promoter can be a promoter naturally associated with a gene or polynucleotide sequence, which can be obtained by isolating a 5 'non-coding sequence located upstream of a coding segment and / or exon. Such promoters can be referred to as “endogenous”. Similarly, an enhancer can also be an enhancer naturally associated with a polynucleotide sequence, which is located either downstream or upstream of the sequence. Instead, placing a polynucleotide coding segment under the control of a recombinant or heterologous promoter provides certain advantages, where the promoter is not normally associated with the polynucleotide sequence in its natural environment. Point to. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers include promoters or enhancers of other genes, as well as any other prokaryotic, viral or eukaryotic cell, and promoters or enhancers that are not “naturally occurring” That is, it can include promoters or enhancers that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthetically generating promoter and enhancer nucleic acid sequences, recombinant cloning and / or nucleic acid amplification techniques can also be used to generate sequences, which can be incorporated into the compositions disclosed herein. Includes relevant PCR ™ (US Pat. No. 4,683,202, US Pat. No. 5,928,906). It is further contemplated that regulatory sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, etc., can be utilized as well.

  Of course, it is important to utilize a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle and organism chosen for expression. Those skilled in the art of molecular biology generally know how to use a combination of promoters, enhancers and cell types for the expression of proteins, see, for example, Sambrook et al. (2012). The promoter utilized may be a constitutive promoter, a tissue-specific promoter, an inducible promoter, and / or a condition suitable to direct high level expression of the DNA segment to be introduced, such as a recombinant protein and / or peptide large It may be a useful promoter under conditions that favor the production on a scale. The promoter may be heterologous or endogenous.

  The promoter sequences exemplified in the experimental examples presented herein are those of the cytomegalovirus (CMV) earliest promoter. This promoter sequence is a strong constitutive promoter sequence and can drive high level expression of any polynucleotide sequence operably linked thereto. However, other constitutive promoter sequences can also be used, including but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) Long terminal repeat (LTR) promoter, Moloney virus promoter, avian leukemia virus promoter, Epstein-Barr virus earliest promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter , Hemoglobin promoter and muscle creatine promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. In the present invention, by using an inducible promoter, the expression of the polynucleotide sequence to which the inducible promoter is operably linked is turned on if such expression is desired, or if expression is not desired. Provides a molecular switch that can turn off expression. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. Furthermore, the present invention also includes the use of tissue specific promoters, which are only active in the desired tissue.

  In order to evaluate the expression of a nucleotide sequence encoding the peptide of the present invention, the expression vector to be introduced into the cell also contains either or both of a selectable marker gene and a reporter gene, and is transfected with a viral vector. Identification and selection of cells that are expressed from a population of cells targeted for infection or infection can also be facilitated. In other embodiments, the selectable marker can be mounted on a separate piece of DNA and used in a cotransfection procedure. Both the selectable marker gene and the reporter gene can be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers are known in the art and include, for example, antibiotic resistance genes such as neo and the like.

  Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Reporter genes that encode proteins that are readily assayable are well known in the art. In general, a reporter gene is present in some form of easily detectable property, such as enzymatic activity, without being present in the recipient organism or tissue or expressed by the recipient organism or tissue. It is a gene that encodes a protein. Reporter gene expression is assayed at an appropriate time after DNA is introduced into the recipient cell.

  Suitable reporter genes can include luciferase, beta-galactosidase, chloramphenicol acetyltransferase, genes encoding secreted alkaline phosphatases, or green fluorescent protein genes (eg, Ui-Tei et al., 2000, FEBS). Lett. 479: 79-82). Suitable expression systems are well known and may be prepared using well known techniques or obtained commercially. Constructs with internal deletions can be generated using unique internal restriction sites or by partial digestion of non-unique restriction sites. The construct can then be transfected into cells that exhibit high levels of expression of siRNA polynucleotides and / or polypeptides. In general, constructs with minimal 5 'flanking regions that show the highest level of expression of the reporter gene are identified as promoters. Such a promoter region can be linked to a reporter gene and used to evaluate a drug for the ability to modulate promoter driven transcription.

In one embodiment of the vaccine , the subject of the invention is a vaccine comprising a peptide of the invention. The vaccines of the present invention can provide any combination of specific peptides for the specific prevention or treatment of cancer in a subject in need of treatment.

  The vaccines of the invention are capable of inducing an antigen-specific T cell response and / or a high titer antibody response, thereby being directed against or reactive against an antigen-expressing cancer or tumor An immune response is induced or elicited. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or both cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α).

  In one embodiment, the subject of the present invention is an anti-cancer vaccine. A vaccine can include one or more cancer antigens. Vaccines can prevent tumor growth. Vaccines can reduce tumor growth. The vaccine can block tumor cell metastasis. Depending on the cancer antigen, targeting vaccine, breast cancer, liver cancer, prostate cancer, melanoma, blood cancer, head and neck cancer, glioblastoma, recurrent respiratory papilloma, anal cancer, cervical cancer, brain tumor, etc. Can be treated.

In certain embodiments, the vaccine comprises (1) humoral immunity via a B cell response that produces the desired antibody; (2) cytotoxic T lymphocytes that attack and kill tumor cells, such as CD8 ⁺ ( (3) increased T helper cell response; and (4) increased inflammatory response via IFN-γ and TFN-α, or preferably by inducing all of the above, Can mediate growth cleanup or inhibition. Vaccines have tumor-free survival of 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, It can be increased by 44% and 45%. Vaccines have a tumor volume of 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42% after immunization. 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59 % And 60% can be reduced.

  The vaccine has a cellular immune response in a subject who has not received the vaccine of about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold. Times to about 5000 times, about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times, or about 300 times It can be increased by about 6000 times. In some embodiments, a vaccine has a cellular immune response in a subject that has been administered a vaccine that is about 50-fold, 100-fold, 150-fold, 200-fold, compared to a cellular immune response in a subject that has not been administered the vaccine. Times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times 2800 times 2900 times 3000 times 3100 times 3200 times 3300 times 3400 times 3500 times 3600 times 370 Times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, It can be increased by 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

  Vaccines have about 50 times to about 6000 times, about 50 times the level of IFN-γ in subjects who have received the vaccine, compared to the level of interferon gamma (IFN-γ) in subjects who have not received the vaccine. To about 5500 times, about 50 times to about 5000 times, about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about It can be increased 6000 times, or about 300 times to about 6000 times. In some embodiments, the vaccine has a level of IFN-γ in a subject that has been administered the vaccine that is about 50-fold, 100-fold greater than the level of IFN-γ in the subject that has not been administered the vaccine. 150x, 200x, 250x, 300x, 350x, 400x, 450x, 500x, 550x, 600x, 650x, 700x, 750x, 800x, 850x, 900x, 950x 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times 2700 times 2800 times 2900 times 3000 times 3100 times 3200 times 3300 times 3400 times 3500 times 3600 times 3700 times Times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, It can be increased by 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

  The vaccines of the present invention have the characteristics necessary for an effective vaccine, for example, the vaccines of the present invention are safe and thus the vaccine itself does not cause illness or death; provides protection against illness; neutralizing antibodies Induces a protective T cell response; is easy to administer, has few side effects, is biologically stable, and has a low cost per dose. A vaccine can achieve some or all of these characteristics by containing cancer antigens as discussed below.

Generation of Loaded (Pulsed) Immune Cells The invention includes cells that have been exposed to or otherwise “pulsed” with an antigen of the invention or other peptide of the invention. For example, APC, such as DC, can be loaded with Ag in vitro, for example, by culturing ex vivo in the presence of the antigen or by exposing to the antigen in vivo.

  Also, APC can be “pulsed” into APC in a manner that exposes the antigen to the APC, and this exposure can occur for a time sufficient to facilitate presenting the antigen on the surface of the APC, Those skilled in the art will readily understand. For example, APCs can be exposed to antigens in the form of small peptide fragments, known as antigenic peptides, which are “pulsed” directly onto the exterior of APC (Mehta-Damani et al. 1994); or APC can be incubated with whole protein or protein particles, which are then ingested by APC. All these proteins are digested by APC into small peptide fragments that are finally transported to the APC surface and presented on the APC surface (Cohen et al., 1994). Antigens in the form of peptides can be exposed to cells by the standard “pulsed” technique described herein.

  While not intending to be bound by any particular theory, foreign antigens or antigens in the form of autoantigens are processed by the APCs of the present invention to retain the immunogenic form of the antigen. An immunogenic form of an antigen means that the antigen is processed by fragmentation to produce a form of the antigen that can be recognized and activated by immune cells, eg, T cells. Preferably, such foreign antigens or autoantigens are proteins that are processed into peptides by APC. Related peptides produced by APC can be extracted, purified and used as an immunogenic composition. APC processed peptides can also be used to induce tolerance to APC processed proteins.

  Antigen-loaded APC, according to another expression, is known as “pulsed APC” of the present invention, which is generated by exposing APC to an antigen either in vitro or in vivo. The When pulsing APCs in vitro, APCs can be plated on culture dishes and exposed to the antigen for a sufficient amount of time to allow the antigen to bind to the APC. . The amount and time required to achieve binding of the antigen to APC can be determined by using methods known in the art or other methods disclosed herein. Other methods known to those skilled in the art, such as immunoassays or binding assays, can be used to detect the presence of the antigen on the APC after exposure to the antigen.

  In a further embodiment of the invention, a vector that allows expression of a specific protein by APC can be transfected into APC. The protein expressed by APC can then be processed and presented on the cell surface. The transfected APC can then be used as an immunogenic composition to generate an immune response against the vector encoded protein.

  As discussed elsewhere herein, vectors can be prepared to include specific polynucleotides that encode and express proteins for which an immunogenic response is desired. Preferably, retroviral vectors are used to infect cells. More preferably, an adenoviral vector is used to infect cells.

  In another embodiment, the viral vector can be directed to the target APC by modifying the viral vector to encode a protein or portion thereof recognized by a receptor on the APC, whereby the vector is Occupying the APC receptor initiates vector endocytosis, allowing the antigen encoded by the viral vector nucleic acid to be processed and presented. The nucleic acid delivered by the virus can be native to the virus, and this nucleic acid encodes the viral protein, even if expressed on APC, which is then processed and presented on the MHC receptor of APC Is done.

  As contemplated herein, a variety of methods can be used to transfect a polynucleotide into a host cell. These methods include, but are not limited to, calcium phosphate precipitation, lipofection, particle gun, microinjection, electroporation, colloidal dispersion systems (ie, macromolecular complexes, nanocapsules, microspheres, beads, and oil-in-water types) Lipid based systems including emulsions, micelles, mixed micelles and liposomes). These methods are understood in the art and are described in the published literature and thus allow those skilled in the art to perform these methods.

  In another embodiment, the polynucleotide encoding the antigen can be cloned into an expression vector and introduced into the APC by another method to produce a loaded APC. become. Various types of vectors and methods for introducing nucleic acids into cells are discussed in the available published literature. For example, the expression vector can be transferred into a host cell by physical, chemical or biological means. For example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and Ausubel et al. (1997, Current Protocols in Mol. It is easily understood that a pulsed cell can be obtained by introducing an expression vector containing a polynucleotide encoding an antigen.

  The present invention includes various methods for pulsing APCs, including, but not limited to, loading APCs with the entire antigen in the form of protein, cDNA or mRNA. However, the present invention should not be construed as limited to the particular form of antigen used to pulse the APC. Rather, the present invention encompasses other methods known in the art for generating APC loaded with antigen. Preferably, APCs are transfected with mRNA encoding a defined antigen. Using appropriate primers and reverse transcriptase-polymerase chain reaction (RT-PCR) coupled to transcription reactions, mRNAs corresponding to gene products of known sequence can be rapidly generated in vitro. it can. When generating pulsed APCs, transfection with APC mRNA offers advantages over other techniques for loading antigen. For example, the ability to amplify RNA from minute amounts of tissue, ie tumor tissue, leads to vaccination of a large number of patients using APC.

  In order for an antigen composition to be useful as a vaccine, the antigen composition must induce an immune response against the antigen in a cell, tissue or mammal (eg, a human). As used herein, an “immunological composition” expresses or presents an antigen (eg, a peptide or polypeptide), a nucleic acid encoding the antigen (eg, an antigen expression vector), or an antigen or cellular component. Cells can be included. In certain embodiments, the antigen composition comprises or encodes all or a portion of any antigen described herein, or an immunological functional equivalent thereof. In other embodiments, the antigen composition comprises a mixture comprising additional immunostimulatory agents or nucleic acids encoding such agents. Immunostimulatory agents include, but are not limited to, additional antigens, immunomodulators, antigen presenting cells or adjuvants. In other embodiments, one or more of the additional agents are covalently bound in any combination to the antigen or immunostimulatory agent. In certain embodiments, the antigen composition is conjugated to or comprises amino acids of the HLA anchor motif.

  A vaccine may vary in its composition of nucleic acids and / or cellular components, as contemplated herein. In a non-limiting example, the nucleic acid encoding the antigen could also be formulated with an adjuvant. Of course, it will be understood that the various compositions described herein may further comprise additional components. For example, one or more vaccine components can be included in a lipid or liposome. In another non-limiting example, the vaccine can include one or more adjuvants. The vaccine of the present invention and its various components can be prepared and / or administered by any method disclosed herein, or will be understood by one of ordinary skill in the art in light of the present disclosure. Will.

  It is understood that the antigen compositions of the present invention can be made by methods well known in the art, including, but not limited to, chemical synthesis by solid phase synthesis and chemical reaction by HPLC. A method of purifying and extracting from other products, or expressing a nucleic acid sequence (eg, DNA sequence) encoding a peptide or polypeptide containing the antigen of the present invention in an in vitro translation system or in a living cell There is a method to generate by. In addition, the antigen composition can include cellular components isolated from a biological sample. The antigen composition is isolated and dialyzed sufficiently to remove one or more undesirable low molecular weight molecules and / or lyophilized in the desired vehicle to enhance the ready-to-use formulation. . It should be further understood that any additional amino acids, mutations, chemical modifications, etc. that result in the vaccine component preferably do not substantially prevent the antibody from recognizing the epitope sequence.

Antigen-presenting cell therapy The present invention encompasses methods of generating a population of APCs (eg, dendritic cells; DC) that present a peptide of the present invention on a surface and subsequently using the population in therapy. Such methods can be performed ex vivo on a sample of cells obtained from a patient. Thus, the APC thus produced forms a pharmaceutical agent that can be used in the treatment or prevention of cancer. These cells should be accepted by the individual's immune system because such cells are derived from that individual. The cells thus generated are delivered to the individual from whom the cells were originally obtained, thus forming a therapeutic embodiment of the present invention.

DCs are derived from pluripotent mononuclear cells that act as antigen presenting cells (APCs). DCs are ubiquitous in peripheral tissues where they are waiting to capture antigen. When the DC captures the antigen, it processes the antigen into small peptides and moves towards the secondary lymphoid organs. It is within the lymphoid organ that the DC presents the antigenic peptide to naive T cells, and the presentation of the peptide initiates a signal cascade that biases T cell differentiation. When exposed, DCs present antigen molecules bound to MHC class I or class II binding peptides and activate CD8 ⁺ T cells or CD4 ⁺ T cells, respectively (Steinman, 1991, Annu. Rev. Immunol.9: 271-296; Banchereau et al., 1998, Nature 392, 245-252; Steinman et al., 2007, Nature 449: 419-426; Ginhouux et al., 2007, J. Exp. 204: 3133-3146; Banerjee et al., 2006, Blood 108: 2655-2661; Sallusto et al., 1999, J. Exp. Med. 189: 611-614; Reid et al., 2000, Curr. Opin. Im munol.12: 114-121; Bykovskaia et al., 1999, J. Leukoc.Biol.66: 659-666; Clark et al., 2000, Microbes Infect. 2: 257-272).

  DCs are involved in the induction, coordination and regulation of adaptive immune responses and also serve to integrate communication between the natural and adaptive arms of the immune system's effectors. These features make DC a strong candidate for immunotherapy. DC has the unique ability to identify the environment by macropinocytosis and receptor-mediated endocytosis (Gerner et al., 2008, J. Immunol. 181: 155-164; Stoitzner et al., 2008, Cancer Immunol). Immunother 57: 1665-1673; Lanzevecchia A., 1996, Curr.Opin.Immunol.8: 348-354; Delamarre et al., 2005, Science, 307 (5715): 1630-1634).

  DC also requires a maturation signal to enhance its antigen presentation ability. By providing additional maturation signals such as TNF-α, CD40L or calcium signaling substances, DCs can be surface molecules such as CD80 and CD86 (also known as second signal molecules). Expression is upregulated (Czerniecki et al., 1997, J. Immunol. 159: 3823-3837; Bedrosian et al., 2000, J. Immunother. 23: 311-320; Maillard et al., 2004, Cancer Res. 64. 5934-5937; Brossart et al., 1998, Blood 92: 4238-4247; Jin et al., 2004, Hum. Immunol. 65: 93-103). It has been established that a mixture of cytokines including TNF-α, IL-1β, IL-6 and prostaglandin E2 (PGE2) has the ability to mature DC (Jonuleit et al., 2000, Arch. Derm. Res. 292: 325-332). DCs can also be matured with calcium ionophores prior to pulsing the antigen.

  DC are receptors that recognize pathogens, such as PKR and MDA-5 (Kalali et al., 2008, J. Immunol. 181: 2694-2704; Nallagatla et al., 2008, RNA Biol. 5 (3): 140-144) and also contains a series of receptors known as Toll-like receptors (TLRs), which are also capable of sensing risks from pathogens. . When these TLRs are stimulated, a series of activity changes are induced in DC, which results in maturation and T cell signaling (Boullart et al., 2008, Cancer Immunol. Immunother. 57 (11): 1589). Kaisho et al., 2003, Curr.Mol.Med.3 (4): 373-385; Pulendran et al., 2001, Science 293 (5528): 253-256; Napolitani et al., 2005, Nat. Immunol.6 (8): 769-776). DC can activate and prolong the action of various arms of cell-mediated responses, such as natural killer γ-δ T cells and α-β T cells, and once DCs are activated, Retain their immunization capacity (Steinman, 1991, Annu. Rev. Immunol. 9: 271-296; Banchereau et al., 1998, Nature 392: 245-252; Reid et al., 2000, Curr. Opin. Immunol.12: 114-121; Bykovskaya et al., 1999, J. Leukoc.Biol.66: 659-666; Clark et al., 2000, Microbes Infect.2: 257-272).

  The present invention also provides methods for inducing antigen presenting cells (APCs) using one or more of the peptides of the present invention. Inducing APC by inducing dendritic cells from peripheral blood mononuclear cells in vitro, ex vivo or in vivo and then contacting them with one or more peptides of the invention (activation) be able to. When the peptides of the present invention are administered to a mammal in need thereof, APCs to which the peptides of the present invention are immobilized are induced in the mammal's body. Alternatively, the peptides of the present invention can be immobilized on APC and then the cells can be administered to the subject as a vaccine. For example, ex vivo administration can include collecting APC from a mammal and contacting the APC with a peptide of the invention.

  The present invention also provides an APC that presents a complex formed between an HLA antigen and one or more peptides of the present invention. APC is obtained by contact with a peptide of the invention or a nucleotide encoding such a peptide, but is preferably derived from a subject to be treated and / or prevented, as a vaccine alone or as a peptide. Including other drugs, exosomes or the T cells of the present invention can be administered.

  The present invention provides compositions and methods for stimulating APC, preferably DC, in the context of immunotherapy for stimulating an immune response in a mammal. As DC activates anti-tumor immunity in a mammal in need thereof, the DC is activated using a peptide of the present invention or a combination of peptides of the present invention to cause DC maturation, thereby causing DC maturation. Can be operated.

  In one embodiment, the present invention includes a method for inducing a T cell response in a mammal. This method comprises the step of administering APC, for example DC, wherein APC has been activated by contacting APC with a peptide of the invention or a combination of peptides of the invention, whereby APC loaded with peptide.

  In one embodiment, the present invention has, among other things, for the purpose of expanding and expanding desired T cells, for the purpose of activating T cells, for the purpose of expanding and expanding specific T cells, and for those purposes. It relates to methods for using APC, and also to a number of therapeutic uses related to T cell expansion and activation using peptide loaded APCs and peptides of the invention. In some cases, DCs activated by OCT4 can be used to expand and propagate T cells specific for the peptide.

  The present invention relates to the discovery that DC contacted with a peptide of the present invention or a combination of peptides of the present invention can be used to induce expansion of T cells specific for the peptide. One skilled in the art will recognize that a DC contacted with a peptide of the invention is considered primed or, according to another expression, loaded with a peptide. The peptide-loaded DCs of the present invention are useful for eliciting an immune response against a desired antigen, such as HER-3. Thus, peptide-loaded DCs of the present invention can be used to treat diseases associated with disordered expression of HER-3.

Methods for Treating Diseases The present invention also encompasses methods for treating and / or preventing diseases caused by pathogenic microorganisms, autoimmune disorders and / or hyperproliferative diseases.

  Diseases that can be treated or prevented by using the present invention include diseases caused by viruses, bacteria, yeast, parasites, protozoa, cancer cells and the like. The pharmaceutical composition of the present invention can be used as a general immune enhancing agent (composition or system that activates DC) and as such has utility in the treatment of disease. Exemplary diseases that can be treated and / or prevented using the pharmaceutical compositions of the present invention include, but are not limited to, infections caused by viruses such as HIV, influenza, herpes, viral hepatitis. Epstein Bar, polio, viral encephalitis, measles, blisters, papillomavirus, etc .; or infections caused by bacteria, eg pneumonia, tuberculosis, syphilis, etc .; or infections caused by parasites, eg malaria, Trypanosomiasis, leishmaniasis, trichomoniasis, amoebiasis and the like.

  Pre-tumor or hyperplastic conditions that can be treated or prevented using the pharmaceutical compositions of the invention (transduced DCs, expression vectors, expression constructs, etc. of the invention) include, but are not limited to, colon Pre-tumor or hyperplastic conditions such as polyps, Crohn's disease, ulcerative colitis, breast lesions and others.

  Cancers that can be treated using the compositions of the present invention include, but are not limited to, primary or metastatic melanoma, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, thymoma, lymphoma, Sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, gastrointestinal cancer, brain tumor, bladder cancer And cervical cancer.

  Other hyperproliferative diseases that can be treated using the DC activation system of the present invention include, but are not limited to, rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyoma, adenoma, lipoma, blood vessel Tumor, fibroma, vascular occlusion, restenosis, atherosclerosis, preneoplastic lesions (eg, adenomatous hyperplasia and prostate neoplasia), epithelial carcinoma, oral hairy leukoplakia or psoriasis Can be mentioned.

  Autoimmune disorders that can be treated using the compositions of the present invention include, but are not limited to, AIDS, Addison's disease, adult respiratory distress syndrome, allergy, anemia, asthma, atherosclerosis, bronchitis, cholecystitis , Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes, emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritable bowel syndrome, Lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome and autoimmune thyroiditis; cancer, hemodialysis And complications of extracorporeal circulation; infection by viruses, bacteria, fungi, parasites, protozoa and protozoa; and trauma.

  In the method of treatment, administration of the composition of the invention may be either for “prevention” or for “treatment”. The compositions of the invention, when provided for prevention, are provided in advance of any symptom, but in certain embodiments, the vaccine is provided after one or more symptoms have developed. To prevent further symptoms from developing or to prevent worsening of existing symptoms. Prophylactic administration of the composition serves to prevent or ameliorate any subsequent infection or disease. When provided for treatment, the pharmaceutical composition is provided at or after the onset of symptoms of infection or disease. Thus, the present invention can be provided before exposure to a disease-causing substance is expected or before a disease state occurs or after an infection or disease has begun.

  An effective amount of the composition is that amount that achieves these selected results that enhances the immune response, and such amount can be determined routinely by one of ordinary skill in the art. For example, an effective amount to treat a failure of the immune system against a cancer or pathogen would be that amount necessary to cause activation of the immune system upon exposure to the antigen and generate an antigen-specific immune response. . The term is also synonymous with “sufficient amount”.

  The effective amount for any particular application can vary depending on factors such as the disease or condition being treated, the particular composition being administered, the size of the subject, and / or the severity of the disease or condition. . One skilled in the art can empirically determine the effective amount of a particular composition of the present invention without necessitating undue experimentation.

Vaccine Formulation The present invention further includes a vaccine formulation suitable for use in immunotherapy. In certain embodiments, the vaccine formulation is used to prevent and / or treat diseases such as cancer and infectious diseases. In one embodiment, the administration of a vaccine according to the invention for preventing and / or treating cancer to a patient is performed before or after a surgical procedure for removing the cancer, of chemotherapy for treating the cancer. It can be performed before or after the procedure, and before or after radiation therapy to treat cancer, and any combination thereof. In other embodiments, the vaccine formulation can be administered to the patient simultaneously or in combination with another composition or pharmaceutical product. It should be understood that the present invention can also be used to prevent cancer in individuals who do not have cancer but may be at risk of developing cancer.

  Administration of cancer vaccines prepared in accordance with the present invention is broadly applicable to cancer prevention or treatment that is determined in part by the selection of the portion of the cancer vaccine that forms the antigen. Cancers that can be appropriately treated according to the practice of the present invention include, but are not limited to, lung, breast, ovary, cervix, colon, head and neck, pancreas, prostate, stomach, bladder, kidney, bone , Cancer of the liver, esophagus, brain, testis, uterus, and various leukemias and lymphomas.

  In one embodiment, the vaccine according to the invention may be derived from the tumor or cancer cell to be treated. For example, in the treatment of lung cancer, lung cancer cells will be treated as described hereinabove to produce a lung cancer vaccine. Similarly, breast cancer vaccines, colon cancer vaccines, pancreatic cancer vaccines, gastric cancer vaccines, bladder cancer vaccines, kidney cancer vaccines and the like are also produced as immunotherapeutic agents to prevent and / or prevent tumors or cancer cells from which vaccines were generated. Utilized according to the execution procedure to treat.

  In another embodiment, the vaccine according to the invention is also collected, as described, by collecting the appropriate antigens that the pathogen excreted in the medium to treat various infectious diseases affecting mammals. It could also be prepared. Because the types of immunogenic and protective antigens expressed by different organisms causing the same disease are heterogeneous, multivalent vaccines can be prepared by preparing vaccines from a pool of organisms that express different important antigens. Can be prepared.

  In another embodiment of the invention, the vaccine can be administered by intranodal injection into the inguinal lymph node. Alternatively, and depending on the target of the vaccine, the vaccine can be administered intradermally or subcutaneously to the limbs, arms and legs of the patient being treated. This approach is generally satisfactory for melanoma as well as for other cancers, but other routes of administration, including the prevention or treatment of infectious diseases, such as intramuscular or intravascular routes, are also possible. Can also be used.

  In addition, vaccines can be administered with adjuvants and / or immunomodulators to boost vaccine activity and patient response. Such adjuvants and / or immunomodulators are understood by those skilled in the art and are readily described in the available published literature.

  As contemplated herein, depending on the type of vaccine being produced, the cells are optionally cultured in a bioreactor or fermentor or other such vessel or device suitable for growing cells in large quantities. By doing so, vaccine production can be scaled up. In such devices, the culture medium is collected periodically, frequently or continuously in order to recover any material or antigen from the culture medium before such material or antigen is degraded in the culture medium. Will be done.

  A device or composition containing a vaccine or antigen that is produced, recovered and suitable for sustained or intermittent release according to the present invention is actually implanted in the body as desired, or An external application to the body would allow such materials to be released into the body relatively slowly or at a defined time.

  Another step in the preparation of a vaccine may be personalization to meet specific vaccine requirements. Those skilled in the art will appreciate such additional steps. For example, certain collected antigenic material can be concentrated and optionally treated with a detergent and ultracentrifuged to remove transplanted alloantigen.

HER3 expression as a biomarker for disease diagnosis and treatment In another embodiment, HER3 expression may serve as a biomarker for potentially invasive disease in patients with Barrett's esophagus and high grade dysplasia (HGD) . In addition, therapeutic agents for targeting HER3 or CMET that can provide secondary prevention of gastroesophageal cancer in some patients are contemplated herein.

  These methods described herein are in no way exhaustive, and additional methods suitable for particular applications will be apparent to those skilled in the art. Furthermore, the effective amount of the composition can be estimated more accurately by analogy with compounds known to exert the desired effect.

<Experimental Example>
The invention will be further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to limit the invention unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed as including all modifications that will become apparent as a result of the teaching provided herein. Should.

  Without further explanation, one of ordinary skill in the art will be able to use the foregoing description and the following illustrative examples to make, use, and perform the claimed methods of the present invention. Conceivable. Accordingly, the following examples specifically point out preferred embodiments of the present invention and should not be construed as limiting the remainder of the disclosure in any way.

Creation of peptide vaccines against other receptor tyrosine kinases that cause breast cancer and other solid tumors . Experiments were designed to develop alternative therapies for patients designated as carriers of BRCA mutations. That is, it seeks an alternative to bilateral mastectomy and does not meet the need for younger patients at the genetic risk of breast cancer.

Women with the breast cancer gene mutation BRCA1 / BRCA2 have a 70% lifetime risk of developing breast cancer, and carriers of the BRCA1 mutation often develop triple-negative breast cancer. Experiments were designed to develop vaccines for this group and to assess their safety in immunity induction trials; this is the first ever vaccination for primary prevention of breast cancer Is an attempt. It will also be observed whether estrogen receptor- ^positive breast cancer, including carriers of BRCA2 mutations, can be prevented using the multivalent vaccine of the present invention.

  Experiments were designed to study receptor tyrosine kinase expression in breast cancer and DCIS from carriers of BRCA mutations. Tumors from carriers of BRCA mutations frequently overexpress the c-MET oncogene and HER-3 early, whereas tumors from non-mutated or sporadic patients are HER-2 and HER- 3 was observed to express. This is important; the targets presented for tumor immunotherapy that can be used to develop vaccines for carriers of sporadic and BRCA mutations are presented herein. This is because it became known now. This is the first distinguishing feature and can be targeted using the immune response for prevention. Accordingly, the present invention includes compositions and methods for developing vaccines and their use for prevention as an alternative to bilateral mastectomy.

Creation of peptide vaccines The HER family consists of four related signaling molecules, HER-1, HER-2, HER-3 and HER-4, which are involved in a variety of cancers. It is known that HER-2 overexpression is found in 20-30% of breast cancers. The results presented herein indicate that other HER family members are involved in both early and invasive breast cancer, as well as other cancers. For example, HER-1 is expressed on a small number of breast cancers, and those cancers are generally triple negative. c-MET is a growth factor receptor involved in the recurrence of many cancers, and this receptor activates HER-3. HER-3 is overexpressed in the colon, prostate, breast and melanoma. HER-3 is expressed in many DCIS lesions and breast cancer. In some patients receiving HER-2 vaccine, HER-3 may be detected in the remaining DCIS at the time of surgery. As a result of these findings, the possibility of targeting these molecules in addition to HER-2 in breast cancer would be beneficial.

Immunogenic peptides derived from HER-3 have been identified (FIGS. 1 and 2) and are shown below:
p11-13 (peptides 51-75): KLYERCEVVMGNLEIVLTGHNADLFSFLQW (SEQ ID NO: 1);
p81-83 (peptides 401-425): SWPPHMMHNFSVSNLTTIGGRSLYN (SEQ ID NO: 2);
p84-86 (peptides 416-440): TTIGGRSLYNRGFSLLIMKNLNVTS (SEQ ID NO: 3);
p12 (peptides 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4);
p81 (peptides 401-415): SWPPHMMHNFSVFSNL (SEQ ID NO: 5);
p84 (peptides 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6); and p91 (peptides 451-465): AGRIYISANRRQLCYH (SEQ ID NO: 7).

The results presented herein indicate that these peptides can activate CD4 T cells across many patients. Peptides can be pulsed into dendritic cells and T cells can be trained to recognize HER-3. HER-3 is expressed in triple-negative breast cancer and may result in resistance to antiestrogens in ER ^-positive breast cancer. HER-3 is also expressed in other cancers, including melanoma, lung cancer, colon cancer, prostate cancer and metastatic brain tumor. While not intending to be bound by any particular theory, peptides derived from the intracellular portion of the molecule may also be advantageous.

  Based on the disclosure presented herein, immunogenic peptides for the receptor tyrosine kinase molecules HER-1 and c-MET are based on the procedure that identified the immunogenic peptides for HER-3, Can be screened and identified. The immunogenic peptides of the present invention can be used to prepare multivalent prophylactic vaccines for breast cancer and other cancers.

  The results presented herein demonstrate the identification of the role of the HER-2 sister protein in breast cancer. These sister proteins can be effective targets and vaccines can be developed for other solid tumors. It has led to the development of peptides that can be used to target HER-1 and HER-3. Specifically, in DCIS, specific anti-HER-1, anti-HER-2 and anti-HER-3 responses in patients have been identified before and after vaccination, which Support the development of multivalent vaccines that can be used to prevent early or treat women with DCIS. The compositions of the present invention are useful for treating other cancers including, but not limited to, colon cancer, melanoma, brain tumors, lung cancer, ovarian cancer and other tumors.

Melanoma Melanoma is an invasive skin cancer that can be life-threatening if not detected early. In mice, experiments were performed using a standard dendritic cell vaccine and the dendritic cells were engineered to exhibit a mutated protein (BRAF) that causes about 70% melanoma. . When vaccinated with these dendritic cells, mice were protected from challenge with melanoma cells, indicating that a vaccine for melanoma can be developed. Without intending to be bound by any particular theory, targeting BRAF and HER-3 in combination is not limited to, but is limited to solid cancers such as colon cancer, pancreatic cancer and lung cancer and It may be useful to treat other cancers, including other gastrointestinal tumors.

  In addition, melanoma tumors have also been shown to use B cells to avoid immune surveillance, and thus it is believed that therapy can be improved by eliminating specific B cells. Experiments can be designed to assess whether changing the tumor microenvironment to a Th1-type response can help prevent evasion.

  In some cases, the vaccines of the present invention can be used to treat melanoma that has spread. In some cases, the present invention also provides a therapy for eliminating residual cells that often becomes resistant to drug therapy.

A novel strategic cytotoxic CD8 + T lymphocyte (CTL) for identifying MHC class II-promiscuous CD4 ⁺ peptides from tumor antigens for use in vaccination has historically been a major antitumor immunity Although considered to be an effector, simply boosting the CTL response with the CD8 + vaccine in various tumor types has yielded unpredictable clinical results, probably because CTL This may be because sub-optimal functions will be lacking without the help of new CD4 + T lymphocytes. CD4 + T helper type 1 (Th1) cells secrete INF-γ / TNF-α to induce tumor senescence and apoptosis. However, it remains a challenge to successfully incorporate CD4 + epitopes in the construction of cancer vaccines and generate durable, antigen-specific CD4 + immunity. An immunogenic HER3 CD4 + that exhibits class II promiscuous and generates anti-HER3 CD4 + immunity for the purpose of inclusion in a vaccine construct using the extracellular domain (ECD) of HER3 as a candidate “oncogenic driver” tumor antigen. Experiments were performed to identify peptides.

  The materials and methods utilized in these experiments are described here.

Materials and Methods To generate anti-HER3 Th1 cellular immunity, experiments were designed to identify immunogenic, class II-promiscuous HER3 CD4 + peptides using HER3 ECD as a tumor antigen.

Protocol Overview A library of 15-mer peptides was generated from the HER3 ECD, overlapping by 5 amino acids. These peptides were pulsed onto mononuclear cell-derived DCs obtained from donors and matured to a type 1 polarized (DC1; secreting IL-12) phenotype. DC1 was collected and obtained from a subject belonging to our DCIS vaccine study and known to have an anti-HER-3 Th1 response and co-cultured with purified CD4 + T cells. Using a large pool of 10 peptides, the identification process was gradually refined to obtain a single reactive epitope as measured by CD4 + T cell interferon gamma (IFN-γ) secretion. Screening 5-6 subjects and 4 peptides that appear to react across most donors: HER3 _56-70 (SEQ ID NO: 4), HER3 _401-415 (SEQ ID NO: 5), HER3 _416-430 (SEQ ID NO: 6) and HER3 _451-465 (SEQ ID NO: 7) were identified. Subjects were identified that did not show evidence of responsiveness to CD4 + T cell recognition of the HER-3 extracellular domain, 4 HER-3 peptides were pulsed to the DC1 of those subjects, and the pulsed DC1 together with CD4 T cells Cultured for 1 week and then tested for reactivity to HER-2 peptide and response to extracellular HER-3 protein. In either case, at least one peptide resulted in recognition of both the pulsed peptide on mononuclear cells and the total protein of HER-3, suggesting that primary sensitization occurred ex vivo. To do. It was also shown that healthy donors can respond to these peptides and that anti-HER-3 Th1 responses are lost in patients with triple-negative breast cancer. Gala, K .; Et al., Clin. Cancer Res 2014; 20: 1410-1416, and Datta, J. et al. Et al., “Progressive Loss of Anti-HER2 CD4 + T-helper Type 1 Response in Breast Tumorgenesis and the Potential for Immunity”, published by OncoImmunol.

Key points of the protocol further illustrated in FIG. 19:
-A library containing 123 peptide fragments 15 amino acids long, overlapping by 5 amino acids, was generated from the extracellular domain (ECD) of HER3.
-Autologous mononuclear cell-derived dendritic cells (DCs) obtained from donors are rapidly converted to type 1 (secreting DC1 → IL-12) phenotype by GM-CSF, IFN-γ and LPS Maturated and pulsed with the relevant peptide (eg, HER3 ECD or HER3 CD4 + peptide as specified). DC1 biases the Th1 response by the production of IL-12.
-Collected DC1 was allosensitized by co-culture with purified CD4 + T cells for 8-10 days.
-Sensitive CD4 + T cells (most of them antigens) against immature DCs (iDCs) pulsed with specific CD4 + peptides of interest (eg clusters of HER3 library peptides) or irrelevant class II peptide controls. Reactivate (expected to be specific).
-The supernatant was then collected from these co-cultures. A Th1 response, measured by IFN-γ ELISA, was considered antigen-specific if IFN-γ production was at least twice that produced by an irrelevant control.
-HLA-DR, DP, DQ typing on donors was performed by the Clinical of the University of Pennsylvania Clinical Immunology Laboratory to assess MHC class II promiscuousness of the CD4 ⁺ Th1 response.

  A library containing 123 overlapping 15 amino acid long peptide fragments was generated from HER3-ECD. Autologous mononuclear cell-derived DCs obtained from donors were matured to DC1 and pulsed with HER3-ECD. Harvested DC1 was co-cultured with purified CD4 T cells. Ten days later, sensitized CD4 T cells were re-activated against immature DC (iDC) pulsed with a cluster of HER3 library peptides or an irrelevant CD4 control peptide 1. A Th1 response, measured by IFN-γ ELISA, was considered antigen-specific if IFN-γ production was at least twice that produced by an irrelevant control.

  1) To identify immunogenic CD4 + peptides, obtain breast cancer patients known to show anti-HER3 ECD reactivity after DC1 vaccine pulsed with HER2; 2) immunogenicity of these peptides Confirmed by the process of “reverse” sensitization in the same patient; 3) Obtain a patient known to have demonstrated anti-HER3 ECD non-responsiveness after vaccination and use it to identify CD4 + peptides Thus, the experiment was performed in a three-step process of observing whether cells are sensitized to native HER3 ECD and thus tolerance to autoantigen (ie, HER3) is overcome / invalidated.

  The results of the experiment are described here.

Sequential screening of HER3 ECD peptide library to identify immunogenic epitopes recognized by HER3 ECD-sensitized CD4 + Th1 cells First, to identify a single immunogenic HER3 CD4 + epitope, anti-HER3 ECD Th1 sensitization was performed in 5 breast cancer patients known to be reactive. To achieve this, we start with a cluster of 10 peptides (1-10, 11-20,... Etc.) and a cluster of 3 peptides (1-3, 3-6, 7-10,... Etc.) Finally, we focused on single immunogenic HER3 peptides and reactivated HER3 ECD-sensitized CD4 + Th1 sequentially against them. Representative screens are shown in FIGS. 2, 13 and 14. Four immunogenic peptides, HER3 (56-70) (SEQ ID NO: 4), HER3 (401-415) (SEQ ID NO: 5), HER3 (416-430) (SEQ ID NO: 6) and HER3 (451-465) ) (SEQ ID NO: 7) was reproducibly identified and these were promiscuous across HLA-DR, DP and DQ subtypes. Th1 cells obtained from 4 non-HER3-responsive donors were sensitized using DC1 pulsed with 4 identified HER3 peptides, and these cells were subsequently immunized with iDC pulsed with HER3 ECD. When challenged to recognize, all donors not only showed good sensitization to individual immunogenic HER3 peptides, but also recognized native HER3-ECD.

  The results presented herein show that DC1 pulsed with a library of overlapping tumor antigen-derived peptides can identify promiscuous class II peptides for CD4 T cell vaccine development Yes. In this study, the immunogenic HER3 CD4 peptide effectively overcomes immune tolerance against self tumor antigens. Vaccines that utilize these HER3 CD4 peptides in construction can be applied to patients with cancers that overexpress HER3. Furthermore, these results quickly and reproducibly identify class II promiscuous immunogenic CD4 epitopes derived from any tumor antigen for cancer immunotherapy using the DC1-Th1 platform. A new strategy is also presented. Table 1 below shows the initial identification of immunogenic CD4 + HER3 ECD peptides in patients known to have anti-HER3 reactivity. Table 2 shows the amino acid sequences of four immunogenic HER3 CD4 + epitopes identified by sequential screening.

Confirmation of immunogenicity of identified CD4 + HER3 ECD epitopes by “reverse” sensitization, ie, the ability of individual epitope-sensitized CD4 + Th1 to recognize native HER3 ECD FIG. 15 is found to have HER3 ECD reactivity CD4 + T cells are sensitized by DC1 pulsed with each donor-specific, immunogenic HER3 epitope and reactivated against iDC pulsed with each HER3 epitope and native HER3 ECD It shows that.

  20 and 21 show the additional results of “reverse” sensitization.

CD4 + Th1 sensitized with DC1 pulsed with immunogenic HER3 epitopes was obtained from 4 HER3 ECD non-reactive donors, as seen in FIG. 16, which appears to abolish anti-HER3 immune self-tolerance . All of the resulting CD4 + Th1 cells were sensitized using DC1 pulsed with four identified HER3 peptides and subsequently challenged to recognize iDC pulsed with HER3 ECD. Donors not only showed good sensitization to individual HER3 epitopes, but also recognized native HER3 ECD.

The CD4 + HER3 epitope was tested using the HER3 extracellular domain (ECD) as a candidate “oncogenic driver” tumor antigen, as seen in FIG. Immunogenic HER3 CD4 + peptides have been identified that show differentiation and generate anti-HER3 CD4 + immunity; these peptides can be used in vaccine constructs.

Peptides derived from tumor antigens The results presented herein are:
The ability to identify promiscuous MHC class II peptides by DC1 pulsed with a library of overlapping peptides from tumor antigens to develop a CD4 + T cell vaccine;
The immunogenic HER3 CD4 + peptide effectively overcomes immune tolerance against self tumor antigens,
-To quickly and reproducibly identify class II promiscuous immunogenic CD4 + epitopes derived from any tumor antigen for cancer immunotherapy using the DC1-CD4 + Th1 platform from these results The new strategy of
-Vaccines constructed using these HER3 CD4 + peptides have shown to be worth studying in patients with cancers that overexpress HER3.

HER3 expression is a marker of tumor progression in premalignant lesions of the gastroesophageal junction Overexpression of receptor tyrosine kinases (RTKs), including members of the HER family, has prognostic and therapeutic significance in invasive esophageal gastric cancer Have. RTK expression in premalignant gastroesophageal lesions has not been extensively studied previously.

  The presence of Barrett's esophagus, or metaplastic columnar epithelium of the distal esophagus predisposes to the development of esophageal adenocarcinoma. While histological metastases from dysplasia to invasive malignancies are well characterized, carcinogenesis in metaplastic cells is accompanied by incompletely understood genetic changes (Cameron, AJ). Gastroenterology 109 (5): 1541-6 (1995)). Several recent reports have identified human epidermal growth factor receptor 2 (HER2) expression in a subset of Barrett's esophageal lesions with dysplasia. (Almhanna, K. et al., Appl. Immunohistochem. Mol. Morpol. July 16 2015) (Almhanna, et al.); Et al. Histopathology 61 (5): 769-76 (2012) (Fassan, et al.); And Rossi, E .; Et al. Cell. Mol. Med. 13 (9B): 3826-33 (2009) (Rossi et al.). Furthermore, the rate of HER2 expression correlates with the degree of dysplasia, suggesting an associated pathway in tumorigenesis.

  Overexpression of receptor tyrosine kinase (RTK) molecules, including HER family members (HER1, HER2, and HER3) and cMET (mesenchymal epithelial transition factor) is useful for the treatment of breast, lung and gastrointestinal cancers (Yokata J. et al., Lancet 1: 765-767 (1986), and identification of HER2 overexpression in a subset of breast cancer in esophageal gastric cancer, HER2 overexpression and aggressive biology and effective targeting of HER2 by monoclonal antibodies This was an important event in the development of targeted therapy for the treatment of solid tumors (Joensuu, H. et al., N. Eng. J. Med. 354 (8): 809-20 (2006)). Provide the basis for further efforts to target RTK molecules in the treatment of other malignancies I am serving.

  Overexpression of HER2 has been demonstrated in a small number of gastric cancers and has a minor impact on the results and is a target for trastuzumab in a metastatic environment (Bang, YJ et al., Lancet 376 (9742) 687). -97 (2010) (Bang et al.)). Overexpression of HER2 is more frequently expressed in the proximal stomach and gastroesophageal junction compared to more distal gastric adenocarcinoma (Rajagopal et al., I., et al., J. Clin. Diagn. Res.9 (3): EC06-10 (2015)), trastuzumab was targeted for translocation and moderately affected outcome (Bang et al. And Fichter, CD et al., Int. J. Cancer 135 (7): 1517-30 (2014) (Fichter et al.)). HER1 and HER3 expression in gastric cancer has a poor prognosis in most related studies. (Kandel, C. et al., J. Clin. Pathol. 67 (4): 307-12 (2014) and Hayashi, M. et al., Clin. Cancer Res. 14 (23): 7843-9 (2008)). Overexpression of CMET is associated with poor prognosis of esophageal adenocarcinoma, and inhibition of cMET-dependent signaling regulates HER1 and HER3 activity. These data provide the basis for further efforts to characterize RTK expression in esophageal gastric cancer and precursor lesions (Liu, X. et al., Clin. Cancer Res. 17 (22): 7127-38 (2011)). The studies described below aim to characterize RTK expression in dysplastic lesions of the gastroesophageal junction in an effort to identify potential targets for treatment and primary prevention.

Methods After clinical trial review board approval, clinical records and histological specimens of 73 Barrett's esophageal patients with dysplasia (low grade dysplasia (n = 32), high grade dysplasia (n = 59)) Reviewed retrospectively. Formalin-fixed, paraffin-embedded tissue blocks from 2003-2012 stored endoscopic biopsies and mucosal resection specimens were sectioned at 5 μm on a plus slide (Fisher Scientific, Waltham, Mass.) Followed by deparaffinization. And rehydrated. All biopsies were obtained from HER1 (clone H11; 1:50; DAKO), HER2 (HerceptTest, DAKO, Carpinteria, Calif.) And HER3 (clone RTJ.2; 1:30; Santa Cruz Biotechnology, Dallas, TX) Bond. -III apparatus), evaluated by a single pathologist under the microscope (Leica Bond-III). Membrane 3 + HER staining was considered positive as well as membrane 2 + HER2 staining in more than 10% of the tumor cells as seen in FIG. CMET immunohistochemistry was performed in 42 cases where sufficient tissue was available. A moderate or scleral staining of more than 50% of the tumor cells was considered positive. RTK overexpression was correlated with clinical data to assess the association with either invasive cancer, dysplastic adenocarcinoma biopsy specimens or subsequent diagnosis of adenocarcinoma in biopsy specimens.

Statistical analysis Two tail tests were used for all analyses. Descriptive statistics are shown as continuous variable categorical variables and median (interquartile range (IQR)) frequency. Categorical and continuous variables were analyzed using Pearson's χ2 or Fisher exact test and Wilcoxon rank sum test, respectively. A P value ≦ 0.05 was considered statistically significant. All tests were two-sided. Analysis was performed using SPSS v22.0 (IBM, Armonk, NY).

Results 73 Barrett's esophageal patients with low-grade dysplasia (n = 32) or high-grade dysplasia (n = 59) were identified and analyzed for HER1, HER2, HER3 and cMET expression by immunohistochemistry. The median age of the cohort was 65 years (IQR 60-73 years). 81.9% were male and 87.5% were white. Alcohol usage in the cohort was 14.3% and active tobacco usage was 6.3%, compared to 55.6% for former smokers. 26.4% had a family history of malignancy. As shown in Table 1 below, there was no significant difference between the LGD and HGD cohorts in measured clinical and demographic variables.

  High grade dysplasia (HGD) is HER1 overexpression (22.4% vs. 3.1%, p = 0.016), HER2 (5.3% vs. 0.0%, p = 0.187) And HER3 (45.6% vs. 9.4%, p <0.001) compared to low grade dysplasia (LGD).

  Lesions of invasive esophageal adenocarcinoma are associated with dysplastic lesions in 6 cases, all of which are associated with HGD (HGD: 10.2% vs. LGD: 0.0%, p <0.001). Occurred. An additional 9 patients were diagnosed with invasive esophageal adenocarcinoma in subsequent biopsy specimens (HGD: 17.0% vs. LGD: 0.0%, p = 0.177). Increased HER1 (increased HER1 (26.7% vs 20.5%, p = 0.616) and HER2 (14.3% vs 2.3%, p = 0.077), overexpression in HGD lesions There was a significant association of HER3, as seen in FIGS. 23A and 23B, patients without invasive cancer lesions (71.4% vs. 38.6%, p = 0.032).

  cMET overexpression was observed in 42 HGD samples (42.9%) compared to 18 samples compared to LGD samples (58.3% vs 36.7%, p = 0.200), with HER3 being the most Of the frequently expressed 62.5% HER3 positive specimens versus 38.2% of HER3 negative specimens (p = 0.212). Similar trends were not observed for HER1-positive (p = 0.729) or HER2-positive (p = NA) samples. Invasive cancer was confirmed in 1 out of 42 (5.6%) patients. In this patient, cMET was overexpressed (p = 0.243).

Discussion This analysis of RTK expression in dysplastic lesions at the gastroesophageal junction shows that (1) HER family proteins up-regulate dysplasia in Barrett's esophagus, (2) HER family and cMET overexpression frequencies vary. There is a positive correlation with the degree of formation; (3) HER protein upregulation is associated with an increased incidence of associated invasive cancers, especially in dysplastic lesions.

Thus, HER3 may serve as a biomarker for potential invasive disease in Barrett's esophagus and HGD patients. Furthermore, therapeutic agents that target HER3 or CMET can provide secondary prevention of gastroesophageal cancer in a subset of patients.
Previous assessment of HER expression in Barrett's esophagus is overexpressed in a few cases

  Previous assessments of HER expression in Barrett's esophagus were limited to assessments of HER2 overexpressed in a few cases. See Almhanna et al., Fassan et al., And Rossi et al. Overexpression of HER2 in this study was present in 3.3% of biopsy specimens and was lower than the overexpression rate of HER1 or HER3. This pattern is consistent with HER family protein expression in invasive gastroesophageal junction cancer where HER3 is more commonly overexpressed than HER2. Fichter, et al. Increased overexpression of HER3 protein with progression from LGD to HGD and especially frequent overexpression of HER3 is an unexpected new finding. HER receptor homodimerization and heterodimerization drive signal activation. Clustered overexpression of multiple members of the HER family has been observed in other tumor types. In addition, activated c-MET positively regulates the activity of HER1 and HER311. Indeed, the interaction between these receptors provides the rationale for a multivalent therapeutic approach that targets multiple receptor tyrosine kinases. (Baselga, J. et al., N. Eng. J. Med. 366 (2): 109-19 (2012); Waddell, T. et al., Lancet Oncol. 14 (6): 481-489 (2013))

  Current data suggest an opportunity for targeted secondary prevention of gastroesophageal cancer that has not yet been investigated. Previously targeted HER2 expression in breast ductal carcinoma (DCIS) targeted promising results. U.S. Patent Application No. 14 / 658,095 filed March 13, 2015, U.S. Patent Application No. 14 / 985,303 filed December 30, 2019, Datta, J. et al. Et al., Onco Immunology 4: 8 e1022301 (2015) DOI: 10.0802 / 216402X. 2015.1022301; Datta, J .; Et al., Breast Cancer Res. 17 (1): 71 (2015). Such an approach remains a farther target for gastrointestinal malignancies. Nevertheless, all current treatment options for Barrett's esophagus, including endoscopic resection and excision modes and radical surgery, have significant limitations. Alternative strategies are needed to save morbidity and reduce the risk of invasive cancer.

  This analysis of RTK expression in dysplastic lesions at the gastroesophageal junction shows that (1) the HER family protein upregulates dysplasia in Barrett's esophagus, (2) the frequency of HER family and cMET overexpression is dysplasia (3) HER protein upregulation is associated with an increased incidence of associated invasive cancers, particularly in dysplastic lesions.

  Thus, HER3 may serve as a biomarker for potential invasive disease in Barrett's esophagus and HGD patients. Furthermore, therapeutic agents that target HER3 or CMET can provide secondary prevention of gastroesophageal cancer in a subset of patients.

  In summary, the data show that HER3 is frequently overexpressed in high-grade dysplastic lesions at the gastroesophageal junction, particularly those with occult invasive cancer and malignant transformation, and these findings indicate that HER3-expressing dysplasia It may justify a more aggressive approach to lesion management and provides a rationale for future applications of HER3-targeted therapeutics in early disease settings that will be readily understood by those skilled in the art.

  The disclosures and publications of any and all patents, patent applications cited herein are hereby incorporated by reference in their entirety. Although the invention has been disclosed with reference to specific embodiments, it is to be understood that other embodiments and modifications of the invention may be devised without departing from the true spirit and scope of the invention. It is clear to the contractor. The appended claims are to be construed to include all such embodiments and equivalent variations.

(Cross-reference of related applications)
This application is a continuation of application number PCT / US15 / 41034, filed July 17, 2015. The application claims priority to US Provisional Patent Application No. 62 / 076,789 filed on November 7, 2014 and US Provisional Patent Application No. 62 / 025,681 filed July 17, 2014. . The entire contents of each patent application are incorporated herein by reference.

  In 25-30% of breast cancers, amplification and overexpression of the growth factor receptor gene HER2 (also known as human epidermal growth factor receptor-2, neu / erbB2) results in enhanced tumor invasiveness and recurrence and Associated with a high risk of death (Slamon, D. et al., 1987, Science 235: 177; Yarden, Y., 2001, Oncology 1: 1). This oncogene encodes a 185 kilodalton (kDa) transmembrane receptor tyrosine kinase. HER2 stands out from the others in several ways as one of the four members of the human epidermal growth factor receptor (EGFR) family. First, HER2 is an orphan receptor. No high affinity ligand has been identified. Second, HER2 becomes a preferred partner for other EGFR family members (HER1 / EGFR, HER3 and HER4), forming heterodimers, which have high ligand affinity and excellent Signaling activity. Third, full-length HER2 undergoes proteolytic cleavage and releases a soluble extracellular domain (ECD). ECD shedding has been shown to be an alternative activation mechanism for full-length HER2, both in vitro and in vivo, because a membrane anchor fragment with kinase activity remains. The central role of HER2 in EGFR family signaling correlates with its involvement in the development of several types of cancer, including breast cancer, ovarian cancer, colon cancer and gastric cancer, regardless of its expression level (Slamon, D Et al., 1989, Science 244: 707; Hynes, N. et al., 1994, Biochem. Biophys. Acta. 1198: 165). HER2 may also confer resistance to certain chemotherapy on tumor cells (Pegram, M. et al., 1997, Oncogene 15: 537). HER2 is an important target for cancer therapeutics given its very important role in tumorigenesis.

  The human EGF receptor (HER) family of receptor tyrosine kinases regulate a great variety of biological processes, including cell proliferation, migration, invasion and survival. This family consists of four members: EGFR (HER1), HER2 (neu or ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Currently, 11 including epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor alpha (TGFα), amphiregulin (AR), epiregulin, betacellulin and heregulin The ligands have been reported. These ligands bind directly to their cognate receptors, which leads to the formation of receptor homodimers or heterodimers that cause activation of multiple signaling pathways. Dysregulation of HER-family members, either through activation of mutations, receptor overexpression or abnormal ligand release, leads to diverse human tumor development. HER3 is overexpressed in breast, ovarian and lung cancers and this genetic feature correlates with poor prognosis. HER3, when activated by heregulin, dimerizes with HER2 and EGFR to form a potent oncogenic receptor heterodimer. Within this complex, HER3 preferentially recruits PI3 kinase to its cytoplasmic docking site, thereby regulating cell growth and survival. To date, HER3 is inactive as a kinase due to the apparently aberrant sequence characteristics in its kinase domain, and the HER-family kinase activity is impaired to initiate a signaling event. It has been speculated that it requires heterodimerization with a non-member. Consistent with this, it was shown that HER2 requires HER3 to drive breast tumor cell proliferation. However, recent findings have shown that HER3 can also phosphorylate Pyk2 and consequently activate the MAPK pathway in human glioma cells. Furthermore, monoclonal antibodies specific for HER3 can also inhibit the growth and migration of cancerous cell lines. Interestingly, recently, cancer cells circumvent HER-family inhibitor therapy by upregulating HER3 signaling, and HER3 inhibition abrogates HER2-driven tamoxifen resistance in breast cancer cells Indicated. Furthermore, resistance to EGFR small molecule inhibitor gefitinib (Iressa) therapy has also been shown to be linked to signal activation of HER3.

  HER3 is a receptor protein and plays an important role in regulating normal cell growth. HER3 lacks its own kinase activity and transduces signals across the cell membrane depending on the presence of HER2. At the beginning of transcription, the HER3 mRNA precursor contains 28 exons and 27 introns. The fully spliced HER3 mRNA is composed of 28 exons after splicing introns.

  During the past decade, targeted therapies have emerged as the basis for cancer therapeutics. Members of the EGF receptor family, ie EGFR (or HER1) and ErbB2 (or HER2 / neu), are becoming particularly interesting targets, because these receptor tyrosine kinases ( This is because RTK) is deregulated in many cancers. The oncogenic function of ErbB3 or HER3, another member of the EGF receptor family, has only recently been examined due to mediating resistance to HER2 and its major role in therapies targeting the PI3K pathway It came to be. Activation of mutations in HER3 and / or its overexpression has been identified in several different tumor types, including breast cancer, gastric cancer, colon cancer, bladder cancer and melanoma, and worse overall in these tumors A prognostic prognosis.

Slamon, D.C. Et al., 1987, Science 235: 177. Yarden, Y.M. 2001, Oncology 1: 1. Slamon, D.C. Et al., 1989, Science 244: 707. Hynes, N .; Et al., 1994, Biochem. Biophys. Acta. 1198: 165 Pegram, M .; Et al., 1997, Oncogene 15: 537.

  Despite advances in the art, it remains uncertain whether effective immune responses can be generated in humans using cell or protein-based vaccine strategies that target HER-3. Accordingly, there is a need in the art for additional immunotherapy approaches to treat or prevent breast cancer and other malignancies associated with HER-3 protein overexpression. The present invention satisfies this need.

  The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 3 is a graph showing immunogenic peptides derived from HER-3 showing the ability to activate CD4 T cells across many patients (SEQ ID NOs: 1-3 , respectively, in the order shown ) . . FIG. 5 is a graph showing an overall screen of HER3 using a group of 10 peptide fragments. Also, a HER3 screen graph using a single peptide is shown (SEQ ID NOs: 4-7 , respectively, in the order shown ). FIG. 5 is a graph showing an overall screen of HER3 using a group of 10 peptide fragments. Also shown is a HER3 screen graph using a single peptide (SEQ ID NOs: 4 and 7 , respectively, in the order shown ) . FIG. 5 is a graph showing an overall screen of HER3 using a group of 10 peptide fragments. Also shown is a HER3 screen graph using a single peptide (SEQ ID NOS: 1-3 , respectively, in the order shown ) . It is a graph which shows the production amount of IFN-γ from different HER3 peptides. It is a graph which shows the production amount of IFN-γ from different HER3 peptides. FIG. 5 is a graph showing the amount of IFN-γ produced from a “reverse” screen that is sensitized to the peptide and HER3 extracellular domain after previously identifying the peptide. FIG. 5 is a graph showing the amount of IFN-γ produced from a “reverse” screen that is sensitized to the peptide and HER3 extracellular domain after previously identifying the peptide. FIG. 6 is a graph showing the amount of IFN-γ produced from a “reverse” screen starting with a peptide and sensitizing to the peptide and the HER3 extracellular domain in a patient who has not been presensitized to the HER extracellular domain. . Shows the amount of IFN-γ produced from a “reverse” screen starting with a full peptide library and sensitized to peptides and HER3 extracellular domain in patients who have not previously sensitized to the HER extracellular domain It is a graph. Shows the amount of IFN-γ produced from a “reverse” screen starting with a full peptide library and sensitized to peptides and HER3 extracellular domain in patients who have not previously sensitized to the HER extracellular domain It is a graph. FIG. 6 is a graph showing the amount of IFN-γ produced from a “reverse” screen starting with a peptide and sensitizing to the peptide and the HER3 extracellular domain in a patient who has not been presensitized to the HER extracellular domain. . FIG. 4 is a graph showing a continuous peptide screen in UPCC 15107-24 donor. FIG. 4 is a graph showing a continuous peptide screen in UPCC 15107-38 donor. FIG. 6 is a graph showing “reverse” sensitization in UPCC 15107-38 and UPCC 15107-24 donors. Immunogenicity in UPCC 15107-30 and UPCC 15107-32 donors (both patients are known not to show anti-HER3 reactivity to identified HER3 peptides and / or native HER3 ECD) FIG. 3 is a graph showing that DC1 pulsed with a HER3 epitope sensitized CD4 + Th1 and overcome anti-HER3 immune tolerance. It is a graph which shows that a CD4 + HER3 epitope shows MHC class II promiscuousness. Placing activated HER-3 CD4 + cells next to cells expressing HER-3 in the chamber causes the HER-3 CD4 + cells to cause apoptosis or death of breast cancer cells expressing HER-3 FIG. FIG. 6 shows a method for identifying immunogenic, class II promiscuous HER3 CD4 + peptides using HER3 ECD as a tumor antigen to generate anti-HER3 Th1 cellular immunity. FIG. 6 is a graph showing that the immunogenicity of the identified CD4 + HER3 ECD epitope was confirmed by “reverse” sensitization. A HER3 ECD screen was performed with the single peptide indicated. FIG. 6 is a graph showing additional results confirming the immunogenicity of identified CD4 + HER3 ECD epitopes by “reverse” sensitization. 3 shows photographs of immunohistochemical scoring of HER staining Histogram showing overexpression of HER family in low dysmorphic (LGD) or high dysmorphic (HGD) Barrett's esophagus (Figure 23A) and highly dysplastic Barrett's lesion with associated invasive cancer (with carcinoma) FIG. 23B is a histogram (FIG. 23B) showing the overexpression rate of highly dysplastic Barrett's lesion (HGD) without HGD) or associated invasive cancer .

  The present invention provides isolated peptides of HER family proteins and other receptor tyrosine kinases. In one embodiment, the present invention provides isolated peptides from one or more of HER-1, HER-3 and c-MET proteins. In one embodiment, the peptide of the invention corresponds to an epitope of HER-1. In one embodiment, the peptide of the invention corresponds to an epitope of HER-3. In one embodiment, the peptide of the invention corresponds to an epitope of c-MET.

  In some embodiments, the corresponding HER family of proteins and other receptor tyrosine kinase epitopes are immunogenic. The present invention further provides compositions comprising one or more peptides of the present invention. In one embodiment, the invention provides a chimeric peptide, the chimeric peptide comprising one or more peptides of the invention.

  In one embodiment, the invention includes a composition comprising a multivalent peptide. A multivalent peptide includes two or more peptides of the invention.

  Further provided are methods of stimulating an immune response and methods of treating cancer in a subject. Also provided are vaccines for use in therapy and prevention. The peptides of the invention can elicit an immune response, either alone or in the context of a chimeric peptide, as described herein. In one embodiment, the immune response is a humoral response. In another embodiment, the immune response is a cell-mediated response. According to some embodiments, the peptides of the invention confer a protective effect.

  In another embodiment, HER3 expression can be used as a marker of tumor progression in precancerous lesions at the gastroesophageal junction.

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

  In general, both the nomenclature used herein and laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization are well known and commonly used in the art. .

  Standard techniques are used for nucleic acid and peptide synthesis. Techniques and procedures are generally described in the prior art in the art and various general references (eg, Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, Y As well as Ausubel et al., 2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout this description.

  The nomenclature used herein and the laboratory procedures used in analytical chemistry and organic synthesis described below are well known in the art and commonly used. Chemical synthesis and analysis are performed using standard techniques or modifications thereof.

  As used herein, each of the following terms has the meaning associated with it in this section.

  As used herein, the articles “a” and “an” are used to refer to one or more (ie, at least one) of the grammatical objects of the article. By way of example, “an element” means one element or more than one element.

  “About” when referring to a measurable value, for example, an amount, a time period, etc., as used herein, is ± 20% from a particular value, or ± 10%, or ± It is intended to encompass variations of 5%, or ± 1%, or ± 0.1%, and such variations are appropriate for performing the disclosed methods.

  When used in the context of an organism, tissue, cell or component thereof, the term “abnormal” refers to at least one observable or detectable characteristic (eg, age, treatment, time of day, etc.) An organism, tissue, cell or component thereof that differs from an organism, tissue, cell or component thereof that exhibits “normal” (expected) characteristics. The characteristics that are normal or expected for one cell or tissue type may be abnormal for different cell or tissue types.

As used herein, “adjuvant therapy” for breast cancer refers to any treatment given after primary therapy (ie, surgery) to increase the probability of long-term survival. “Neoadjuvant therapy” is a treatment given before primary therapy.

  The term “antigen” or “ag” as used herein is defined as a molecule that causes an immune response. This immune response can be involved in either the production of antibodies or the activation of certain immunologically competent cells or both. One skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. In addition, antigens can be obtained from recombinant or genomic DNA. Those skilled in the art will understand that any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will thus encode the term “antigen” as used herein. Will. Furthermore, one skilled in the art will understand that the antigen need not be encoded solely by the full-length nucleotide sequence of the gene. The present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences can be easily placed in various combinations to elicit a desired immune response. Becomes clear. Furthermore, those skilled in the art will also appreciate that an antigen need not be encoded by a “gene” at all. It will be readily apparent that the antigen may be produced synthetically or obtained from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

  An “antigen-presenting cell” (APC) is a cell that is capable of activating T cells, including but not limited to mononuclear cells / macrophages, B cells and dendritic cells (DC).

“APC incorporating antigen” or “APC pulsed with antigen” includes APC that has been exposed to and activated by an antigen. For example, APC can be antigen uptake in vitro, eg, during culturing in the presence of antigen . It can also be incorporated into APCs by exposure to the antigen in vivo. “PC incorporating antigen” has traditionally been in two ways: (1) “pulsing” a small peptide fragment known as an antigenic peptide directly onto the exterior of the APC, or (2) APC, It is prepared by either incubating with total protein or protein particles and then allowing the APC to ingest the total protein or protein particles. These proteins are digested by APC into small peptide fragments and finally transported to the APC surface where they are presented. Furthermore, APCs incorporating antigens can also be generated by introducing a polynucleotide encoding the antigen into the cell.

  The term “anti-tumor effect” as used herein refers to various physiological symptoms associated with decreased tumor volume, decreased number of tumor cells, decreased number of metastases, increased life expectancy, or cancerous condition. It refers to the biological effects that can be revealed by remission. In the first place, “anti-tumor activity” can also be revealed by the ability of the peptides, polynucleotides, cells and antibodies of the present invention in preventing tumor development.

  The term “autoimmune disease” as used herein is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self antigens. In particular, examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), nutrition Impaired epidermolysis bullosa, epididymis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis , Rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthritis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis.

  As used herein, the term “self” is intended to refer to any material that is obtained from an individual and that is subsequently being reintroduced into the same individual.

  The term “B cell” as used herein is defined as a cell derived from bone marrow and / or spleen. B cells can develop into plasma cells, which produce antibodies.

  The term “cancer”, as used herein, is defined as cell hyperproliferation, resulting in aberrant growth, lack of differentiation, local tissue invasion as a result of the abandonment of normal control, a unique trait of cancer. And / or metastasis occurs. Examples include but are not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain tumor, lymphoma, leukemia, lung cancer, germ cell tumor, etc. Can be mentioned.

  A “disease” is an animal health condition in which the animal's health condition continues to deteriorate if the animal is unable to maintain homeostasis and the disease does not ameliorate.

  A “disorder” in an animal refers to a case where the animal is capable of maintaining homeostasis, but the health status of the animal is less favorable than in the absence of the disorder. Disorders that are left untreated do not necessarily cause a further decline in the animal's health.

  A disease or disorder is “reduced” if the severity or frequency of at least one sign or symptom of the disease or disorder experienced by the patient is reduced.

  “Effective amount” or “therapeutically effective amount” is used interchangeably herein and, as described herein, is a compound, formulation, material or composition effective to achieve a particular biological result. Refers to the amount of a thing. Such results can include, but are not limited to, inhibition of viral infection as determined by any suitable means in the art.

  As used herein, “endogenous” refers to any material belonging to or produced within an organism, cell, tissue or system.

  As used herein, the term “exogenous” refers to any material that is introduced from or produced outside an organism, cell, tissue or system.

  “HER receptor” is a receptor protein tyrosine kinase belonging to the HER receptor family, EGFR (ErbB1, HER1) receptor, HER2 (ErbB2) receptor, HER3 (ErbB3) receptor and HER4 (ErbB4) receptor including. HER receptors generally bind to HER ligands and / or can dimerize with another HER receptor molecule; an extracellular domain; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain As well as a carboxyl-terminal signaling domain with several tyrosine residues that can be phosphorylated. The HER receptor may be a “native sequence” HER receptor or an “amino acid sequence variant” thereof. Preferably, the HER receptor is a native sequence human HER receptor.

  “HER pathway” refers to a signaling network mediated by the HER receptor family.

  “HER activation” refers to activation or phosphorylation of any one or more HER receptors. In general, activation of HER results in signal transduction (eg, signal transduction caused by the intracellular kinase domain of the HER receptor that phosphorylates tyrosine residues or substrate polypeptides in the HER receptor). HER activation can be mediated by binding of a HER ligand to a HER dimer containing the HER receptor of interest. Binding of a HER ligand to a HER dimer can activate the kinase domain of one or more HER receptors in the dimer, thereby in one or more HER receptors. Phosphorylation of tyrosine residues and / or phosphorylation of tyrosine residues in additional substrate polypeptides, eg, Akt intracellular kinase or MAPK intracellular kinase.

  “HER3” and “ErbB3” are disclosed, for example, in US Pat. Nos. 5,183,884 and 5,480,968, and Kraus et al., PNAS (USA) 86: 9193-9197 (1989). Refers to a receptor polypeptide.

  “HER3 extracellular domain” or “HER3ECD” refers to a domain of HER3 that is outside the cell and that is anchored or circulating in the cell membrane, including fragments thereof. In one embodiment, the extracellular domain of HER3 may comprise four domains: domain I, domain II, domain III and domain IV. In one embodiment, the HER3 ECD comprises amino acids 1-636 (numbering including the signal peptide). In one embodiment, HER3 domain III comprises amino acids 328-532 (numbering including signal peptide).

  “Homologous” as used herein is a subunit between two macromolecular molecules, eg, two nucleic acid molecules, eg, two DNA molecules or two RNA molecules, or two polypeptide molecules. Refers to sequence similarity. If the same monomer subunit occupies a position of a subunit in both two molecules, for example if adenine occupies a position in each of the two DNA molecules, these molecules Is completely homologous or 100% homologous. The percent homology between two sequences is a direct function of the number of matching or homologous positions, for example, in a sequence of two compounds, half of the positions (eg, 10 subunits high in length) If the 5 positions in the molecule are homologous, the two sequences are 50% identical, and if 90% of the positions, eg 9 out of 10 are matched or homologous, the two sequences The sequences share 90% homology. As an example, the DNA sequences 5'ATTGCC3 'and 5'TATGGC3' share 50% homology.

  Furthermore, when the term “homology” or “identity” is used herein to refer to nucleic acids and proteins, it is understood that it applies to homology or identity at both the nucleic acid and amino acid sequence levels. Should.

  The term “hyperproliferative disease” is defined as a disease that results from cellular hyperproliferation. Exemplary hyperproliferative diseases include, but are not limited to, cancer or autoimmune diseases. Other hyperproliferative diseases can include, for example, vascular occlusion, restenosis, atherosclerosis or inflammatory bowel disease.

  As used herein, “instruction material” includes publications, records, schematics or any other representational medium that can be used to convey the usefulness of the compositions and methods of the invention. Instructions for use of the kit of the present invention are attached to, for example, a container containing the nucleic acid, peptide and / or composition of the present invention, or shipped together with a container containing the nucleic acid, peptide and / or composition. be able to. Alternatively, the instructional material and the compound may be shipped separately from the container, with the intention that the instructional material and the compound be used together by the recipient.

  “Immune response” as used herein means the activation of a host immune system, eg, a mammalian immune system, in response to the introduction of an antigen. The immune response can take the form of a cellular response or a humoral response, or both.

  “Isolated” means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not in an “isolated state”, but the same nucleic acid or peptide that is partially or completely separated from coexisting material in its natural state is “isolated. It is. The isolated nucleic acid or protein may be present in substantially purified form, or may be present in an externally applied environment such as, for example, a host cell.

  By the term “modulating” as used herein, as compared to the level of response in a subject in the absence of treatment or compound, and / or otherwise identical, but not yet. By mediating a detectable increase or decrease in response level in a subject when compared to the response level in a treated subject. The term encompasses perturbing and / or affecting a native signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

  “Peptide”, “protein” or “polypeptide” as used herein can mean amino acid-linked sequences and can be natural, synthetic, or natural and synthetic variants or combinations. .

  As used herein, “population” includes reference to an isolated culture comprising a homogeneous, substantially homogeneous, or heterogeneous cell culture. In general, a “population” can also be considered an “isolated” cell culture.

  As used herein, a “recombinant cell” is a host cell that contains a recombinant polynucleotide.

  A “sample” or “biological sample” as used herein is obtained from a subject, including but not limited to organs, tissues, exosomes, blood, plasma, saliva, urine and other body fluids. Means biological material. The sample can be material from the subject of any source.

  “Signal 1” as used herein generally refers to the first biochemical signal passed from activated DCs to T cells. Signal 1 is provided by an antigen expressed on the surface of the DC and is sensed by the T cell through the T cell receptor.

  “Signal 2” as used herein generally refers to a second signal provided to the T cell by the DC. Signal 2 is provided by “costimulatory” molecules on activated DCs, usually CD80 and / or CD86 (but other costimulatory molecules are also known) and sensed by the T cell through the surface receptor CD28. Is done.

  “Signal 3” as used herein generally refers to a signal generated from a soluble protein (usually a cytokine) produced by activated DC. These are sensed through receptors on T lymphocytes. The third signal instructs the T cell as to what phenotypic or functional characteristics it should acquire in order to best cope with the current threat.

  By the term “specifically binds” as used herein, recognizes and binds to another molecule or feature in a sample, but substantially recognizes another molecule or feature. Means a molecule that does not bind to them, for example, an antibody.

  The terms “subject”, “patient”, “individual” and the like are used interchangeably herein, whether any animal or those suitable for the methods described herein, whether in vitro or in situ. Refers to cells. In certain non-limiting embodiments, the patient, subject or individual is a human.

  The term “T cell” as used herein is defined as a cell derived from the thymus that is involved in a variety of cell-mediated immune responses.

  The term “T-helper”, when used herein with reference to cells, includes a subgroup of lymphocytes (white blood cells or leukocyte types) that includes different cell types that can be identified by one skilled in the art. Indicates. In particular, T-helper cells according to the present disclosure include effector Th cells (eg, Th1, Th2 and Th17). These Th cells secrete cytokines, proteins or peptides that activate or interact with other leukocytes.

  “Th1 T cells”, as used herein, produce the cytokine IFN-γ at high levels and survive in host cells, against certain disease-causing microorganisms, and also against cancer. Refers to T cells that are believed to be extremely effective.

  “Th17 T cells” as used herein are considered highly effective against disease-causing microorganisms that produce cytokines IL-17 and IL-22 at high levels and survive on mucosal surfaces. Refers to the T cell being

  A “therapeutically effective amount” is the amount of a compound of the invention that, when administered to a patient, ameliorates the symptoms of the disease. The amount of a compound of the invention that constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and severity of the disease, the age of the patient to be treated, and the like. A therapeutically effective amount can be routinely determined by one of ordinary skill in the art in view of the skilled person's own knowledge and the present disclosure.

  The terms “treat”, “treating” and “treatment” refer to the therapeutic or prophylactic strategies described herein. The method of “treatment” exceeds the expected survival for prevention, cure, delay, reduced severity or remission of one or more symptoms of a disorder or relapse disorder, or when no such treatment is given, In order to prolong the survival of a subject, the composition of the present invention can be used to ultimately acquire a subject in need of such treatment, eg, a subject suffering from or suffering from a disease or disorder Take advantage of administering to subjects at risk.

  The term “vaccine” as used herein is defined as a material used to elicit an immune response after administration to an animal, preferably a mammal, more preferably a human. Once introduced into a subject, a vaccine can elicit an immune response including, but not limited to, the generation of antibodies, cytokines and / or other cellular responses.

  A “variant” with respect to a peptide or polypeptide retains at least one biological activity, although the amino acid sequence differs by amino acid insertion, deletion or conservative substitution. A variant may also mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of amino acids, ie, replacing amino acids with different amino acids with similar properties (eg, hydrophilicity, charged region extent and distribution) typically results in minor changes in the art. It is recognized that These minor changes can be identified, in part, by examining the hydrophobic hydrophilicity index of amino acids according to understanding in the art. Kyte et al. Mol. Biol. 157: 105-132 (1982). The hydrophobic hydrophilicity index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydrophobic hydrophilicity index can be substituted and still retain protein function. In one aspect, amino acids having a hydrophobic hydrophilicity index of ± 2 are substituted. The hydrophilicity of amino acids can also be used to indicate substitutions that would result in a protein that retains biological function. In the peptide context, examining the hydrophilicity of an amino acid makes it possible to calculate the largest local average hydrophilicity of the peptide, which can be well correlated with antigenicity and immunogenicity. It is a useful measure that has been reported. U.S. Pat. No. 4,554,101, which is incorporated herein by reference in its entirety. As understood in the art, substitution of amino acids with similar hydrophilicity values can yield peptides that retain biological activity, eg, immunogenicity. Substitutions can be performed with amino acids having hydrophilic values within ± 2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with such observations, amino acid substitutions corresponding to biological functions are related to the relative similarity of amino acids, particularly those amino acids, indicated by hydrophobicity, hydrophilicity, charge, size and other properties. It is understood that it depends on the side chain.

  Scope: Throughout this disclosure, various aspects of this invention can be presented as a range format. It should be understood that the description as a range format is merely for convenience and brevity and should not be construed as a firm limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges and individual numerical values within that range. For example, a description of a range, for example, 1-6 is a partial range, for example, 1-3, 1-4, 1-5, 2-4, 2-6, 3-6 etc., and within that range Individual numbers, such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6, should be considered specifically disclosed. This applies regardless of the range width.

DESCRIPTION The present invention provides immunological compositions comprising HER family proteins and other receptor tyrosine kinase (RTK) peptides. In one embodiment, the present invention provides isolated peptides from one or more of HER-1, HER-3 and c-MET proteins. In one embodiment, the peptides of the invention are useful for eliciting an immune response. Compositions comprising the peptides of the present invention are also useful as prophylactic therapeutic agents for initial protection and as therapeutic agents for the treatment of ongoing conditions.

  The present invention also provides a method for treating or preventing cancer. Such methods include the step of administering a peptide of the invention or a combination of peptides of the invention to a subject in need thereof. As a result of the administration of such peptides, anti-tumor immunity is induced. Accordingly, the present invention provides methods for inducing anti-tumor immunity in a subject, such methods comprising a peptide of the invention or a combination of peptides of the invention, and pharmaceutical and cell compositions derived therefrom. Administering to a subject.

  The present invention includes a method for inducing a T cell response in a mammal. The method includes administering an antigen presenting cell (APC) that specifically induces T cell proliferation. In one embodiment, the method comprises administering a dendritic cell vaccine pulsed with a peptide of the invention, thereby specifically inducing T cell proliferation against an antigen corresponding to the peptide.

  In one embodiment, APC pulsed with a peptide of the invention can be used to culture and expand T cells. Once APC is used to expand and proliferate the T cells to obtain a sufficient number of antigen-specific T cells, the antigen-specific T cells so obtained are administered to the mammal, whereby the antigen in the mammal Induces a specific T cell response.

  The present invention includes preparations of activated DC. In one embodiment, the DC preparation is greater than 90% pure. In another embodiment, the DC preparation is fully activated. For example, the DC is activated using a DC activation regimen that involves contacting the DC with a TLR agonist (eg, LPS). In another embodiment, the process of mobilizing calcium is used in conjunction with other DC activation regimens (eg, activators) that enhance cytokines of different third signals to activate DCs.

The present invention includes mature, antigen- incorporated DCs that have been activated by any DC activation regimen. The DCs of the present invention produce desirable levels of cytokines and chemokines. In one embodiment, the present invention provides a method of pulsing and activating cells, whereby the cells remain active after cryopreservation. The benefits of the DC preparation of the invention are that from a single leukocyte apheresis (collection from a patient), the cells are efficient in multiple (eg, 10 or more) “booster” doses in addition to the initial vaccine. These frozen doses can be thawed as needed at a remote treatment site, without the need for any specialized cell processing facility or further quality control testing.

  The present invention also relates to cryopreservation of these activated DCs, which are cryopreserved to retain efficacy and function in antigen presentation and production of various cytokines and chemokines upon thawing, Thus, activated DCs that have been cryopreserved and subsequently thawed are clinically effective at the same level as freshly collected and activated DCs.

  The present invention contemplates that the present invention provides a method for generating and cryopreserving DCs that have superior function in generating stronger signals to T cells, and thus more A powerful, DC-based vaccine is obtained. By effectively cryopreserving such cells, samples can be stored, thawed and used later, thereby reducing the need to repeat the pheresis and erutation processes during vaccine production. Let It is an advantage that the DCs can be frozen and then later thawed and removed, which means that the vaccine produced in a single shot can be subdivided and frozen, and then for weeks This means that it can be administered to patients one at a time over a period of months or years, resulting in a “booster” vaccination that enhances immunity.

This embodiment also includes the use of HER3 expression as a marker of tumor progression in a premalignant lesion of the gastroesophageal junction, also known as Barrett's esophagus. This marker has a prognostic and therapeutic applications definitive invasive esophageal gastric cancer.

Compositions The present invention provides isolated peptides of HER family proteins and other receptor tyrosine kinases. In one embodiment, the present invention provides isolated peptides from one or more of HER-1, HER-3 and c-MET proteins. In one embodiment, the peptides of the invention correspond to epitopes of the corresponding HER protein or c-MET protein. In some embodiments, the corresponding HER protein or c-MET protein epitope is immunogenic.

  The present invention provides a composition comprising one or more peptides of the present invention. The invention also provides a composition comprising one or more chimeric peptides. In one embodiment, the chimeric peptide further comprises one epitope of the corresponding HER protein or c-MET protein.

  Further provided are compositions having one or more multivalent peptides. These multivalent peptides contain more than one epitope of the invention.

  Methods of stimulating an immune response using the compositions of the present invention and methods of treating cancer in a subject are included in the present invention. Vaccines are also provided for use in therapy and prevention. The epitopes of the invention can elicit an immune response, either alone or in the context of a chimeric peptide, as described herein. In one embodiment, the immune response is a humoral response. In another embodiment, the immune response is a cell-mediated response. According to some embodiments, the epitope or peptide of the invention confers a protective effect.

In one embodiment, the epitope of HER-3 or other peptide of the invention is
p11-13 (peptides 51-75): KLYERCEVVMGNLEIVLTGHNADLFSFLQW (SEQ ID NO: 1);
p81-83 (peptides 401-425): SWPPHMMHNFSVSNLTTIGGRSLYN (SEQ ID NO: 2);
p84-86 (peptides 416-440): TTIGGRSLYNRGFSLLIMKNLNVTS (SEQ ID NO: 3);
p12 (peptides 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4);
p81 (peptides 401-415): SWPPHMMHNFSVFSNL (SEQ ID NO: 5);
p84 (peptides 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6);
p91 (peptides 451 to 465): AGRIYISANRQLCYH (SEQ ID NO: 7)
including.

  The HER-3 peptide or any peptide of the present invention may be cyclic or linear. If the epitope is circular, it can be circular in any suitable manner. For example, disulfide bonds can be formed between selected pairs of cysteines (Cys) to provide the desired conformation. It is believed that the formation of a cyclic epitope can result in a conformation that improves the humoral response and thus improves the protective effect.

  The HER-3 epitope identified by SEQ ID NO: 4 corresponds to positions 56-70 of the HER-3 protein. The HER-3 epitope identified by SEQ ID NO: 5 corresponds to positions 401-415 of the HER-3 protein. The HER-3 epitope identified by SEQ ID NO: 6 corresponds to positions 416-430 of the HER-3 protein. The HER-3 epitope identified by SEQ ID NO: 7 corresponds to positions 451-465 of the HER-3 protein.

  As described herein, an epitope of HER-3 of the present invention also encompasses peptides that are functional equivalents of the peptide identified by SEQ ID NO. Such functional equivalents have an altered sequence, in which case one or more amino acids in the sequence of the corresponding HER-3 epitope are substituted, or one or more amino acids are It has been deleted from or added to the corresponding reference sequence. For example, 1 to 3 amino acids can be added to the amino terminus, the carboxy terminus, or both. In some examples, the HER-3 epitope is glycosylated.

  In other examples, the HER-3 epitope can be a retro-inverso isomer of the HER-3 epitope. Retro-inverso modification involves the reversal of all amide bonds within the peptide backbone. This reversal can be achieved by reversing the direction of the sequence and reversing the asymmetry of each amino acid residue by using D-amino acids instead of L-amino acids. This retro-inverso isomer form can retain at least some peptide bond planarity and conformational constraints.

  Non-conservative amino acid substitutions and / or conservative substitutions can be made. A substitution is a conservative amino acid substitution if the substituted amino acid has structural or chemical properties similar to the corresponding amino acid in the reference sequence. By way of example, conservative amino acid substitutions include replacing one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine and isoleucine with another such amino acid; one hydroxyl-containing amino acid, such as serine and When replacing threonine with another such amino acid; when replacing one acidic residue such as glutamic acid or aspartic acid with another such residue; replacing one amide-containing residue such as asparagine and glutamine When exchanging with another such residue; when exchanging one aromatic residue such as phenylalanine and tyrosine with another such residue; one basic residue such as lysine, arginine and histidine In exchange for another such residue; and one small amino acid Acid, such as alanine, serine, threonine, methionine and glycine, which may be replaced with another such amino acids.

  In some examples, deletions and additions are located at the amino terminus, carboxy terminus, or both of one sequence of the peptides of the invention. For example, an equivalent of a HER-3 epitope is at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91%, at least 92%, at least with the corresponding HER-3 epitope sequence Have an amino acid sequence that is 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical. Sequences that are at least 90% identical have only one change, ie, any combination of deletions, additions or substitutions, per 10 amino acids of the reference sequence. Percent identity is determined by comparing the variant amino acid sequence to a reference sequence using programs known or developed in the art.

  In the case of a functional equivalent longer than the sequence of the corresponding HER-3 epitope, the functional equivalent is a sequence that is at least 90% identical to the sequence of the HER-3 epitope and the wild-type HER-3 protein It may have a sequence within which is adjacent to the sequence of the HER-3 epitope.

  By altering the sequence of the epitope and then assaying the resulting polypeptide for the ability to stimulate an immune response, eg, the production of antibodies, the functional equivalent of a HER-3 epitope can be identified . Such antibodies can be found in a variety of body fluids, including serum and ascites. Briefly, a body fluid sample is isolated from a warm-blooded animal, such as a human, for which it is desired to determine whether an antibody specific for a HER-3 polypeptide is present. Incubating the body fluid with the HER-3 polypeptide under conditions sufficient for and for a time sufficient to form an immune complex between the polypeptide and the antibody specific for the protein; Preferably, the assay is performed using an ELISA method.

  According to other embodiments of the invention, a chimeric peptide and a composition comprising one or more chimeric peptides are provided. According to various embodiments, the chimeric peptide comprises a HER-3 epitope, another epitope, and a linker that connects the HER-3 epitope to other epitopes. In one embodiment, other epitopes can include, but are not limited to, another HER-3 epitope, HER-1 epitope, HER-2 epitope, and c-MET epitope. It is further understood that any suitable linker may be used. For example, depending on the epitope used, the HER-3 epitope can be linked to either the amino terminus or the carboxy terminus of other epitopes. The location and selection of other epitopes depends on the structural features of the HER-3 epitope, ie whether it is an alpha helix or beta-turn or chain.

  In one embodiment, the linker can be a peptide about 2 to about 15 amino acids, about 2 to about 10 amino acids, or about 2 to about 6 amino acids in length. The chimeric peptide may be linear or cyclic. Furthermore, the HER-3 epitope, other epitopes and / or linkers may be in the form of retro-inverso. Thus, the HER-3 epitope alone may be in retro-inverso form. Alternatively, the HER-3 epitope and other epitopes may be in retro-inverso form. In another example, the HER-3 epitope, other epitopes and linkers may be in the form of retro-inverso.

  In another embodiment, the peptides of the present invention can be combined into a mixture instead of taking the form of a chimeric peptide. In any event, a composition of the invention comprising a peptide can be a useful agent for pulsing antigen presenting cells (eg, dendritic cells) to produce a cellular vaccine. In another embodiment, a composition of the invention comprising a peptide can be an immunogen useful for inducing the production of antibodies. The compositions of the invention can also be used to immunize a subject and delay or prevent tumor development. The compositions of the invention can be used in vaccines to provide a protective effect.

  According to a further embodiment of the invention, a composition comprising a mixture of two or more of the peptides or chimeric peptides of the invention is provided. In some examples, each of the two or more chimeric peptides has a different HER-3 epitope. In other examples, one of the HER-3 epitopes is selected from SEQ ID NOs: 1-7.

  The peptides of the invention, including chimeric peptides, can be prepared using well-known techniques. For example, peptides can be synthesized and prepared using either recombinant DNA technology or chemical synthesis. The peptides of the present invention may be synthesized individually or as longer polypeptides composed of two or more peptides. The peptides of the present invention are preferably isolated, ie, substantially free of other naturally occurring host cell proteins and fragments thereof.

  The peptide and chimeric peptide of the present invention can be synthesized using a commercially available peptide synthesizer. For example, Kaumaya et al., “De Novo” Engineering of Peptide Immunogenic and Antigenic Determinants as Biogenetics as Biogenetics, Biosciences, Peptides, Peptides, Peptides, Peptides, Peptides, Peptides. The chemical methods described in can be used. For example, a HER-3 epitope can be synthesized co-linearly with other epitopes to form a chimeric peptide. Peptide synthesis can be performed using Fmoc / t-But chemistry. Peptides and chimeric peptides can be made circular in any suitable manner. For example, using a method that differentially protects cysteine residues, performs iodine oxidation, adds water to facilitate removal of Acm groups, and forms disulfide bonds simultaneously, and / or a silyl chloride-sulfoxide method. Thus, a disulfide bond can be obtained.

  Peptides and chimeric peptides can also be generated using cell-free translation systems and RNA molecules derived from DNA constructs encoding epitopes or peptides. Instead, the epitope or chimeric peptide is generated by transfecting a host cell with an expression vector containing a DNA sequence encoding the respective epitope or chimeric peptide, and then inducing expression of the polypeptide in the host cell. You can also In the case of production of recombinants, recombinant constructs containing one or more of the sequences encoding epitopes, chimeric peptides or variants thereof can be obtained using conventional methods such as calcium phosphate transfection, DEAE-dextran mediated transfection. , Introduced into host cells by microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, or infection.

  The peptides of the present invention may contain modifications, such as glycosylation, side chain oxidation, or phosphorylation, but only if the modification does not destroy the biological activity of the peptide. . Other modifications include, for example, the incorporation of D-amino acids or other amino acid mimetics that can be used to increase the serum half-life of the peptide.

  The peptides of the invention can be prepared as a combination, the combination comprising two or more peptides of the invention for use as a vaccine for a disease, eg, cancer. The peptides may be in cocktails or conjugated to each other using standard techniques. For example, a peptide can be expressed as a single polypeptide sequence. The peptides in the combination may be the same or different.

  The present invention should also be construed to include “mutants”, “derivatives” and “variants” of the peptides of the present invention (or the DNA encoding said peptides). A variant is a peptide in which one or more amino acids are changed (or one or more base pairs are changed when referring to a nucleotide sequence encoding said amino acid) and thus obtained The resulting peptide (or DNA) is not identical to the sequences listed herein, but has the same biological properties as the peptides disclosed herein.

  The present invention also provides a polynucleotide encoding at least one peptide selected from peptides having any one or more of SEQ ID NOs: 1-7. Nucleic acid sequences include both DNA sequences that are transcribed into RNA and RNA sequences that are translated into peptides. According to another embodiment, the polynucleotide of the present invention is deduced from the amino acid sequence of the peptide of the present invention. As is known in the art, several alternative polynucleotides are possible due to overlapping codons, but these retain the biological activity of the translated peptide.

  In addition, the invention also encompasses isolated nucleic acids that encode peptides having substantial homology to the peptides disclosed herein. Preferably, the nucleotide sequence of the isolated nucleic acid encoding the peptide of the present invention is “substantially homologous”, ie about 60% homologous to the nucleotide sequence of the isolated nucleic acid encoding the peptide of the present invention, more preferably , About 70% homology, even more preferably about 80% homology, more preferably about 90% homology, even more preferably about 95% homology, even more preferably about 99% homology.

  The scope of the present invention includes homologues, analogs, variants, derivatives and salts, including shorter and longer peptides and polynucleotides; and analogs of peptides and polynucleotides having one or more amino acid or nucleic acid substitutions As well as amino acids or nucleic acid derivatives known in the art, non-natural amino acids or nucleic acids and synthetic amino acids or nucleic acids, provided that these modifications preserve the biological activity of the original molecule. I want you to clearly understand what must be done. Specifically, in accordance with the principles of the present invention, any active fragments of active peptides, as well as extensions, conjugates and mixtures are disclosed.

  The present invention includes any isolated nucleic acid that is homologous to the nucleic acid described or referred to herein, provided that these homologous DNAs are the biology of the peptides disclosed herein. Should be construed as having active activity.

  The nucleic acid of the present invention includes a sequence of RNA or DNA encoding the peptide of the present invention, which makes the nucleotide sequence more stable, whether with or without cells. Or, those skilled in the art will appreciate that any modified form thereof, including chemical modifications of RNA, is also encompassed. Nucleotide chemical modifications can also be used to enhance the efficiency with which a cell incorporates a nucleotide sequence or the expression of a nucleotide in a cell. The present invention contemplates any combination of nucleotide sequence modifications.

  Furthermore, mutants, derivatives of the proteins of the invention using methods of recombinant DNA well known in the art, such as those described in Sambrook and Russell, supra, and Ausubel et al., Supra. Alternatively, any number of procedures can be used to generate variant forms. Procedures for introducing amino acid changes into a peptide or polypeptide by altering the DNA sequence encoding the polypeptide are well known in the art and are also described in these and other articles. Yes.

  Nucleic acids encoding the peptides of the invention can be incorporated into suitable vectors, eg, retroviral vectors. These vectors are well known in the art. Nucleic acids, or vectors usefully containing nucleic acids, can be transferred into the desired cells, and such cells are preferably obtained from the patient. Advantageously, the present invention provides an off-the-shelf composition that allows rapid modification of a patient's own cells (or another mammal's own cells). Thus, it becomes possible to quickly and easily generate modified cells having excellent cancer cell killing properties.

In vectors and other related aspects, the invention includes an isolated nucleic acid encoding one or more of the peptides having a sequence selected from the group consisting of SEQ ID NOs: 1-7.

  In one embodiment, the present invention comprises a nucleic acid sequence encoding one or more peptides of the present invention, which sequence is operably linked to a nucleic acid comprising a promoter / regulatory sequence, and thus the latter The nucleic acid is preferably capable of directing the expression of the protein encoded by the former nucleic acid. Accordingly, the present invention provides an expression vector and method for introducing exogenous DNA into a cell and simultaneously expressing the exogenous DNA in the cell, such as Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold. Spring Harbor Laboratory, New York), and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York). Integration of the desired polynucleotide into the vector and selection of the vector is well known in the art, as described, for example, in Sambrook et al., Supra, and Ausubel et al., Supra.

  Polynucleotides can also be cloned into several types of vectors. However, this invention should not be construed as limited to any particular vector. Instead, the present invention should be construed to encompass a very wide range of vectors that are readily available and / or well known in the art. For example, the polynucleotides of the invention can be cloned into vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

  In certain embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector, and a mammalian cell vector. There are numerous expression vector systems that include at least some or all of the compositions discussed above. Systems based on prokaryotic and / or eukaryotic vectors can be utilized and used with the present invention to produce polynucleotides or their cognate polypeptides. Many such systems are commercially available and widely available.

  Furthermore, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012) and Ausubel et al. (1997) and other virology and molecular biology manuals. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, eg, WO 01/96584; WO 01/29058; and US Pat. No. 6,326,193.

  In order to express the desired nucleotide sequence of the present invention, at least one module in each promoter functions to position the start site for RNA synthesis. The best known example is the TATA box, but in some promoters lacking the TATA box, for example, the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 gene, a separate overlay that overlaps the start site. The elements themselves help to fix the starting location.

  Additional promoter elements, ie enhancers, regulate the frequency of transcription initiation. Typically these are located in the region 30-110 bp upstream from the start site, but recently several promoters have been shown to contain functional elements also downstream of the start site. ing. The spacing between promoter elements is often flexible, so that the function of the promoter is preserved even when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can work together or function independently to activate transcription.

  A promoter can be a promoter naturally associated with a gene or polynucleotide sequence, which can be obtained by isolating a 5 'non-coding sequence located upstream of a coding segment and / or exon. Such promoters can be referred to as “endogenous”. Similarly, an enhancer can also be an enhancer naturally associated with a polynucleotide sequence, which is located either downstream or upstream of the sequence. Instead, placing a polynucleotide coding segment under the control of a recombinant or heterologous promoter provides certain advantages, where the promoter is not normally associated with the polynucleotide sequence in its natural environment. Point to. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers include promoters or enhancers of other genes, as well as any other prokaryotic, viral or eukaryotic cell, and promoters or enhancers that are not “naturally occurring” That is, it can include promoters or enhancers that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthetically generating promoter and enhancer nucleic acid sequences, recombinant cloning and / or nucleic acid amplification techniques can also be used to generate sequences, which can be incorporated into the compositions disclosed herein. Includes relevant PCR ™ (US Pat. No. 4,683,202, US Pat. No. 5,928,906). It is further contemplated that regulatory sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, etc., can be utilized as well.

  Of course, it is important to utilize a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle and organism chosen for expression. Those skilled in the art of molecular biology generally know how to use a combination of promoters, enhancers and cell types for the expression of proteins, see, for example, Sambrook et al. (2012). The promoter utilized may be a constitutive promoter, a tissue-specific promoter, an inducible promoter, and / or a condition suitable to direct high level expression of the DNA segment to be introduced, such as a recombinant protein and / or peptide large It may be a useful promoter under conditions that favor the production on a scale. The promoter may be heterologous or endogenous.

  The promoter sequences exemplified in the experimental examples presented herein are those of the cytomegalovirus (CMV) earliest promoter. This promoter sequence is a strong constitutive promoter sequence and can drive high level expression of any polynucleotide sequence operably linked thereto. However, other constitutive promoter sequences can also be used, including but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) Long terminal repeat (LTR) promoter, Moloney virus promoter, avian leukemia virus promoter, Epstein-Barr virus earliest promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter , Hemoglobin promoter and muscle creatine promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. In the present invention, by using an inducible promoter, the expression of the polynucleotide sequence to which the inducible promoter is operably linked is turned on if such expression is desired, or if expression is not desired. Provides a molecular switch that can turn off expression. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. Furthermore, the present invention also includes the use of tissue specific promoters, which are only active in the desired tissue.

  In order to evaluate the expression of a nucleotide sequence encoding the peptide of the present invention, the expression vector to be introduced into the cell also contains either or both of a selectable marker gene and a reporter gene, and is transfected with a viral vector. Identification and selection of cells that are expressed from a population of cells targeted for infection or infection can also be facilitated. In other embodiments, the selectable marker can be mounted on a separate piece of DNA and used in a cotransfection procedure. Both the selectable marker gene and the reporter gene can be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers are known in the art and include, for example, antibiotic resistance genes such as neo and the like.

  Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Reporter genes that encode proteins that are readily assayable are well known in the art. In general, a reporter gene is present in some form of easily detectable property, such as enzymatic activity, without being present in the recipient organism or tissue or expressed by the recipient organism or tissue. It is a gene that encodes a protein. Reporter gene expression is assayed at an appropriate time after DNA is introduced into the recipient cell.

  Suitable reporter genes can include luciferase, beta-galactosidase, chloramphenicol acetyltransferase, genes encoding secreted alkaline phosphatases, or green fluorescent protein genes (eg, Ui-Tei et al., 2000, FEBS). Lett. 479: 79-82). Suitable expression systems are well known and may be prepared using well known techniques or obtained commercially. Constructs with internal deletions can be generated using unique internal restriction sites or by partial digestion of non-unique restriction sites. The construct can then be transfected into cells that exhibit high levels of expression of siRNA polynucleotides and / or polypeptides. In general, constructs with minimal 5 'flanking regions that show the highest level of expression of the reporter gene are identified as promoters. Such a promoter region can be linked to a reporter gene and used to evaluate a drug for the ability to modulate promoter driven transcription.

In one embodiment of the vaccine , the subject of the invention is a vaccine comprising a peptide of the invention. The vaccines of the present invention can provide any combination of specific peptides for the specific prevention or treatment of cancer in a subject in need of treatment.

  The vaccines of the invention are capable of inducing an antigen-specific T cell response and / or a high titer antibody response, thereby being directed against or reactive against an antigen-expressing cancer or tumor An immune response is induced or elicited. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or both cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α).

  In one embodiment, the subject of the present invention is an anti-cancer vaccine. A vaccine can include one or more cancer antigens. Vaccines can prevent tumor growth. Vaccines can reduce tumor growth. The vaccine can block tumor cell metastasis. Depending on the cancer antigen, targeting vaccine, breast cancer, liver cancer, prostate cancer, melanoma, blood cancer, head and neck cancer, glioblastoma, recurrent respiratory papilloma, anal cancer, cervical cancer, brain tumor, etc. Can be treated.

In certain embodiments, the vaccine comprises (1) humoral immunity via a B cell response that produces the desired antibody; (2) cytotoxic T lymphocytes that attack and kill tumor cells, such as CD8 ⁺ ( (3) increased T helper cell response; and (4) increased inflammatory response via IFN-γ and TFN-α, or preferably by inducing all of the above, Can mediate growth cleanup or inhibition. Vaccines have tumor-free survival of 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, It can be increased by 44% and 45%. Vaccines have a tumor volume of 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42% after immunization. 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59 % And 60% can be reduced.

  The vaccine has a cellular immune response in a subject who has not received the vaccine of about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold. Times to about 5000 times, about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times, or about 300 times It can be increased by about 6000 times. In some embodiments, a vaccine has a cellular immune response in a subject that has been administered a vaccine that is about 50-fold, 100-fold, 150-fold, 200-fold, compared to a cellular immune response in a subject that has not been administered the vaccine. Times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times 2800 times 2900 times 3000 times 3100 times 3200 times 3300 times 3400 times 3500 times 3600 times 370 Times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, It can be increased by 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

  Vaccines have about 50 times to about 6000 times, about 50 times the level of IFN-γ in subjects who have received the vaccine, compared to the level of interferon gamma (IFN-γ) in subjects who have not received the vaccine. To about 5500 times, about 50 times to about 5000 times, about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about It can be increased 6000 times, or about 300 times to about 6000 times. In some embodiments, the vaccine has a level of IFN-γ in a subject that has been administered the vaccine that is about 50-fold, 100-fold greater than the level of IFN-γ in the subject that has not been administered the vaccine. 150x, 200x, 250x, 300x, 350x, 400x, 450x, 500x, 550x, 600x, 650x, 700x, 750x, 800x, 850x, 900x, 950x 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times 2700 times 2800 times 2900 times 3000 times 3100 times 3200 times 3300 times 3400 times 3500 times 3600 times 3700 times Times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, It can be increased by 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

  The vaccines of the present invention have the characteristics necessary for an effective vaccine, for example, the vaccines of the present invention are safe and thus the vaccine itself does not cause illness or death; provides protection against illness; neutralizing antibodies Induces a protective T cell response; is easy to administer, has few side effects, is biologically stable, and has a low cost per dose. A vaccine can achieve some or all of these characteristics by containing cancer antigens as discussed below.

Generation of antigen uptake (antigen pulsed) immune cells The invention includes cells that have been exposed to or otherwise “pulsed” with an antigen of the invention or other peptide of the invention. For example, APCs, such as DCs, can be made uptake of antigens in vitro, for example, by culturing ex vivo in the presence of the antigen or by exposing to the antigen in vivo.

  Also, APC can be “pulsed” into APC in a manner that exposes the antigen to the APC, and this exposure can occur for a time sufficient to facilitate presenting the antigen on the surface of the APC, Those skilled in the art will readily understand. For example, APCs can be exposed to antigens in the form of small peptide fragments, known as antigenic peptides, which are “pulsed” directly onto the exterior of APC (Mehta-Damani et al. 1994); or APC can be incubated with whole protein or protein particles, which are then ingested by APC. All these proteins are digested by APC into small peptide fragments that are finally transported to the APC surface and presented on the APC surface (Cohen et al., 1994). Antigens in the form of peptides can be exposed to cells by the standard “pulsed” technique described herein.

  While not intending to be bound by any particular theory, foreign antigens or antigens in the form of autoantigens are processed by the APCs of the present invention to retain the immunogenic form of the antigen. An immunogenic form of an antigen means that the antigen is processed by fragmentation to produce a form of the antigen that can be recognized and activated by immune cells, eg, T cells. Preferably, such foreign antigens or autoantigens are proteins that are processed into peptides by APC. Related peptides produced by APC can be extracted, purified and used as an immunogenic composition. APC processed peptides can also be used to induce tolerance to APC processed proteins.

Antigen- incorporated APC, according to another expression, is known as “pulsed APC” of the present invention, which is generated by exposing APC to the antigen either in vitro or in vivo. . When pulsing APCs in vitro, APCs can be plated on culture dishes and exposed to the antigen for a sufficient amount of time to allow the antigen to bind to the APC. . The amount and time required to achieve binding of the antigen to APC can be determined by using methods known in the art or other methods disclosed herein. Other methods known to those skilled in the art, such as immunoassays or binding assays, can be used to detect the presence of the antigen on the APC after exposure to the antigen.

  In a further embodiment of the invention, a vector that allows expression of a specific protein by APC can be transfected into APC. The protein expressed by APC can then be processed and presented on the cell surface. The transfected APC can then be used as an immunogenic composition to generate an immune response against the vector encoded protein.

  As discussed elsewhere herein, vectors can be prepared to include specific polynucleotides that encode and express proteins for which an immunogenic response is desired. Preferably, retroviral vectors are used to infect cells. More preferably, an adenoviral vector is used to infect cells.

  In another embodiment, the viral vector can be directed to the target APC by modifying the viral vector to encode a protein or portion thereof recognized by a receptor on the APC, whereby the vector is Occupying the APC receptor initiates vector endocytosis, allowing the antigen encoded by the viral vector nucleic acid to be processed and presented. The nucleic acid delivered by the virus can be native to the virus, and this nucleic acid encodes the viral protein, even if expressed on APC, which is then processed and presented on the MHC receptor of APC Is done.

  As contemplated herein, a variety of methods can be used to transfect a polynucleotide into a host cell. These methods include, but are not limited to, calcium phosphate precipitation, lipofection, particle gun, microinjection, electroporation, colloidal dispersion systems (ie, macromolecular complexes, nanocapsules, microspheres, beads, and oil-in-water types) Lipid based systems including emulsions, micelles, mixed micelles and liposomes). These methods are understood in the art and are described in the published literature and thus allow those skilled in the art to perform these methods.

In another embodiment, the polynucleotide encoding the antigen can be cloned into an expression vector and the vector can be introduced into the APC to produce APC incorporating the antigen by another method. It becomes possible. Various types of vectors and methods for introducing nucleic acids into cells are discussed in the available published literature. For example, the expression vector can be transferred into a host cell by physical, chemical or biological means. For example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and Ausubel et al. (1997, Current Protocols in Mol. It is easily understood that a pulsed cell can be obtained by introducing an expression vector containing a polynucleotide encoding an antigen.

The present invention includes various methods for pulsing APCs, including, but not limited to, incorporating an entire antigen in the form of a protein, cDNA or mRNA into APC. However, the present invention should not be construed as limited to the particular form of antigen used to pulse the APC. Rather, the present invention encompasses other methods known in the art for generating APCs incorporating antigens. Preferably, APCs are transfected with mRNA encoding a defined antigen. Using appropriate primers and reverse transcriptase-polymerase chain reaction (RT-PCR) coupled to transcription reactions, mRNAs corresponding to gene products of known sequence can be rapidly generated in vitro. it can. When generating pulsed APCs, transfection with APC mRNA offers advantages over other techniques for incorporating antigens. For example, the ability to amplify RNA from minute amounts of tissue, ie tumor tissue, leads to vaccination of a large number of patients using APC.

  In order for an antigen composition to be useful as a vaccine, the antigen composition must induce an immune response against the antigen in a cell, tissue or mammal (eg, a human). As used herein, an “immunological composition” expresses or presents an antigen (eg, a peptide or polypeptide), a nucleic acid encoding the antigen (eg, an antigen expression vector), or an antigen or cellular component. Cells can be included. In certain embodiments, the antigen composition comprises or encodes all or a portion of any antigen described herein, or an immunological functional equivalent thereof. In other embodiments, the antigen composition comprises a mixture comprising additional immunostimulatory agents or nucleic acids encoding such agents. Immunostimulatory agents include, but are not limited to, additional antigens, immunomodulators, antigen presenting cells or adjuvants. In other embodiments, one or more of the additional agents are covalently bound in any combination to the antigen or immunostimulatory agent. In certain embodiments, the antigen composition is conjugated to or comprises amino acids of the HLA anchor motif.

  A vaccine may vary in its composition of nucleic acids and / or cellular components, as contemplated herein. In a non-limiting example, the nucleic acid encoding the antigen could also be formulated with an adjuvant. Of course, it will be understood that the various compositions described herein may further comprise additional components. For example, one or more vaccine components can be included in a lipid or liposome. In another non-limiting example, the vaccine can include one or more adjuvants. The vaccine of the present invention and its various components can be prepared and / or administered by any method disclosed herein, or will be understood by one of ordinary skill in the art in light of the present disclosure. Will.

  It is understood that the antigen compositions of the present invention can be made by methods well known in the art, including, but not limited to, chemical synthesis by solid phase synthesis and chemical reaction by HPLC. A method of purifying and extracting from other products, or expressing a nucleic acid sequence (eg, DNA sequence) encoding a peptide or polypeptide containing the antigen of the present invention in an in vitro translation system or in a living cell There is a method to generate by. In addition, the antigen composition can include cellular components isolated from a biological sample. The antigen composition is isolated and dialyzed sufficiently to remove one or more undesirable low molecular weight molecules and / or lyophilized in the desired vehicle to enhance the ready-to-use formulation. . It should be further understood that any additional amino acids, mutations, chemical modifications, etc. that result in the vaccine component preferably do not substantially prevent the antibody from recognizing the epitope sequence.

Antigen-presenting cell therapy The present invention encompasses methods of generating a population of APCs (eg, dendritic cells; DC) that present a peptide of the present invention on a surface and subsequently using the population in therapy. Such methods can be performed ex vivo on a sample of cells obtained from a patient. Thus, the APC thus produced forms a pharmaceutical agent that can be used in the treatment or prevention of cancer. These cells should be accepted by the individual's immune system because such cells are derived from that individual. The cells thus generated are delivered to the individual from whom the cells were originally obtained, thus forming a therapeutic embodiment of the present invention.

DCs are derived from pluripotent mononuclear cells that act as antigen presenting cells (APCs). DCs are ubiquitous in peripheral tissues where they are waiting to capture antigen. When the DC captures the antigen, it processes the antigen into small peptides and moves towards the secondary lymphoid organs. It is within the lymphoid organ that the DC presents the antigenic peptide to naive T cells, and the presentation of the peptide initiates a signal cascade that biases T cell differentiation. When exposed, DCs present antigen molecules bound to MHC class I or class II binding peptides and activate CD8 ⁺ T cells or CD4 ⁺ T cells, respectively (Steinman, 1991, Annu. Rev. Immunol.9: 271-296; Banchereau et al., 1998, Nature 392, 245-252; Steinman et al., 2007, Nature 449: 419-426; Ginhouux et al., 2007, J. Exp. 204: 3133-3146; Banerjee et al., 2006, Blood 108: 2655-2661; Sallusto et al., 1999, J. Exp. Med. 189: 611-614; Reid et al., 2000, Curr. Opin. Im munol.12: 114-121; Bykovskaia et al., 1999, J. Leukoc.Biol.66: 659-666; Clark et al., 2000, Microbes Infect. 2: 257-272).

  DCs are involved in the induction, coordination and regulation of adaptive immune responses and also serve to integrate communication between the natural and adaptive arms of the immune system's effectors. These features make DC a strong candidate for immunotherapy. DC has the unique ability to identify the environment by macropinocytosis and receptor-mediated endocytosis (Gerner et al., 2008, J. Immunol. 181: 155-164; Stoitzner et al., 2008, Cancer Immunol). Immunother 57: 1665-1673; Lanzevecchia A., 1996, Curr.Opin.Immunol.8: 348-354; Delamarre et al., 2005, Science, 307 (5715): 1630-1634).

  DC also requires a maturation signal to enhance its antigen presentation ability. By providing additional maturation signals such as TNF-α, CD40L or calcium signaling substances, DCs can be surface molecules such as CD80 and CD86 (also known as second signal molecules). Expression is upregulated (Czerniecki et al., 1997, J. Immunol. 159: 3823-3837; Bedrosian et al., 2000, J. Immunother. 23: 311-320; Maillard et al., 2004, Cancer Res. 64. 5934-5937; Brossart et al., 1998, Blood 92: 4238-4247; Jin et al., 2004, Hum. Immunol. 65: 93-103). It has been established that a mixture of cytokines including TNF-α, IL-1β, IL-6 and prostaglandin E2 (PGE2) has the ability to mature DC (Jonuleit et al., 2000, Arch. Derm. Res. 292: 325-332). DCs can also be matured with calcium ionophores prior to pulsing the antigen.

  DC are receptors that recognize pathogens, such as PKR and MDA-5 (Kalali et al., 2008, J. Immunol. 181: 2694-2704; Nallagatla et al., 2008, RNA Biol. 5 (3): 140-144) and also contains a series of receptors known as Toll-like receptors (TLRs), which are also capable of sensing risks from pathogens. . When these TLRs are stimulated, a series of activity changes are induced in DC, which results in maturation and T cell signaling (Boullart et al., 2008, Cancer Immunol. Immunother. 57 (11): 1589). Kaisho et al., 2003, Curr.Mol.Med.3 (4): 373-385; Pulendran et al., 2001, Science 293 (5528): 253-256; Napolitani et al., 2005, Nat. Immunol.6 (8): 769-776). DC can activate and prolong the action of various arms of cell-mediated responses, such as natural killer γ-δ T cells and α-β T cells, and once DCs are activated, Retain their immunization capacity (Steinman, 1991, Annu. Rev. Immunol. 9: 271-296; Banchereau et al., 1998, Nature 392: 245-252; Reid et al., 2000, Curr. Opin. Immunol.12: 114-121; Bykovskaya et al., 1999, J. Leukoc.Biol.66: 659-666; Clark et al., 2000, Microbes Infect.2: 257-272).

  The present invention also provides methods for inducing antigen presenting cells (APCs) using one or more of the peptides of the present invention. Inducing APC by inducing dendritic cells from peripheral blood mononuclear cells in vitro, ex vivo or in vivo and then contacting them with one or more peptides of the invention (activation) be able to. When the peptides of the present invention are administered to a mammal in need thereof, APCs to which the peptides of the present invention are immobilized are induced in the mammal's body. Alternatively, the peptides of the present invention can be immobilized on APC and then the cells can be administered to the subject as a vaccine. For example, ex vivo administration can include collecting APC from a mammal and contacting the APC with a peptide of the invention.

  The present invention also provides an APC that presents a complex formed between an HLA antigen and one or more peptides of the present invention. APC is obtained by contact with a peptide of the invention or a nucleotide encoding such a peptide, but is preferably derived from a subject to be treated and / or prevented, as a vaccine alone or as a peptide. Including other drugs, exosomes or the T cells of the present invention can be administered.

  The present invention provides compositions and methods for stimulating APC, preferably DC, in the context of immunotherapy for stimulating an immune response in a mammal. As DC activates anti-tumor immunity in a mammal in need thereof, the DC is activated using a peptide of the present invention or a combination of peptides of the present invention to cause DC maturation, thereby causing DC maturation. Can be operated.

In one embodiment, the present invention includes a method for inducing a T cell response in a mammal. This method comprises the step of administering APC, for example DC, wherein APC has been activated by contacting APC with a peptide of the invention or a combination of peptides of the invention, whereby It is an APC incorporating a peptide.

In one embodiment, the present invention has, among other things, for the purpose of expanding and expanding desired T cells, for the purpose of activating T cells, for the purpose of expanding and expanding specific T cells, and for those purposes. It relates to methods for using APC, and also relates to a number of therapeutic uses related to T cell expansion and activation using APCs and peptides incorporating the peptides of the present invention. In some cases, DCs activated by OCT4 can be used to expand and propagate T cells specific for the peptide.

The present invention relates to the discovery that DC contacted with a peptide of the present invention or a combination of peptides of the present invention can be used to induce expansion of T cells specific for the peptide. One skilled in the art will recognize that a DC contacted with a peptide of the invention is considered primed or, according to another expression, incorporated a peptide. The DCs incorporating the peptides of the present invention are useful for eliciting an immune response against a desired antigen, eg, HER-3. Thus, DCs incorporating the peptides of the present invention can be used to treat diseases associated with disordered expression of HER-3.

Methods for Treating Diseases The present invention also encompasses methods for treating and / or preventing diseases caused by pathogenic microorganisms, autoimmune disorders and / or hyperproliferative diseases.

  Diseases that can be treated or prevented by using the present invention include diseases caused by viruses, bacteria, yeast, parasites, protozoa, cancer cells and the like. The pharmaceutical composition of the present invention can be used as a general immune enhancing agent (composition or system that activates DC) and as such has utility in the treatment of disease. Exemplary diseases that can be treated and / or prevented using the pharmaceutical compositions of the present invention include, but are not limited to, infections caused by viruses such as HIV, influenza, herpes, viral hepatitis. Epstein Bar, polio, viral encephalitis, measles, blisters, papillomavirus, etc .; or infections caused by bacteria, eg pneumonia, tuberculosis, syphilis, etc .; or infections caused by parasites, eg malaria, Trypanosomiasis, leishmaniasis, trichomoniasis, amoebiasis and the like.

  Pre-tumor or hyperplastic conditions that can be treated or prevented using the pharmaceutical compositions of the invention (transduced DCs, expression vectors, expression constructs, etc. of the invention) include, but are not limited to, colon Pre-tumor or hyperplastic conditions such as polyps, Crohn's disease, ulcerative colitis, breast lesions and others.

  Cancers that can be treated using the compositions of the present invention include, but are not limited to, primary or metastatic melanoma, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, thymoma, lymphoma, Sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, gastrointestinal cancer, brain tumor, bladder cancer And cervical cancer.

  Other hyperproliferative diseases that can be treated using the DC activation system of the present invention include, but are not limited to, rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyoma, adenoma, lipoma, blood vessel Tumor, fibroma, vascular occlusion, restenosis, atherosclerosis, preneoplastic lesions (eg, adenomatous hyperplasia and prostate neoplasia), epithelial carcinoma, oral hairy leukoplakia or psoriasis Can be mentioned.

  Autoimmune disorders that can be treated using the compositions of the present invention include, but are not limited to, AIDS, Addison's disease, adult respiratory distress syndrome, allergy, anemia, asthma, atherosclerosis, bronchitis, cholecystitis , Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes, emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritable bowel syndrome, Lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome and autoimmune thyroiditis; cancer, hemodialysis And complications of extracorporeal circulation; infection by viruses, bacteria, fungi, parasites, protozoa and protozoa; and trauma.

  In the method of treatment, administration of the composition of the invention may be either for “prevention” or for “treatment”. The compositions of the invention, when provided for prevention, are provided in advance of any symptom, but in certain embodiments, the vaccine is provided after one or more symptoms have developed. To prevent further symptoms from developing or to prevent worsening of existing symptoms. Prophylactic administration of the composition serves to prevent or ameliorate any subsequent infection or disease. When provided for treatment, the pharmaceutical composition is provided at or after the onset of symptoms of infection or disease. Thus, the present invention can be provided before exposure to a disease-causing substance is expected or before a disease state occurs or after an infection or disease has begun.

  An effective amount of the composition is that amount that achieves these selected results that enhances the immune response, and such amount can be determined routinely by one of ordinary skill in the art. For example, an effective amount to treat a failure of the immune system against a cancer or pathogen would be that amount necessary to cause activation of the immune system upon exposure to the antigen and generate an antigen-specific immune response. . The term is also synonymous with “sufficient amount”.

  The effective amount for any particular application can vary depending on factors such as the disease or condition being treated, the particular composition being administered, the size of the subject, and / or the severity of the disease or condition. . One skilled in the art can empirically determine the effective amount of a particular composition of the present invention without necessitating undue experimentation.

Vaccine Formulation The present invention further includes a vaccine formulation suitable for use in immunotherapy. In certain embodiments, the vaccine formulation is used to prevent and / or treat diseases such as cancer and infectious diseases. In one embodiment, the administration of a vaccine according to the invention for preventing and / or treating cancer to a patient is performed before or after a surgical procedure for removing the cancer, of chemotherapy for treating the cancer. It can be performed before or after the procedure, and before or after radiation therapy to treat cancer, and any combination thereof. In other embodiments, the vaccine formulation can be administered to the patient simultaneously or in combination with another composition or pharmaceutical product. It should be understood that the present invention can also be used to prevent cancer in individuals who do not have cancer but may be at risk of developing cancer.

  Administration of cancer vaccines prepared in accordance with the present invention is broadly applicable to cancer prevention or treatment that is determined in part by the selection of the portion of the cancer vaccine that forms the antigen. Cancers that can be appropriately treated according to the practice of the present invention include, but are not limited to, lung, breast, ovary, cervix, colon, head and neck, pancreas, prostate, stomach, bladder, kidney, bone , Cancer of the liver, esophagus, brain, testis, uterus, and various leukemias and lymphomas.

  In one embodiment, the vaccine according to the invention may be derived from the tumor or cancer cell to be treated. For example, in the treatment of lung cancer, lung cancer cells will be treated as described hereinabove to produce a lung cancer vaccine. Similarly, breast cancer vaccines, colon cancer vaccines, pancreatic cancer vaccines, gastric cancer vaccines, bladder cancer vaccines, kidney cancer vaccines and the like are also produced as immunotherapeutic agents to prevent and / or prevent tumors or cancer cells from which vaccines were generated. Utilized according to the execution procedure to treat.

  In another embodiment, the vaccine according to the invention is also collected, as described, by collecting the appropriate antigens that the pathogen excreted in the medium to treat various infectious diseases affecting mammals. It could also be prepared. Because the types of immunogenic and protective antigens expressed by different organisms causing the same disease are heterogeneous, multivalent vaccines can be prepared by preparing vaccines from a pool of organisms that express different important antigens. Can be prepared.

  In another embodiment of the invention, the vaccine can be administered by intranodal injection into the inguinal lymph node. Alternatively, and depending on the target of the vaccine, the vaccine can be administered intradermally or subcutaneously to the limbs, arms and legs of the patient being treated. This approach is generally satisfactory for melanoma as well as for other cancers, but other routes of administration, including the prevention or treatment of infectious diseases, such as intramuscular or intravascular routes, are also possible. Can also be used.

  In addition, vaccines can be administered with adjuvants and / or immunomodulators to boost vaccine activity and patient response. Such adjuvants and / or immunomodulators are understood by those skilled in the art and are readily described in the available published literature.

  As contemplated herein, depending on the type of vaccine being produced, the cells are optionally cultured in a bioreactor or fermentor or other such vessel or device suitable for growing cells in large quantities. By doing so, vaccine production can be scaled up. In such devices, the culture medium is collected periodically, frequently or continuously in order to recover any material or antigen from the culture medium before such material or antigen is degraded in the culture medium. Will be done.

  A device or composition containing a vaccine or antigen that is produced, recovered and suitable for sustained or intermittent release according to the present invention is actually implanted in the body as desired, or An external application to the body would allow such materials to be released into the body relatively slowly or at a defined time.

  Another step in the preparation of a vaccine may be personalization to meet specific vaccine requirements. Those skilled in the art will appreciate such additional steps. For example, certain collected antigenic material can be concentrated and optionally treated with a detergent and ultracentrifuged to remove transplanted alloantigen.

HER3 Expression as a Biomarker for Disease Diagnosis and Treatment In another embodiment, HER3 expression may serve as a biomarker for potentially invasive disease in patients with Barrett's esophagus and high dysmorphism (HGD). Furthermore, it is possible to bring the secondary prevention of gastroesophageal cancer in some patients, a therapeutic agent for targeting HER3 or CMET is contemplated herein.

  These methods described herein are in no way exhaustive, and additional methods suitable for particular applications will be apparent to those skilled in the art. Furthermore, the effective amount of the composition can be estimated more accurately by analogy with compounds known to exert the desired effect.

<Experimental Example>
The invention will be further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to limit the invention unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed as including all modifications that will become apparent as a result of the teaching provided herein. Should.

  Without further explanation, one of ordinary skill in the art will be able to use the foregoing description and the following illustrative examples to make, use, and perform the claimed methods of the present invention. Conceivable. Accordingly, the following examples specifically point out preferred embodiments of the present invention and should not be construed as limiting the remainder of the disclosure in any way.

Creation of peptide vaccines against other receptor tyrosine kinases that cause breast cancer and other solid tumors . Experiments were designed to develop alternative therapies for patients designated as carriers of BRCA mutations. That is, it seeks an alternative to bilateral mastectomy and does not meet the need for younger patients at the genetic risk of breast cancer.

Women with the breast cancer gene mutation BRCA1 / BRCA2 have a 70% lifetime risk of developing breast cancer, and carriers of the BRCA1 mutation often develop triple-negative breast cancer. Experiments were designed to develop vaccines for this group and to assess their safety in immunity induction trials; this is the first ever vaccination for primary prevention of breast cancer Is an attempt. It will also be observed whether estrogen receptor- ^positive breast cancer, including carriers of BRCA2 mutations, can be prevented using the multivalent vaccine of the present invention.

  Experiments were designed to study receptor tyrosine kinase expression in breast cancer and DCIS from carriers of BRCA mutations. Tumors from carriers of BRCA mutations frequently overexpress the c-MET oncogene and HER-3 early, whereas tumors from non-mutated or sporadic patients are HER-2 and HER- 3 was observed to express. This is important; the targets presented for tumor immunotherapy that can be used to develop vaccines for carriers of sporadic and BRCA mutations are presented herein. This is because it became known now. This is the first distinguishing feature and can be targeted using the immune response for prevention. Accordingly, the present invention includes compositions and methods for developing vaccines and their use for prevention as an alternative to bilateral mastectomy.

Creation of peptide vaccines The HER family consists of four related signaling molecules, HER-1, HER-2, HER-3 and HER-4, which are involved in a variety of cancers. It is known that HER-2 overexpression is found in 20-30% of breast cancers. The results presented herein indicate that other HER family members are involved in both early and invasive breast cancer, as well as other cancers. For example, HER-1 is expressed on a small number of breast cancers, and those cancers are generally triple negative. c-MET is a growth factor receptor involved in the recurrence of many cancers, and this receptor activates HER-3. HER-3 is overexpressed in the colon, prostate, breast and melanoma. HER-3 is expressed in many DCIS lesions and breast cancer. In some patients receiving HER-2 vaccine, HER-3 may be detected in the remaining DCIS at the time of surgery. As a result of these findings, the possibility of targeting these molecules in addition to HER-2 in breast cancer would be beneficial.

Immunogenic peptides derived from HER-3 have been identified (FIGS. 1 and 2) and are shown below:
p11-13 (peptides 51-75): KLYERCEVVMGNLEIVLTGHNADLFSFLQW (SEQ ID NO: 1);
p81-83 (peptides 401-425): SWPPHMMHNFSVSNLTTIGGRSLYN (SEQ ID NO: 2);
p84-86 (peptides 416-440): TTIGGRSLYNRGFSLLIMKNLNVTS (SEQ ID NO: 3);
p12 (peptides 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4);
p81 (peptides 401-415): SWPPHMMHNFSVFSNL (SEQ ID NO: 5);
p84 (peptides 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6); and p91 (peptides 451-465): AGRIYISANRRQLCYH (SEQ ID NO: 7).

The results presented herein indicate that these peptides can activate CD4 T cells across many patients. Peptides can be pulsed into dendritic cells and T cells can be trained to recognize HER-3. HER-3 is expressed in triple-negative breast cancer and may result in resistance to antiestrogens in ER ^-positive breast cancer. HER-3 is also expressed in other cancers, including melanoma, lung cancer, colon cancer, prostate cancer and metastatic brain tumor. While not intending to be bound by any particular theory, peptides derived from the intracellular portion of the molecule may also be advantageous.

  Based on the disclosure presented herein, immunogenic peptides for the receptor tyrosine kinase molecules HER-1 and c-MET are based on the procedure that identified the immunogenic peptides for HER-3, Can be screened and identified. The immunogenic peptides of the present invention can be used to prepare multivalent prophylactic vaccines for breast cancer and other cancers.

  The results presented herein demonstrate the identification of the role of the HER-2 sister protein in breast cancer. These sister proteins can be effective targets and vaccines can be developed for other solid tumors. It has led to the development of peptides that can be used to target HER-1 and HER-3. Specifically, in DCIS, specific anti-HER-1, anti-HER-2 and anti-HER-3 responses in patients have been identified before and after vaccination, which Support the development of multivalent vaccines that can be used to prevent early or treat women with DCIS. The compositions of the present invention are useful for treating other cancers including, but not limited to, colon cancer, melanoma, brain tumors, lung cancer, ovarian cancer and other tumors.

Melanoma Melanoma is an invasive skin cancer that can be life-threatening if not detected early. In mice, experiments were performed using a standard dendritic cell vaccine and the dendritic cells were engineered to exhibit a mutated protein (BRAF) that causes about 70% melanoma. . When vaccinated with these dendritic cells, mice were protected from challenge with melanoma cells, indicating that a vaccine for melanoma can be developed. Without intending to be bound by any particular theory, targeting BRAF and HER-3 in combination is not limited to, but is limited to solid cancers such as colon cancer, pancreatic cancer and lung cancer and It may be useful to treat other cancers, including other gastrointestinal tumors.

  In addition, melanoma tumors have also been shown to use B cells to avoid immune surveillance, and thus it is believed that therapy can be improved by eliminating specific B cells. Experiments can be designed to assess whether changing the tumor microenvironment to a Th1-type response can help prevent evasion.

  In some cases, the vaccines of the present invention can be used to treat melanoma that has spread. In some cases, the present invention also provides a therapy for eliminating residual cells that often becomes resistant to drug therapy.

A novel strategic cytotoxic CD8 + T lymphocyte (CTL) for identifying MHC class II-promiscuous CD4 ⁺ peptides from tumor antigens for use in vaccination has historically been a major antitumor immunity Although considered to be an effector, simply boosting the CTL response with the CD8 + vaccine in various tumor types has yielded unpredictable clinical results, probably because CTL This may be because sub-optimal functions will be lacking without the help of new CD4 + T lymphocytes. CD4 + T helper type 1 (Th1) cells secrete INF-γ / TNF-α to induce tumor senescence and apoptosis. However, it remains a challenge to successfully incorporate CD4 + epitopes in the construction of cancer vaccines and generate durable, antigen-specific CD4 + immunity. An immunogenic HER3 CD4 + that exhibits class II promiscuous and generates anti-HER3 CD4 + immunity for the purpose of inclusion in a vaccine construct using the extracellular domain (ECD) of HER3 as a candidate “oncogenic driver” tumor antigen. Experiments were performed to identify peptides.

  The materials and methods utilized in these experiments are described here.

Materials and Methods To generate anti-HER3 Th1 cellular immunity, experiments were designed to identify immunogenic, class II-promiscuous HER3 CD4 + peptides using HER3 ECD as a tumor antigen.

Protocol Overview A library of 15-mer peptides was generated from the HER3 ECD, overlapping by 5 amino acids. These peptides were pulsed onto mononuclear cell-derived DCs obtained from donors and matured to a type 1 polarized (DC1; secreting IL-12) phenotype. DC1 was collected and obtained from a subject belonging to our DCIS vaccine study and known to have an anti-HER-3 Th1 response and co-cultured with purified CD4 + T cells. Using a large pool of 10 peptides, the identification process was gradually refined to obtain a single reactive epitope as measured by CD4 + T cell interferon gamma (IFN-γ) secretion. Screening 5-6 subjects and 4 peptides that appear to react across most donors: HER3 _56-70 (SEQ ID NO: 4), HER3 _401-415 (SEQ ID NO: 5), HER3 _416-430 (SEQ ID NO: 6) and HER3 _451-465 (SEQ ID NO: 7) were identified. Subjects were identified that did not show evidence of responsiveness to CD4 + T cell recognition of the HER-3 extracellular domain, 4 HER-3 peptides were pulsed to the DC1 of those subjects, and the pulsed DC1 together with CD4 T cells Cultured for 1 week and then tested for reactivity to HER-2 peptide and response to extracellular HER-3 protein. In either case, at least one peptide resulted in recognition of both the pulsed peptide on mononuclear cells and the total protein of HER-3, suggesting that primary sensitization occurred ex vivo. To do. It was also shown that healthy donors can respond to these peptides and that anti-HER-3 Th1 responses are lost in patients with triple-negative breast cancer. Gala, K .; Et al., Clin. Cancer Res 2014; 20: 1410-1416, and Datta, J. et al. Et al., “Progressive Loss of Anti-HER2 CD4 + T-helper Type 1 Response in Breast Tumorgenesis and the Potential for Immunity”, published by OncoImmunol.

Key points of the protocol further illustrated in FIG. 19:
-A library containing 123 peptide fragments 15 amino acids long, overlapping by 5 amino acids, was generated from the extracellular domain (ECD) of HER3.
-Autologous mononuclear cell-derived dendritic cells (DCs) obtained from donors are rapidly converted to type 1 (secreting DC1 → IL-12) phenotype by GM-CSF, IFN-γ and LPS Maturated and pulsed with the relevant peptide (eg, HER3 ECD or HER3 CD4 + peptide as specified). DC1 biases the Th1 response by the production of IL-12.
-Collected DC1 was allosensitized by co-culture with purified CD4 + T cells for 8-10 days.
-Sensitive CD4 + T cells (most of them antigens) against immature DCs (iDCs) pulsed with specific CD4 + peptides of interest (eg clusters of HER3 library peptides) or irrelevant class II peptide controls. Reactivate (expected to be specific).
-The supernatant was then collected from these co-cultures. A Th1 response, measured by IFN-γ ELISA, was considered antigen-specific if IFN-γ production was at least twice that produced by an irrelevant control.
-HLA-DR, DP, DQ typing on donors was performed by the Clinical of the University of Pennsylvania Clinical Immunology Laboratory to assess MHC class II promiscuousness of the CD4 ⁺ Th1 response.

  A library containing 123 overlapping 15 amino acid long peptide fragments was generated from HER3-ECD. Autologous mononuclear cell-derived DCs obtained from donors were matured to DC1 and pulsed with HER3-ECD. Harvested DC1 was co-cultured with purified CD4 T cells. Ten days later, sensitized CD4 T cells were re-activated against immature DC (iDC) pulsed with a cluster of HER3 library peptides or an irrelevant CD4 control peptide 1. A Th1 response, measured by IFN-γ ELISA, was considered antigen-specific if IFN-γ production was at least twice that produced by an irrelevant control.

  1) To identify immunogenic CD4 + peptides, obtain breast cancer patients known to show anti-HER3 ECD reactivity after DC1 vaccine pulsed with HER2; 2) immunogenicity of these peptides Confirmed by the process of “reverse” sensitization in the same patient; 3) Obtain a patient known to have demonstrated anti-HER3 ECD non-responsiveness after vaccination and use it to identify CD4 + peptides Thus, the experiment was performed in a three-step process of observing whether cells are sensitized to native HER3 ECD and thus tolerance to autoantigen (ie, HER3) is overcome / invalidated.

  The results of the experiment are described here.

Sequential screening of HER3 ECD peptide library to identify immunogenic epitopes recognized by HER3 ECD-sensitized CD4 + Th1 cells First, to identify a single immunogenic HER3 CD4 + epitope, anti-HER3 ECD Th1 sensitization was performed in 5 breast cancer patients known to be reactive. To achieve this, we start with a cluster of 10 peptides (1-10, 11-20,... Etc.) and a cluster of 3 peptides (1-3, 3-6, 7-10,... Etc.) Finally, we focused on single immunogenic HER3 peptides and reactivated HER3 ECD-sensitized CD4 + Th1 sequentially against them. Representative screens are shown in FIGS. 2, 13 and 14. Four immunogenic peptides, HER3 (56-70) (SEQ ID NO: 4), HER3 (401-415) (SEQ ID NO: 5), HER3 (416-430) (SEQ ID NO: 6) and HER3 (451-465) ) (SEQ ID NO: 7) was reproducibly identified and these were promiscuous across HLA-DR, DP and DQ subtypes. Th1 cells obtained from 4 non-HER3-responsive donors were sensitized using DC1 pulsed with 4 identified HER3 peptides, and these cells were subsequently immunized with iDC pulsed with HER3 ECD. When challenged to recognize, all donors not only showed good sensitization to individual immunogenic HER3 peptides, but also recognized native HER3-ECD.

  The results presented herein show that DC1 pulsed with a library of overlapping tumor antigen-derived peptides can identify promiscuous class II peptides for CD4 T cell vaccine development Yes. In this study, the immunogenic HER3 CD4 peptide effectively overcomes immune tolerance against self tumor antigens. Vaccines that utilize these HER3 CD4 peptides in construction can be applied to patients with cancers that overexpress HER3. Furthermore, these results quickly and reproducibly identify class II promiscuous immunogenic CD4 epitopes derived from any tumor antigen for cancer immunotherapy using the DC1-Th1 platform. A new strategy is also presented. Table 1 below shows the initial identification of immunogenic CD4 + HER3 ECD peptides in patients known to have anti-HER3 reactivity. Table 2 shows the amino acid sequences of four immunogenic HER3 CD4 + epitopes identified by sequential screening.

Confirmation of immunogenicity of identified CD4 + HER3 ECD epitopes by “reverse” sensitization, ie, the ability of individual epitope-sensitized CD4 + Th1 to recognize native HER3 ECD FIG. 15 is found to have HER3 ECD reactivity CD4 + T cells are sensitized by DC1 pulsed with each donor-specific, immunogenic HER3 epitope and reactivated against iDC pulsed with each HER3 epitope and native HER3 ECD It shows that.

  20 and 21 show the additional results of “reverse” sensitization.

CD4 + Th1 sensitized with DC1 pulsed with immunogenic HER3 epitopes was obtained from 4 HER3 ECD non-reactive donors, as seen in FIG. 16, which appears to abolish anti-HER3 immune self-tolerance . All of the resulting CD4 + Th1 cells were sensitized using DC1 pulsed with four identified HER3 peptides and subsequently challenged to recognize iDC pulsed with HER3 ECD. Donors not only showed good sensitization to individual HER3 epitopes, but also recognized native HER3 ECD.

The CD4 + HER3 epitope was tested using the HER3 extracellular domain (ECD) as a candidate “oncogenic driver” tumor antigen, as seen in FIG. Immunogenic HER3 CD4 + peptides have been identified that show differentiation and generate anti-HER3 CD4 + immunity; these peptides can be used in vaccine constructs.

Peptides derived from tumor antigens The results presented herein are:
The ability to identify promiscuous MHC class II peptides by DC1 pulsed with a library of overlapping peptides from tumor antigens to develop a CD4 + T cell vaccine;
The immunogenic HER3 CD4 + peptide effectively overcomes immune tolerance against self tumor antigens,
-To quickly and reproducibly identify class II promiscuous immunogenic CD4 + epitopes derived from any tumor antigen for cancer immunotherapy using the DC1-CD4 + Th1 platform from these results The new strategy of
-Vaccines constructed using these HER3 CD4 + peptides have shown to be worth studying in patients with cancers that overexpress HER3.

HER3 expression is a marker of tumor progression in premalignant lesions of the gastroesophageal junction Overexpression of receptor tyrosine kinases (RTKs), including members of the HER family, has prognostic and therapeutic significance in invasive esophageal gastric cancer Have. RTK expression in premalignant gastroesophageal lesions has not been extensively studied previously.

The presence of Barrett's esophagus, or metaplastic columnar epithelium of the distal esophagus, predisposes to the development of esophageal adenocarcinoma (Cameron, AJ et al., Gastroenterology 109 (5): 1541-6 (1995)) . Although histological metastasis to invasive malignancy from dysplasia has been well characterized, carcinogenesis in metaplastic cells, Ru der those with genetic changes that are incompletely understood. Several recent reports have identified human epidermal growth factor receptor 2 (HER2) expression in a subset of Barrett's esophageal lesions with dysplasia. (Almanna, K. et al., Appl. Immunohistochem. Mol. Morpol. July 16 2015) (Almanna et al. ); Al, Histopathology 61 (5): 769-76 (2012) (Fassan et al); and Rossi, E. Et al. Cell. Mol. Med. 13 (9B): 3826-33 (2009) (Rossi et al.). Furthermore, the rate of HER2 expression correlates with the degree of dysplasia, suggesting an associated pathway in tumorigenesis.

Overexpression of receptor tyrosine kinase (RTK) molecules, including members of the HER family (HER1, HER2, and HER3) and cMET (mesenchymal epithelial transition factor) are more common malignancies including breast cancer, lung cancer and gastrointestinal cancer has been confirmed in a number of tumors (Yokata, J et al., Lancet 1:.. 765-767 ( 1986), also has been confirmed even esophageal gastric identification of HER2 overexpression in a subset of breast cancer, HER2 overexpression more aggressive specific association with ecology and effective targeting of HER2 with monoclonal antibodies, was an important event in the development of targeted therapies for the treatment of solid tumors (Joensuu, H. et al., N.Eng. J. Med. 354 (8): 809-20 (2006)) This experience has been linked to other malignancies. It provides the basis for further efforts to target RTK molecules in the treatment of tumors.

Overexpression of HER2 has been identified in a small number of gastric cancer, impact on the outcomes that Do the target of trastuzumab in just a transition environment (Bang, Y.J., Et al., Lancet 376 (9742) 687-97 ( 2010) (Bang et al.) HER2 overexpression is more frequently expressed in the proximal gastric and gastroesophageal junctions compared to more distant gastric adenocarcinoma (Rajagopal, I. et al., J. Clin. Diagn.Res.9 (3):.. EC06-10 (2015)), effect on the outcome that Do targets slightly trastuzumab (Bang et al., and, Fichter, C D, et al., Int. J. Cancer 135 (7):. 1517-30 (2014 ) (Fichter et al)) HER1 and HER3 expression in gastric carcinoma, pre most studies Associated with failure (Kandel, C, et al., J.Clin.Pathol.67 (4):. 307-12 (2014), and, Hayashi, M et al., Clin.Cancer Res.14 (23):. 7843 -9 (2008) overexpression of .CMET are associated with poor prognosis in esophageal adenocarcinoma, inhibition of cMET dependent signaling modulates the activity of HER1 and HER3 (Liu, X., et al., Clin Res.17 (22):. 7127-38 ( 2011)) studies these data, which you provide a basis for further effort to characterize the RTK expression in esophageal gastric and precursor lesions are described below. in order to identify potential targets for the treatment and primary prevention, in dysplastic lesions gastroesophageal junction It aims to characterize the kick RTK expression.

After approval of the way Institutional Review Board, dysplasia (low grade dysplasia (LGD) (n = 32) , advanced dysplasia (HGD) (n = 59) ) clinical records and histology of 73 people Barrett's esophagus patients with Retrospective reviews were made retrospectively. Formalin-fixed, paraffin-embedded tissue blocks from 2003-2012 stored endoscopic biopsies and mucosal resection specimens were sectioned at 5 μm on a plus slide (Fisher Scientific, Waltham, Mass.) Followed by deparaffinization. And rehydrated. All biopsies were collected from HER1 (clone H11; 1:50; DAKO), HER2 (HercepTest, DAKO, Carpinteria, CA) and HER3 (clone RTJ.2; 1:30; Santa Cruz Biotechnology, Dallas, TX) Bond. -III device) and immunostained under a microscope (Leica Bond-III) evaluated by a single pathologist. 3 + HER staining of the membrane, as with the membrane at least 10% of the tumor cells is been 2+ HER2 staining staining as seen in Figure 22, and while looking at the positive. CMET immunohistochemical staining was performed in 42 cases where sufficient tissue was available. Moderate or intensified membrane staining of 50% or more of the tumor cells was considered positive. RTK overexpression was correlated with clinical data of either dysplasia-adenocarcinoma biopsy specimen pair or subsequent diagnosis of adenocarcinoma in the biopsy specimen to assess association with invasive cancer .

Statistical analysis Two-sided tests were performed for all analyses . Descriptive statistics are shown as continuous variable categorical variables and median (interquartile range (IQR)) frequency. Pearson χ2 test or Fisher's test, and using the Wilcoxon rank-sum test, respectively, were analyzed continuous variables and categorical variables. A P value ≦ 0.05 was considered statistically significant. All trials were bilateral . Analysis was performed using SPSS v22.0 (IBM, Armonk, NY).

result
Against patients with Barrett's esophagus total 73 persons having low degree heteromorphic a (n = 32) or high heteromorphic (n = 59), were identified and analyzed the expression of HER1, HER2, HER3 and cMET by immune staining. The median age of the cohort was 65 years (IQR 60 ~ 73 years). 81.9% were male and 87.5% were white. Alcohol use rate in the cohort is 14.3%, utilization of aggressive tobacco but was 6.3%, and 5 5.6% were former smokers. 26.4% had a family history of malignancy. As shown in Table 1 below, there was no significant difference between the LGD and HGD cohorts in measured clinical and demographic variables.

High dysmorphism (HGD) is associated with overexpression of HER1 (22.4% vs. 3.1%, p = 0.016), overexpression of HER2 (5.3 ) compared to low grade dysplasia (LGD). % Vs. 0.0%, p = 0.187) , and HER3 overexpression (45.6% vs. 9.4%, p <0.001) .

Lesions of invasive esophageal adenocarcinoma is associated with dysplastic lesions in 6 cases, all occurred in connection with HG D (HGD: 10.2% vs. LGD: 0.0%, p <0 .001 ) . Additional 9 patients were diagnosed with invasive esophageal adenocarcinoma in subsequent raw Inspection body (HGD: 17.0% vs. LGD: 0.0%, p = 0.017 ). As seen in FIGS. 23A and 23B, overexpression in HGD lesions was significantly associated with HER3 (71.4% vs. 38.6%, compared to HGD lesions without invasive cancer lesions). p = 0.032), not significantly associated with HER1 or HER2 (increased HER1: 26.7% vs. 20.5%, p = 0.616, increased HER2: 14.3% vs. 2. 3%, p = 0.077).

Overexpression of cMET was observed in 18 of 42 evaluated samples (42.9%) and was more observed in HGD samples compared to LGD samples (58.3% vs 36.7%, p = 0.200) and in most cases co-expressed with HER3 (62.5% of HER3 positive specimens vs. 38.2% of HER3 negative specimens (p = 0.212)). HER1 positive (p = 0.729) or HER2-positive similar trend with (p = NA) specimens was observed. Invasive cancer was confirmed in 1 out of 42 (5.6%) patients. In this patient, cMET was overexpressed (p = 0.243).

The analysis of RTK expression in dysplasia lesions debate gastroesophageal junction, (1) HER family proteins are A Ppuregyureto in Barrett's esophagus with dysplasia Rukoto; (2) the frequency of the HER family and cMET overexpression that there is a positive correlation with the degree of dysplasia; upregulation of (3) HER protein, especially in dysplastic lesions, to be associated with increased incidence of associated invasive cancer, to confirm.

Thus, HER3 may serve as a biomarker for potential invasive disease in Barrett's esophagus and HGD patients. Furthermore, therapeutic agents that target HER3 or CMET can provide secondary prevention of gastroesophageal cancer in a subset of patients .

Previous assessments of HER expression in Barrett's esophagus were limited to assessments of HER2 overexpressed in a few cases (see Almhanna et al., Fassan et al., And Rossi et al. ) . Overexpression of HER2 in this study was present in 3.3% of biopsy specimens and was lower than the overexpression rate of HER1 or HER3. This pattern is consistent with HER family protein expression in invasive gastroesophageal junction cancer where HER3 is more commonly overexpressed than HER2 ( Fichter et al. ) . Increased overexpression of HER3 protein with progression from LGD to HGD and especially frequent overexpression of HER3 are not surprising but novel findings. HER receptor homodimerization and heterodimerization drive signal activation, and clustered overexpression of multiple members of the HER family has been observed in other tumor types. In addition, c-MET The activated positively regulate the activity of HER1 and HER 3 ^11. Indeed, the interaction between these receptors that have multiple receptor tyrosine kinases provide rationale of multivalent therapeutic approaches targeting (Baselga, J., Et al., N. Eng.J.Med. 366 (2): 109-19 (2012); Waddell, T. et al., Lancet Oncol.14 (6): 481-489 (2013) ).

This data is also, still suggests the opportunity of targeting secondary prevention of gastro-esophageal cancer that has not been investigated. Previously targeted HER2 expression in breast non-invasive ductal carcinoma (DCIS) targeted promising results ( US patent application Ser. No. 14 / 658,095 filed Mar. 13, 2015, U.S. Patent Application No. 14 / 985,303, filed December 30, 2019, Datta, J. et al., Onco Immunology 4: 8 e1022301 (2015) DOI: 10.080 / 216402X.2015.1022301; J. et al., Breast Cancer Res. 17 (1): 71 (2015) ) . Such an approach remains a farther target for gastrointestinal malignancies. Nevertheless, all current treatment options for Barrett's esophagus, including endoscopic resection and excision modes and radical surgery, have significant limitations. Alternative strategies are needed that eliminate pathological conditions and reduce the risk of invasive cancer.

The analysis of RTK expression in dysplastic lesions gastroesophageal junction (1) the HER family proteins are A Ppuregyureto in Barrett's esophagus with dysplasia, (2) Frequency of HER family and cMET overexpression different that there is a degree positively correlated with formation; (3) upregulation of HER proteins, especially in dysplastic lesions, to be associated with increased incidence of associated invasive cancer, to confirm.

  Thus, HER3 may serve as a biomarker for potential invasive disease in Barrett's esophagus and HGD patients. Furthermore, therapeutic agents that target HER3 or CMET can provide secondary prevention of gastroesophageal cancer in a subset of patients.

In summary, the data show the relationship between frequent overexpression of HER3 and malignant transformation in highly dysplastic lesions at the gastroesophageal junction, especially in highly dysplastic lesions with occult invasive cancer . These findings are likely to justify a more aggressive management approach HER3 expressing dysplastic lesions, as will be readily appreciated by those skilled in the art, for future application of HER3 targeted therapy in early disease Environment Provides a rationale for

  The disclosures and publications of any and all patents, patent applications cited herein are hereby incorporated by reference in their entirety. Although the invention has been disclosed with reference to specific embodiments, it is to be understood that other embodiments and modifications of the invention may be devised without departing from the true spirit and scope of the invention. It is clear to the contractor. The appended claims are to be construed to include all such embodiments and equivalent variations.

Claims (24)

  p11-13 (Peptides 51-75): KLYERCEVVMGNLEIVLTGHNADLFSFLQW (SEQ ID NO: 1), p81-83 (Peptides 401-425): SWPPHMNFSFSNLTTIGRGSLYN (SEQ ID NO: 2), p84-86 (Peptides 416-440ML) , P12 (peptides 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4), p81 (peptides 401-415): SWPPHMHNFSVFSNL (SEQ ID NO: 5), p84 (peptides 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6), and p91 (peptide) 451-465): selected from the group consisting of AGRIYISANRQLCYH (SEQ ID NO: 7) Away peptide.   An immunomodulator comprising one or more peptides according to claim 1.   A vaccine comprising one or more peptides according to claim 1 and a pharmaceutically acceptable salt.   The vaccine of claim 3 further comprising an adjuvant.   A cell that has been contacted with one or more of the peptides of claim 1.   The cell according to claim 5, which is an antigen-presenting cell.   The cell according to claim 5, which is a T cell.   A method for raising an immune response in a subject comprising administering to the subject a composition according to claim 1.   A method of treating cancer in a subject comprising administering to the subject one or more of the peptides of claim 1.   The method of claim 9, wherein the subject is a human and has cancer.   11. The method of claim 10, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, lung cancer, prostate cancer, colon cancer, melanoma, pancreatic cancer, gastrointestinal cancer, brain tumor and any combination thereof.   A method of activating a cell comprising contacting said cell with one or more peptides according to claim 1.   13. The method of claim 12, wherein the cell is an antigen presenting cell.   The method of claim 12, wherein the cell is a T cell. A method for generating activated dendritic cells (DCs) loaded with peptides for use in immunotherapy comprising the steps of:
Pulsing the DC with one or more of the peptides of claim 1;
Activating said DC with at least one TLR agonist.   16. The method of claim 15, comprising contacting the DC with an agent that increases intracellular calcium concentration in the DC.   The method of claim 15, wherein the agent comprises a calcium ionophore.   16. The method of claim 15, further comprising cryopreserving the DC, wherein when the DC is thawed, the DC generates an effective amount of at least one cytokine to generate a T cell response.   A cell produced from the method of claim 15.   16. A vaccine comprising cells produced from the method of claim 15.   21. The vaccine of claim 20, wherein the vaccine takes the form of an injectable multi-dose vaccine.   21. A method of eliciting an immune response in a mammal comprising administering to the mammal in need thereof a cell population generated from the method of claim 20.   21. A method of treating a disease or disorder in a mammal, comprising administering to the mammal in need thereof a cell population generated from the method of claim 20.   A biomarker for detecting tumor progression in a premalignant lesion of the gastroesophageal junction in a subject having Barrett's esophagus, comprising detecting overexpression of HER3 in the subject.