JP2018521953A - Identification of immunogenic MHC class II peptides for immunity based therapy - Google Patents

Abstract

The present invention provides compositions, methods and vaccines that can activate the immune system and can be used to treat malignancies associated with overexpression of HER-3 protein. Such compositions comprise an epitope of the HER-3 protein.
[Selected figure] Figure 1

Description

(Cross-reference to related applications)
This application is a continuation-in-part of PCT / US16 / 21042 filed on March 4, 2016, which application is a continuation-in-part of PCT / US15 / 41034 on July 17, 2015 . The application claims priority to US Provisional Patent Application No. 62 / 076,789, filed Nov. 7, 2014, and US Provisional Patent Application No. 62/025, 681, filed Jul. 17, 2014. Do. The entire content of each patent application is incorporated herein by reference.

(Sequence listing)
This application contains a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. This ASCII copy, created on 14 June 2016, is 319-003_PCT_CIP2_SL. It is named txt and its size is 2,666 bytes.

  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) enhances tumor aggressiveness and relapse and It is 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 ("RTK"). As one of the four members of the human epidermal growth factor receptor ("EGFR") family, HER2 stands out from others in several aspects. First, HER2 is an orphan receptor. High affinity ligands have not been identified. Second, HER2 is a preferred partner for other EGFR family members (HER1 / EGFR, HER3 and HER4) to form heterodimers, which have high ligand affinity and superiority. Signal transduction activity. Third, full-length HER2 undergoes proteolytic cleavage to release soluble extracellular domain ("ECD"). Shedding of ECD has been shown to be an alternative activation mechanism for full-length HER2 both in vitro and in vivo, as membrane anchor fragments with kinase activity remain. The central role of HER2 in EGFR family signaling correlates with its involvement in carcinogenesis of several types of cancer, for example 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 cause tumor cells to be resistant to certain chemotherapies (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 RTKs regulate a wide 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). At the moment, epidermal growth factor ("EGF"), heparin-binding EGF-like growth factor ("HB-EGF"), transforming growth factor alpha (TGFα), amphiregulin (AR), epiregulin, betacellulin and heregulin Eleven ligands have been reported, including. These ligands bind directly to their cognate receptors, which leads to the formation of homodimers or heterodimers of the receptor that cause activation of multiple signal transduction pathways. Dysregulation of the HER-family members either by mutational activation, 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. When HER3 is activated by heregulin, it 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 proliferation and survival. To date, HER3 is inactive as a kinase due to the apparently unusual sequence features in its kinase domain, and the kinase activity of the HER-family is impaired to initiate signal transduction events It has been speculated that heterodimerization with members not required is required. Consistent with this, it has been shown that HER2 requires HER3 to drive cell growth of mammary tumors. However, recent findings have also shown that HER3 is also able to phosphorylate Pyk2, resulting in activation of 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 evade HER-family inhibitor therapy by up-regulating HER3 signaling, and HER3 inhibition abrogates HER2-driven tamoxifen resistance in breast cancer cells Indicated. Furthermore, resistance to the EGFR small molecule inhibitor gefitinib (Iressa) therapy has also been shown to be linked to HER3 signal activation.

  HER3 is a receptor protein and plays an important role in regulating normal cell growth. HER3 lacks its intrinsic kinase activity and, depending on the presence of HER2, transduces signals across cell membranes. Upon transcription, HER3 pre-mRNA contains 28 exons and 27 introns. The intron has been spliced out and the fully spliced HER3 mRNA is composed of 28 exons.

  In the past decade, targeted therapies have emerged as the basis for cancer therapeutics. Members of the EGF receptor family, namely EGFR (or HER1) and ErbB2 (or HER2 / neu), are becoming regarded as particularly intriguing targets, since these RTKs It is because it is deregulated in cancer. The oncogenic function of another member of the EGF receptor family, ErbB3 or HER3, has only recently become controversial due to its ability to mediate resistance to HER2 and its major role in therapies directed against the PI3K pathway It came to be done. Mutation activation 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 precursor.

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

  Despite advances in the art, it remains uncertain whether an effective immune response can be generated in humans using cell or protein based vaccine strategies that target HER3. Thus, there is a need in the art to have additional immunotherapeutic approaches to treat or prevent breast cancer and other malignancies associated with overexpression of HER3 protein. The present embodiment meets 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. Presently preferred embodiments are illustrated in the drawings in order to illustrate the present invention. However, it should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 16 is a graph showing an immunogenic peptide derived from HER3 showing the ability to activate CD4 T cells across many patients (each in order of appearance, SEQ ID NO: 1-3). FIG. 10 is a graph showing the overall screen of HER3 using a group of 10 peptide fragments. Also shown is a screen plot of HER3 using a single peptide (SEQ ID NOS: 4-7, respectively, in order of appearance). FIG. 10 is a graph showing the overall screen of HER3 using a group of 10 peptide fragments. Also shown is a graph of the screen of HER3 with a single peptide (SEQ ID NOS: 4 and 7, respectively, in apparent order). FIG. 10 is a graph showing the overall screen of HER3 using a group of 10 peptide fragments. Also shown is a graph of the screen of HER3 with a single peptide (SEQ ID NO: 1-3, respectively, in apparent order). FIG. 5 is a graph showing the generation of IFN-γ from different HER3 peptides. FIG. 5 is a graph showing the generation of IFN-γ from different HER3 peptides. FIG. 16 is a graph showing the generation of IFN-γ from a “reverse” screen sensitized to the peptide and HER3 extracellular domain after previously identifying the peptide. FIG. 16 is a graph showing the generation of IFN-γ from a “reverse” screen sensitized to the peptide and HER3 extracellular domain after previously identifying the peptide. FIG. 16 is a graph showing the generation of IFN-γ from a “reverse” screen initiated with the peptide and sensitized to the peptide and HER3 extracellular domain in patients not previously sensitized to the HER extracellular domain. Graph showing the generation of IFN-γ from a “reverse” screen initiated with the entire peptide library and sensitized to the peptide and HER3 extracellular domain in patients not previously sensitized to the HER extracellular domain It is. Graph showing the generation of IFN-γ from a “reverse” screen initiated with the entire peptide library and sensitized to the peptide and HER3 extracellular domain in patients not previously sensitized to the HER extracellular domain It is. FIG. 16 is a graph showing the generation of IFN-γ from a “reverse” screen initiated with the peptide and sensitized to the peptide and HER3 extracellular domain in patients not previously sensitized to the HER extracellular domain. FIG. 16 is a graph showing a screen of consecutive peptides in the UPCC 15107-24 donor. FIG. 16 is a graph showing a screen of consecutive peptides in the UPCC 15107-38 donor. FIG. 17 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 to show no anti-HER3 reactivity to the identified HER3 peptide and / or native HER3 ECD) FIG. 6 is a graph showing that DC1 pulsed with HER3 epitope sensitized CD4 + Th1 and overcome anti-HER3 immune tolerance. FIG. 16 is a graph showing that CD4 + HER3 epitopes show MHC class II promiscuity. When activated HER-3 CD4 + cells are placed alongside cells expressing HER-3 in a chamber, HER-3 CD4 + cells cause apoptosis or death of breast cancer cells expressing HER-3 FIG. FIG. 1 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. 16 is a graph showing that the immunogenicity of the identified CD4 + HER3 ECD epitope was confirmed by “reverse” sensitization. A screen of HER3 ECD was performed using the single peptide shown. FIG. 7 is a graph showing additional results where the immunogenicity of the identified CD4 + HER3 ECD epitope was confirmed by “reverse” sensitization. The photograph of immunohistochemistry scoring of HER staining is shown. A and B show overexpression rates of the HER family in Barrett's esophagus with low grade dysplasia (LGD) or high grade dysplasia (HGD) (Figure 22A), and high grade dysplastic Barrett lesions (carcinoma FIG. 22B is a histogram showing HGD with or without associated invasive cancer (HGD) (FIG. 22B). Figures 24A-24C show reduced anti-HER3 CD4 Th1 cell response from HD (healthy donors) to ER IBC / ER ^pos IBC (estrogen receptor positive invasive breast cancer (IBC)) and TN IBC (triple negative IBC). The figure shows histograms (left panel) of IFN-γ ELISpot analysis of systemic CD4 + Th1 cell responses. The group of patients studied is HD; BD (benign chest biopsy); DCIS (HER2 positive ("HER2 ^pos ") luminal carcinoma) HER2 IBC / HER2 ^pos IBC; ER IBC / ERpos IBC (estrogen receptor positive IBC) And TN IBC (triple negative IBC). The corresponding table to the right of each histogram is an individual comparison by student's t-test between two groups at a time. One-way ANOVA tests were performed in all groups. FIG. 24A shows that cumulative anti-HER3 CD4 T cell responses measured by IFN-γ spots per million cells via ELISpot assay, from HDS to BD, DCIS to HER2 ^pos IBC to ER ^pos IBC, and finally In the figure, it shows that it decreased from TN IBC (90 vs. 80 vs. 66 vs. 79 vs. 48 vs. 40, respectively p = 0.01). Figure 24B shows that the number of HER3 peptides with repertoire or positive CD4 Th1 response is HD to BD, DCIS to HER2 ^pos IBC to ER ^pos IBC, and finally TN IBC (1.0 vs 0.6 vs 0) .8 vs 0.8 vs 0.5 vs 0.3, respectively p = 0.003). FIG. 24C shows responsiveness, percent of subjects responding to at least one peptide, DCIS to HER2 ^pos IBC-ER ^pos IBC, and finally TN IBC (76.7% vs. 63.6% vs. 53.8). % Vs. 66.7% vs. 45.0% vs. 33.3% (p = 0.02 respectively). Figures 25A and 25B show that loss of CD4 T cell response is specific to HER3 as there is no difference in tetratonus or anti-CD3 / CD28 stimulation among the group of patients tested. The figure shows histograms (left panel) of IFN-γ ELISpot analysis of systemic CD4 + Th1 cell responses. The patient groups studied are: HD; BD; DCIS; HER2 IBC / HER2 ^pos IBC; ER IBC / ER ^pos IBC; and TN IBC. The corresponding table to the right of each histogram is an individual comparison by student's t-test between two groups at a time. One-way ANOVA tests were performed in all groups. FIG. 25A shows tetanus as measured by IFN-γ spots per 200,000 cells via an ELISpot assay between HD, BD, DCIS, HER2 IBC / HER2 ^pos IBC, ER IBC / ER ^pos IBC or TN IBC. It shows that there is no statistically significant difference in response (37 vs. 30 vs. 19 vs. 34 vs. 24 vs. 29, respectively, p = 0.65). FIG. 25B shows anti-CD3 / anti-CD28 polyclonal stimulation measured by IFN-γ spots per 200,000 cells via ELISpot assay between HD, BD, DCIS, HER2 IBC / HER2 ^pos IBC, ER IBC / ER ^pos IBC There is no statistically significant difference between (688 vs. 549 vs. 804 vs. 699 vs. 629 vs. 675, respectively, p = 0.68). 26A-26C show that anti-HER3 CD4 T cell responses correlate with relapse and response to neoadjuvant chemotherapy but not in lymph node metastases. FIG. 26A compares the immune response of IBC patients with lymph node status in initial surgery (lymph node positive (“LN +” or “LN ^pos ” vs. lymph node negative (“LN-” or “LN ^neg ”)) in IBC patients. With histogram (second panel) (0.4 vs. 0.6, respectively p = 0.08), responsiveness (third panel) (35.7% vs. 54.8%, respectively p = 0.19) , Cumulative response (upper panel) (40 vs. 56, respectively p = 0.12), repertoire or tetanus response (lower panel) (22 vs. 29 each, p = 0.35) Figure 26B shows us. Compare the immune response of IBC patients to relapse versus non-relapse (no disease) in at least one year patients with a significantly lower cumulative response (upper panel) (17 vs. 66, p = 0.04) from diagnosis of 4 Two With grams), repertoire (second panel: 0.0 vs 0.6, p <0.05 respectively) and responsiveness (third panel) (0% vs 55.6%, respectively p = 0.01) . There was no difference in tetanus response between relapsed and non-relapsed IBC patients (lower panel) (27 vs. 35, p = 0.65, respectively). 26C has four histograms comparing the immune response of IBC patients with response to neoadjuvant chemotherapy (pathologic complete response ("pCR") vs. residual disease ("<pCR"). Neoadjuvant chemotherapy Among patients receiving pCR, compared to patients with pCR, showed a significantly higher cumulative response (upper panel is 144 vs. 32, p = 0.004 respectively) and Repertoire (second panel) (0.8 vs 0.4, p = 0.05 respectively) Responsiveness (third panel) (80.0% vs. immune response) between pCR and <pCR patients' immune response There was no 27.3%, respectively p = 0.10) or tetanus response (lower panel) (17 vs. 59, each p = 0.15). Figures 27A-27D show that anti-HER3 CD4 T cell responses are significantly higher in postmenopausal HD / BD but do not change with age, race or pregnancy history. FIG. 27A has four histograms comparing the immune response of HD patients with age (<50 (<50) vs. <50). Repertoire (second panel: 0.8 vs 1.1, p = 0.38, respectively), responsiveness (third panel) (second panel): There were no statistically significant differences by age in cumulative response ( 72.0% vs. 75.0%, respectively p = 1.0) or tetanus response (lower panel) (39 vs. 30, p = 0.40 respectively). FIG. 27B has four histograms comparing the immune response of HD patients by race (Caucasus vs. African American vs. Other). There was no statistically significant difference in the races in the cumulative response (upper panel) (87 vs. 83 vs. 95, respectively p = 0.96), the third panel (69.0 vs. 71.4% vs. 100%) (P = 0.35) or tetanus response (lower panel) (second panel) (33 vs. 51 vs. 26 each, p = 0.30). FIG. 27C shows statistically significant differences in cumulative responses (upper panel) by pregnancy history (0 and 1 pregnancy) with 4 histograms comparing the immune response of HD patients by pregnancy history / pregnancy None (82 vs. 91, respectively p = 0.71), (second panel) (1.0 vs. 0.9, respectively p = 0.62) or responsiveness (third panel) (76.5% vs. 70.8%, respectively p = 0.74). Interestingly, the tetanus response (lower panel) was significantly higher in the nanny compared to patients with at least one pregnancy (47 vs. 27, p = 0.04 respectively). FIG. 27D has four histograms comparing the immune response of HD patients with menopausal status (pre-menopausal-post-menopausal). Compared to pre-menopausal HD / BD, compared with pre-menopausal HD / BD, it showed a significantly higher cumulative response (upper panel) (136 spots vs 70 spots per million cells respectively, p = 0.005), repertoire (second panel) (1.4 vs 0.8 peptides, respectively p = 0.03). Postmenopausal and menopause by response (third panel) (90.9% vs 66.7%, respectively p = 0.23) or tetanus response (lower panel) (38 vs. 28, p = 0.37) There was no difference between previous HD / sBD. FIG. 28 is a graph of the determination of the linearity of ELISpot. The ELISpot assay was determined to be linear and accurate under the operator who performed all the assays for this study by serial dilution of known anti-HER3 CD4 T cell responders into media. The cumulative response is from a dilution of 0.01 to 0.001 from 1.0 to 0.1 (230 to 35 to 12 to 5 spots per million cells, respectively p <0.0001, r ² = 0.88) Followed a linear regression curve.

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

  In one embodiment, the invention comprises a composition comprising a multivalent peptide. Multivalent peptides include two or more peptides of the invention.

  Further provided are methods of activating an immune response and methods of treating cancer in a subject. Also provided are vaccines for therapeutic and prophylactic use. The peptides of this example 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 for tumor progression in precancerous lesions of the gastroesophageal junction.

  In another embodiment, anti-HER3CD4 Th1 loss is determined which involves the use of HER3 MHC class II immunogenic peptides.

Definitions Unless otherwise defined, 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 pertains. 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, the nomenclature used herein as well as laboratory procedures in cell culture, molecular genetics, organic chemistry and chemistry and hybridization of nucleic acids are well known and commonly employed in the art .

  Standard techniques are used for nucleic acid and peptide synthesis. Techniques and procedures generally refer to conventional methods in the art and various general references (e.g., Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY, And according to Ausubel et al., 2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout the present description.

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

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

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

  As used herein, “about” when referring to a measurable value, such as amount, time period, etc., is ± 20%, or ± 10%, or ± from a particular value. It is intended to encompass variations of 5%, or ± 1%, or ± 0.1%, such variations being appropriate to practice the disclosed method.

  When used in the context of an organism, a tissue, a cell or components thereof, the term "abnormal" means that at least one observable or detectable feature (eg, age, treatment, time of day, etc.) An organism, tissue, cells or components thereof that differ from the organisms, tissues, cells or components thereof that exhibit the "normal" (expected) respective characteristics. For one cell or tissue type, normal or expected characteristics may be abnormal for different cell or tissue types.

  As used herein, "adjuvant therapy" for breast cancer refers to any therapy given after the primary therapy (ie, surgery) to increase the chance of long-term survival. "Neoadjuvant therapy or neoadjuvant therapy" or treatment given prior to 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 antibody production or activation of specific 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. Furthermore, antigens can also be obtained from recombinant or genomic DNA. It is understood by those skilled in the art that any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes the term "antigen" as used herein Will. Furthermore, one skilled in the art will also 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 various combinations of these nucleotide sequences can be easily arranged to elicit the desired immune response. It becomes clear. Furthermore, one skilled in the art will also understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that the antigen may be produced synthetically or obtained from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

  An "antigen presenting cell" ("APC") is a cell capable of activating T cells, including, but not limited to, mononuclear cells / macrophages, B cells and dendritic cells (DCs).

  "Antigen-loaded APC" or "antigen-pulsed APC" includes APCs that have been exposed to antigen and have been activated by antigen. For example, APC can be loaded with Ag in vitro, eg, during culture in the presence of antigen. The APC can also be loaded by exposure to the antigen in vivo. "Antigen-loaded APC" is conventionally either of two methods: (1) "pulsing" a small peptide fragment known as an antigenic peptide directly onto the exterior of the APC, or (2) APC. Incubate with whole protein or protein particles, and then prepare whole protein or protein particles by either ingesting APC. 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 antigen-encoding polynucleotides into cells.

  "Anti-HER3 response", "anti-HER3 CD4 Th1 response", "anti-HER3 CD4 T cell response" and the like mean an immune response specific to HER3 protein.

  The term "anti-tumor effect" as used herein, refers to a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, a prolongation of life expectancy, or various physiological conditions associated with a cancerous condition Refers to biological effects that can be manifested by amelioration of In the first place, the "anti-tumor effect" can also be revealed by the ability of the peptides, polynucleotides, cells and antibodies of the invention in preventing the development of tumors.

  The term "autoimmune disease" as used herein is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of inappropriate and excessive responses to self antigens. Notably, examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotiditis, Crohn's disease, diabetes (type I), nutrition Failure 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, Sjögren's syndrome, spondyloarthritis, thyroiditis, vasculitis, vitiligo, myxedema, malignant anemia, ulcerative colitis.

  As used herein, the term "autologous" is intended to refer to any material obtained from an individual that is later intended to be reintroduced into the same individual.

  The term "B cells" as used herein is defined as cells 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 hyperproliferation of cells and results from the abandonment of normal control, which is a unique trait of cancer, resulting in unregulated growth, lack of differentiation, invasion of local tissue. 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, renal cancer, liver cancer, brain cancer, brain cancer, lymphoma, leukemia, lung cancer, germ cell tumor, etc. It can be mentioned.

  "CD4 + Th1 cells on the surface", "Th1 cells", "CD4 + T helper type 1 cells", "CD4 + T cells" etc. express the surface protein CD4 and produce high levels of T helper cells Subtype cytokine IFN-γ of helper cells. See also "T helper cells".

  The "cumulative response" is a group of patients represented as the sum of reaction spots (Spot forming cells per 106 cells from IFN-γ ELISpot analysis "SFC") from all MHC class II binding peptides from a given patient Means a complex immune response group.

  "DC vaccination", "DC immunization", "DC1 immunization" and the like use autologous dendritic cells to recognize specific molecules by using the immune system and acquire specific responses against them Point to a strategy.

  The terms "dendritic cells" or "DCs" are antigen presenting cells which may be present in vivo, in vitro, ex vivo, or in a host or subject, or derived from hematopoietic stem cells or monocytes. Dendritic cells and their precursors can be isolated from various lymphoid organs such as spleen, lymph nodes, and bone marrow and peripheral blood. DC have a characteristic morphology with thin sheets (lamellipodia) extending in multiple directions from dendritic cell bodies. Typically, dendritic cells express high levels of MHC and costimulatory (e.g., B7-1 and B7-2) molecules. Dendritic cells can induce antigen-specific differentiation of T cells in vitro and can initiate primary T cell responses in vitro and in vivo. In the context of vaccine production, "activated DC" is DC exposed to a Toll-like receptor agonist such as lipopolysaccharide "LPS". Activated DC may or may not be loaded with antigen.

  A "disease" is a state of health of an animal where the animal can not maintain homeostasis and if the disease does not ameliorate the health of the animal continues to deteriorate.

  A "disorder" in an animal refers to the case where the animal is capable of maintaining homeostasis, but where the animal's health is less favorable than in the absence of the disorder. Left untreated, a disorder does 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.

  An "effective amount" or "therapeutically effective amount" is used interchangeably herein and, as described herein, a compound, formulation, material or composition effective to achieve a particular biological result. It refers to the amount of goods. Such results may 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 introduced from or produced outside of an organism, cell, tissue or system.

  "HER receptor" is a receptor protein tyrosine kinase belonging to the HER receptor family, and is an EGFR (ErbB1, HER1) receptor, a HER2 (ErbB2) receptor, a HER3 (ErbB3) receptor and a HER4 (ErbB4) receptor including. The HER receptor is generally capable of binding to a HER ligand and / or dimerizing with another HER receptor molecule; an extracellular domain; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain As well as the carboxyl terminal signal transduction domain with several tyrosine residues which 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, HER activation results in signal transduction (eg, signal transduction triggered by the intracellular kinase domain of the HER receptor which phosphorylates a tyrosine residue or substrate polypeptide in the HER receptor). HER activation can be mediated by binding of the 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 causing one or more HER receptors to Phosphorylation of tyrosine residues and / or phosphorylation of additional substrate polypeptides, such as, for example, phosphorylation of tyrosine residues in Akt intracellular kinase or MAPK intracellular kinases occur.

  "HER2" is a member of the human epidermal growth factor receptor ("EGFR") family. HER2 is overexpressed in about 20-25% of human breast cancers and is expressed in many other cancers.

  "HER2pos" is a classification or molecular subtype of certain types of breast cancer as well as many other cancers. HER2 positive is currently defined by gene amplification by FISH (fluorescent in situ hybridization) assay and 2+ or 3+ of pathological staining intensity.

  "HER2 neg" is defined by the absence of gene amplification by FISH and can almost always encompass the range of pathological staining form 0-2 +.

  “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). Receptor polypeptide.

  "The extracellular domain of HER3" or "HER3 ECD" refers to a domain of HER3 that is external to the cell and that is anchored or circulating to 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 signal peptide). In one embodiment, HER3 domain III comprises amino acids 328-532 (numbering including signal peptide).

HER3 immunogenic peptides, "HER3 binding peptides", "HER3 epitopes" etc. as used herein are MHC class II peptides derived from or based on the sequences of HER3 proteins, in particular HER3 ECD and Refer to their equivalents. HER3 peptides can activate CD4 T cells across many patients. This peptide can be used to pulse dendritic cells and teach T cells to recognize HER3. HER3 is expressed in triple negative breast cancer and can confer resistance to anti-estrogen in ERpos breast cancer. HER3 is also expressed in other cancers, including melanoma, lung, colon, prostate cancer, and metastatic brain tumors. According to a preferred embodiment, four HER3 immunogenic peptides (epitopes) or binding peptides are identified as follows:
p12 (peptide 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4).
p81 (peptides 401-415): SWPPHMNHFSVFSNL (SEQ ID NO: 5).
p84 (peptides 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6). And p91 (peptides 451-465): AGRIYISANRQLCYH (SEQ ID NO: 7).

  “Homology” as used herein refers to two polymeric molecules, eg, two nucleic acid molecules, eg, a subunit between two DNA molecules or two RNA molecules, or two polypeptide molecules. Refers to sequence similarity. When the same monomer subunit occupies a certain subunit position in both of two molecules, for example, if adenine occupies a certain position in each of two DNA molecules, these molecules Are completely homologous or 100% homologous. The percent homology between two sequences is a direct function of the number of matching or homologous positions, eg, half of the positions (eg, a length of 10 subunits in the two compound sequences) These two sequences are 50% identical if five positions in the molecule are homologous, and these two if 90% of the positions, eg 9 out of 10, are matched or homologous. The sequences share 90% homology. By way of example, the DNA sequences 5 'ATTGCC 3' and 5 'TATTGGC 3' share 50% homology.

  Furthermore, when the terms "homology" or "identity" are used herein to refer to nucleic acids and proteins, it is understood to apply to homology or identity at both the nucleic acid and amino acid sequence levels. Should.

  The term "hyperproliferative disease" is defined as a disease resulting from hyperproliferation of cells. 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 materials" includes publications, records, diagrams or any other presentation media that can be used to convey the utility of the compositions and methods of the present invention. Instructions for use of the kit of the present invention are attached, for example, to a container containing the nucleic acid, peptide and / or composition of the present invention, or shipped together with the container containing the nucleic acid, peptide and / or composition be able to. Alternatively, the instructional material can be shipped separately from the container, with the intention that the instructional material and the compound be used in combination by the recipient.

  By "immune response" as used herein is meant activation of the host's immune system, eg, the mammalian immune system, in response to the introduction of an antigen. The immune response may take the form of a cellular or humoral response, or both.

  "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not in the "isolated state", but the same nucleic acid or peptide partially or completely separated from coexisting materials in its natural state 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, when compared to the level of response in a subject in the absence of a therapeutic or compound, and / or otherwise identical but not identical Means of mediating a detectable increase or decrease in the level of response in the subject when compared to the level of response in the subject of treatment. The term encompasses perturbing and / or affecting the native signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

  For each subject group analyzed for anti-HER3 CD4 + Th1 immune responses, a "metric" of CD4 + Th1 responses or "metrics of immune response" is defined: (a) overall anti-HER3 responsiveness (≧ 1 immunity) (B) Response repertoire (expressed as the average number of immunogenic peptides recognized by each target group); (c) From four MHC class II HER3 immunogenic peptides from each subject group Cumulative response, expressed as the sum of the reactive spots (spot forming cells per 106 cells from IFN-γ ELISpot analysis “SFC”).

  "Peptide", "protein" or "polypeptide" as used herein can mean a sequence of linked amino acids and can be natural, synthetic, or a modified form or combination of natural and synthetic .

  As used herein, "population" includes reference to cultures in isolated form, including homogeneous, substantially homogeneous, or xenogeneic cell cultures. In general, "population" can also be considered as "isolated" cell culture.

  Receptor tyrosine kinases ("RTKs") are high affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. The human EGF receptor ("HER") family of RTKs regulate a wide variety of biological processes including cell proliferation, migration, invasion and survival. The family consists of four members: HER1 (ErbB1), HER2 (neu or ErbB2), HER3 (ErbB3), and HER4 (ErbB4).

  As used herein, a "recombinant cell" is a host cell that comprises a recombinant polynucleotide.

  "Responsive" or "anti-HER3 responsive", as used interchangeably herein, means the percentage of subjects that respond to at least one of the four HER3 immunogenic peptides.

  "Response repertoire" is defined as the average number ("n") of HER3 immunogenic peptides recognized by each subject group.

  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 bodily fluids. Means biological material. The sample may be a 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 DC and is sensed by T cells through T cell receptors.

  "Signal 2" as used herein generally refers to the second signal provided by DC to T cells. Signal 2 is provided by "co-stimulatory" molecules, usually CD80 and / or CD86 (but other co-stimulatory molecules are also known) on activated DCs, sensing by T cells through surface receptor CD28 Be done.

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

  By the term "specifically bind", as used herein, to recognize and bind to another molecule or feature in a sample but to substantially recognize the other molecule or feature And molecules that do not bind to them, such as antibodies.

  The terms "subject", "patient", "individual" etc are used interchangeably herein and any animal or those suitable for the methods described herein, whether in vitro or in situ Point to the cell of In certain non-limiting embodiments, the patient, subject or individual is a human.

  As used herein, the term "targeted therapy" usually refers to drugs or other agents that interfere with specific target molecules involved in the growth of cancer cells, with little damage to normal cells to achieve an anti-tumor effect. Refers to cancer treatment using the substance of In contrast, traditional cytotoxic chemotherapeutic drugs act on all actively dividing cells. Specifically for breast cancer therapeutic monoclonal antibodies, trastuzumab / HERCEPTIN® targets the HER2 / neu receptor.

  The term "T cell" as used herein is defined as a thymus derived cell involved in a variety of cell mediated immune responses.

  The term "T-helper" as used herein with reference to cells refers to a subgroup of lymphocytes (type of white blood cells or white blood cells), including 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 stimulate or interact with other leukocytes.

  As used herein, the terms "helper T cell", "helper T cell", "Th cell" and the like refer to a cell and refer to a subgroup of lymphocytes (a white blood cell or a type of white blood cell) comprising different cells. It includes types that can be identified by those skilled in the art. In particular, T helper cells are effector T cells whose main function is to promote the activation and function of other B and T lymphocytes and / or macrophages. Helper T cells differentiate into two major subtypes of cells known as "Th1" or "type 1" and "Th2" or "type 2" phenotypes. These Th cells secrete cytokines, proteins or peptides that stimulate or interact with other leukocytes.

  As used herein, "Th1 cells," "CD4 + Th1 cells," "CD4 + T helper type 1 cells," "CD4 + T cells," etc. refer to mature T cells that express the surface glycoprotein CD4. . CD4 + T helper cells are activated upon presentation of peptide antigens by MHC class II molecules expressed on the surface of antigen presenting peptides ("APCs") such as dendritic cells. Upon activation of CD4 + T helper cells by the MHC antigen complex, they secrete high levels of cytokines such as interferon gamma ("IFN-gamma"). Such cells are believed to be highly effective against the specific disease causing microbes present inside the host cell, and against the microbes and cancers causing the specific disease within the host cell. Is also important for anti-tumor response in human cancer.

  "Th17 T cells" as used herein are considered to be highly effective against disease causing microorganisms that produce high levels of cytokines IL-17 and IL-22 and survive on mucosal surfaces Refers to T cells that are

  A "therapeutically effective amount" is that amount of a compound of the invention that ameliorates the symptoms of the disease when administered to a patient. The amount of a compound of the invention which constitutes a "therapeutically effective amount" will vary depending upon the compound, the condition of the disease and its severity, the age of the patient to be treated, etc. A therapeutically effective amount can be determined routinely by one of ordinary skill in the art, given the knowledge of one of ordinary skill in the art and the present disclosure.

  The terms "treat", "treating" and "treatment" refer to the therapeutic or prophylactic strategies described herein. The method of “treatment” may be for the prevention, cure, delay, reduction in severity or amelioration of one or more symptoms of the disorder or relapse disorder, or in excess of the expected survival if not receiving such treatment. In order to prolong the survival of a subject, the composition of the present invention is ultimately obtained from a subject in need of such treatment, eg, a subject suffering from a disease or disorder or such disease or disorder Take advantage of administering to a subject at risk.

  "Triple negative" and "TN" breast cancer refers to any breast cancer cell that tests negative for estrogen receptor ("ER"), progesterone receptor ("PR") and HER2.

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

  "Variant" with respect to a peptide or polypeptide differs in amino acid sequence by insertion, deletion or conservative substitution of amino acids but retains at least one biological activity. 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 substitutions of amino acids, ie, replacing an amino acid with an amino acid that differs in similar properties (eg, hydrophilicity, degree and distribution of charged regions), typically produces minor changes in the art. Are recognized. These minor changes can be identified, in part, by examining the hydropathic index of amino acids, as understood in the art. Kyte et al. Mol. Biol. 157: 105-132 (1982). The hydrophobicity index of amino acids is based on examination of their hydrophobicity and charge. It is known in the art that amino acids of similar hydrophobicity index can be substituted and still retain the function of the protein. In one aspect, amino acids with a hydrophobicity index of ± 2 are substituted. Also, the hydrophilicity of amino acids can be used to indicate substitutions that would result in a protein retaining biological function. In the context of peptides, examining the hydrophilicity of amino acids 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 strategy that has been reported. US Patent No. 4,554,101, which is incorporated herein by reference in its entirety. As understood in the art, substitutions of amino acids with similar hydrophilicity values can result in peptides that retain biological activity, eg, immunogenicity. Substitutions can be performed with amino acids having hydrophilicity values within ± 2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. In line with such observations, amino acid substitutions corresponding to biological functions are related to the relative similarity of amino acids, in particular their amino acids, as indicated by their hydrophobicity, hydrophilicity, charge, size and other properties. It is understood to depend on the side chain.

  Scope: Throughout the disclosure, various aspects of the invention can be presented as a scope 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 rigid limitation on the scope of the present invention. Therefore, the description of a range should be considered as specifically disclosing all possible partial ranges and individual numerical values within the range. For example, the description of the 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 the range Individual numbers of, for example, 1, 2, 2.7, 3, 4, 5, 5.3 and 6, should be considered as being specifically disclosed. This applies regardless of the width of the range.

DESCRIPTION The present invention provides immunological compositions comprising peptides of the HER family of proteins and other receptor tyrosine kinases. In one embodiment, the 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 invention are also useful as prophylactic therapeutic agents for initial protection, and also as therapeutic agents for the treatment of ongoing conditions.

  The invention also provides methods for treating or preventing cancer. Such methods comprise the step of administering a peptide of the invention or a combination of peptides of the invention to a subject in need thereof. Administration of such peptides results in the induction of anti-tumor immunity. Thus, the invention provides a method for inducing anti-tumor immunity in a subject, such method comprising the peptide of the invention or the combination of peptides of the invention, and pharmaceutical and cellular compositions derived therefrom Administering to the subject.

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

  In one embodiment, T cells can be cultured and expanded using APCs pulsed with a peptide of the invention. Once APCs have been used to expand T cells to obtain a sufficient number of antigen specific T cells, the antigen specific T cells thus obtained are administered to a mammal, thereby causing the antigen to be present in the mammal. Induce specific T cell responses.

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

  The present invention includes mature, antigen-loaded 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 invention provides a method of pulsing and activating cells, whereby the cells maintain an active state after cryopreservation. The benefit of the DC preparation of the invention is that, from a single leucocyte apheresis (collection from a patient), cells are added to the initial vaccine and efficiency to multiple (eg 10 or more) “booster” doses They are cryopreserved and these doses can be thawed as needed at the remote treatment site, without any specialized cell processing facilities or even required quality control tests.

  The present invention also relates to the cryopreservation of these activated DCs, which, upon thawing, are cryopreserved so as to retain their potency and function in antigen presentation and the production of various cytokines and chemokines, Thus, activated DCs that are cryopreserved and then thawed are clinically effective at the same level as freshly harvested and activated DCs.

  Herein, it is contemplated that the present invention provides a method for generating and cryopreserving DCs with superior function in that they generate stronger signals to T cells, and thus more A strong, DC-based vaccine is obtained. By effectively cryopreserving such cells, the sample can be stored and thawed for later use, thereby reducing the need to repeat the process of pheresis and ertriation while producing the vaccine. Let It is an advantage that the DCs can be frozen and then later allowed to thaw and remove them, which allows the single-produced vaccine to be aliquoted and kept frozen, then several weeks This means that one can be administered to the patient one at a time, for months or years, to provide a "booster" vaccination that enhances immunity.

  This embodiment also involves the use of HER3 expression as a marker of tumor progression in premalignant lesions 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 the HER family of proteins and other receptor tyrosine kinases. In one embodiment, the 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 the corresponding HER protein or c-MET protein. In some embodiments, the epitope of the corresponding HER protein or c-MET protein is immunogenic.

  The invention provides compositions comprising one or more peptides of the 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.

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

  Included in the invention are methods of activating an immune response using the compositions of the invention and methods of treating cancer in a subject. Vaccines are also provided for therapeutic and prophylactic use. 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 HER3 epitope or other peptide of the invention is
p11-13 (peptides 51-75): KLYERCEVVMGNLEIVLTGHNADLSFLQW (SEQ ID NO: 1);
p81-83 (peptides 401-425): SWPPHMHNFSVFSNLTTIGGRSLYN (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): SWPPHM HNFS VFSNL (SEQ ID NO: 5);
p84 (peptides 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6);
p91 (peptides 451-465): AGRIYISANRQLCYH (SEQ ID NO: 7)
including.

  The HER3 peptide or any peptide of the invention may be cyclic or linear. The epitope, if circular, can be circular in any suitable manner. For example, disulfide bonds can be formed between selected pairs of cysteine (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 HER3 epitope identified by SEQ ID NO: 4 corresponds to positions 56 to 70 of the HER3 protein. The HER3 epitope identified by SEQ ID NO: 5 corresponds to positions 401 to 415 of the HER3 protein. The HER3 epitope identified by SEQ ID NO: 6 corresponds to positions 416 to 430 of the HER3 protein. The HER3 epitope identified by SEQ ID NO: 7 corresponds to positions 451-465 of the HER3 protein.

  As described herein, the epitopes of HER3 of the present invention also encompass peptides which are functional equivalents of the peptides identified by SEQ ID NO. Such functional equivalents have altered sequences, in which case one or more amino acids in the sequence of the corresponding HER3 epitope are replaced, or one or more amino acids correspond. It is deleted from or added to the reference sequence. For example, one to three amino acids can be added to the amino terminus, carboxy terminus, or both. In some instances, the HER3 epitope is glycosylated.

  In other examples, the HER3 epitope can be the retro-inverso isomer of the HER3 epitope. Retro-inverso modifications include inversion of all amide bonds in 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 form of retro-inverso isomer can retain the planarity and conformational constraints of at least some peptide bonds.

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

  In some instances, deletions and additions are located at the amino terminus, carboxy terminus, or both, of one sequence of a peptide of the invention. For example, the equivalent of the HER3 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 93%, at least 70% identical to the sequence of the corresponding HER3 epitope 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 deletion, addition or substitution, per 10 amino acids of the reference sequence. Percent identity is determined by comparing the amino acid sequence of the variant to a reference sequence using programs known or developed in the art.

  In the case of a functional equivalent which is longer than the corresponding HER3 epitope sequence, the functional equivalent is a sequence which is at least 90% identical to the HER3 epitope sequence and the sequence of the HER3 epitope in the wild-type HER3 protein May have a sequence adjacent to

  Functional equivalents of the HER3 epitope can be identified 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. Such antibodies can be found in a variety of body fluids, including serum and ascites fluid. Briefly, a fluid sample is isolated from a warm-blooded animal, such as a human, for whom it is desirable to determine whether an antibody specific for HER3 polypeptide is present. The body fluid is incubated with the HER3 polypeptide, under conditions sufficient to form an immune complex between the polypeptide and the antibody specific for the protein and for a time sufficient to allow so, and preferably then , Assay using the ELISA method.

  According to another embodiment of the present invention there is provided a composition comprising a chimeric peptide and one or more chimeric peptides. According to various embodiments, the chimeric peptide comprises a HER3 epitope, another epitope, and a linker connecting the HER3 epitope to the other epitope. In one embodiment, the other epitopes may include, but are not limited to, other HER3 epitopes, HER1 epitopes, HER2 epitopes and c-MET epitopes. It is further understood that any suitable linker may be used. For example, depending on the epitope used, the HER3 epitope can be linked to either the amino or carboxy terminus of the other epitope. The location and selection of the other epitopes depend on the structural features of the HER3 epitope, ie whether it is an alpha helix or a beta-turn or a chain.

  In one embodiment, the linker may be a peptide of about 2 to about 15 amino acids, about 2 to about 10 amino acids, or about 2 to about 6 amino acids in length. Chimeric peptides may be linear or cyclic. Furthermore, the HER3 epitope, other epitopes and / or linkers may be in the form of retro-inverso. Thus, the HER3 epitope alone may be in the form of retro-inverso. Alternatively, the HER3 epitope and other epitopes may be in the form of retro-inverso. In another example, the HER3 epitope, other epitopes and the linker may be in the form of retro-inverso.

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

  According to a further embodiment of the present invention there is provided a composition comprising a mixture of two or more of the peptides or chimeric peptides of the present invention. In some instances, the HER3 epitopes of each of the two or more chimeric peptides are different. In another example, one of the HER3 epitopes is selected from SEQ ID NOs: 1-7.

  The peptides of the invention, including chimeric peptides, can be prepared using 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 invention are preferably isolated, ie, substantially free of other naturally occurring host cell proteins and fragments thereof.

  Commercially available peptide synthesizers can be used to synthesize the peptides and chimeric peptides of the invention. For example, Kaumaya et al., "De Novo" Engineering of Peptide Immunogenics and Antigenic Determinants as Potential Vaccines, Peptides, Design, Synthesis and Biological Activity (1994), pages 133-164, specifically incorporated by reference herein. The chemical methods described in can be used. For example, HER-3 epitopes can be co-linearly synthesized with other epitopes to form chimeric peptides. Peptide synthesis can be performed using Fmoc / t-But chemistry. The peptides and chimeric peptides can be cyclic in any suitable manner. For example, methods of differentially protecting cysteine residues, performing iodine oxidation, adding water to facilitate removal of the Acm group, simultaneously forming disulfide bonds, and / or using the silyl chloride-sulfoxide method Thus, a disulfide bond can be obtained.

  Peptides and chimeric peptides can also be produced using cell-free translation systems and RNA molecules derived from DNA constructs encoding epitopes or peptides. Alternatively, the epitope or chimeric peptide is generated by transfecting the 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 It can also be done. In the case of the production of recombinants, recombinant constructs comprising one or more of the sequences encoding the epitope, the chimeric peptide or a variant thereof, can be carried out in a conventional manner, for example calcium phosphate transfection, DEAE-dextran mediated transfection Transfer into host cells by microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape lading, ballistic transfer, or infection.

  The peptides of the invention may contain modifications, such as glycosylation, oxidation of side chains, or phosphorylation, but this is limited to the case where the modifications do not destroy the biological activity of the peptide. . Other modifications include, for example, incorporation of D-amino acids or other amino acid mimetics that can be used to extend the serum half life of the peptide.

  The peptides of the invention can be prepared as a combination, which comprises two or more peptides of the invention for use as a vaccine for a disease, eg, cancer. The peptides may be made into a cocktail or may be conjugated to one another using standard techniques. For example, peptides 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 encompass “mutants”, “derivatives” and “variants” of the peptides of the present invention (or DNA encoding said peptides), mutants, derivatives thereof and A variant is a peptide in which one or more amino acids are changed (or, when referring to a nucleotide sequence encoding said amino acids, one or more base pairs are changed), and thus The peptides (or DNA) that are not identical to the sequences listed herein have the same biological properties as the peptides disclosed herein.

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

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

  The scope of the present invention includes homologs, 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 And derivatives of amino acids or nucleic acids known in the art, non-naturally occurring amino acids or nucleic acids and synthetic amino acids or nucleic acids, provided that these modifications preserve the biological activity of the original molecule Clearly understand what you have to do. Specifically, in accordance with the principles of the present invention, any active fragments of the active peptide, 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 referenced herein, provided that these homologous DNAs have the biology of the peptides disclosed herein. Should be interpreted as having a specific activity.

  The nucleic acids of the invention encompass sequences of RNA or DNA encoding the peptides of the invention, DNA making the nucleotide sequence more stable, with or without cells. Those skilled in the art will understand that it also includes any of its variants, including chemical modifications of RNA. Chemical modifications of the nucleotides can also be used to enhance the efficiency with which the cells take up the nucleotide sequence or the efficiency with which the nucleotides are expressed in the cells. The present invention contemplates any combination of nucleotide sequence modifications.

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

  The nucleic acid encoding the peptide of the invention can be incorporated into a suitable vector, for example a retroviral vector. These vectors are well known in the art. The nucleic acid, or a vector containing the nucleic acid usefully, can be transferred into the desired cells, which are preferably obtained from the patient. Advantageously, the present invention provides an off-the-shelf composition, which rapidly alters the patient's own cells (or another mammal's own cells). Thus, it becomes possible to rapidly and easily generate modified cells having excellent cancer cell killing properties.

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

  Polynucleotides can also be cloned into several types of vectors. However, it should not be construed that the present invention is limited to any particular vector. Instead, the present invention should be construed to encompass a very wide variety of vectors which are readily available and / or which are 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. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

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

  In addition, expression vectors can also be provided to cells in the form of viral vectors. Techniques for viral vectors are well known in the art and are described, for example, in Sambrook et al. (2012) and Ausubel et al. (1997) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retrovirus, adenovirus, adeno-associated virus, herpes virus and lentivirus. In general, a suitable vector contains an origin of replication functional 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 sequences of the invention, at least one module in each promoter is functioned to position the initiation site for RNA synthesis. The best known example of this is the TATA box, but some promoters lacking the TATA box, such as in the promoter of the mammalian terminal deoxynucleotidyl transferase gene and in the promoter of the SV40 gene, distinctly overlapping the start site The element itself helps to fix the start location.

  Additional promoter elements, ie, enhancers, regulate the frequency of transcription initiation. Typically, they 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 promoter function is preserved, even when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can work together or independently to activate transcription.

  The promoter may be one that is naturally associated with the gene or polynucleotide sequence, which may be obtained by isolating the 5 'non-coding sequence located upstream of the coding segment and / or the exon. Such promoters can be termed "endogenous." Similarly, an enhancer can also be an enhancer that is naturally associated with a polynucleotide sequence, which is either downstream or upstream of that sequence. Alternatively, placing the coding segment of the polynucleotide under the control of a recombinant or heterologous promoter provides certain advantages, in which case 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 the polynucleotide sequence in its natural environment. Such promoters or enhancers can be promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell, and non-naturally occurring promoters or enhancers, 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, sequences may also be generated using recombinant cloning and / or nucleic acid amplification techniques, which can be used in the compositions disclosed herein. The relevant PCRTM is included (US Pat. No. 4,683,202, US Pat. No. 5,928,906). Furthermore, it is also contemplated that regulatory sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, etc., may be utilized as well.

  Of course, it is important to utilize promoters and / or enhancers which effectively direct the expression of the DNA segment in the cell type, organelle and organism chosen for expression. Those skilled in molecular biology are generally aware of how to use combinations of promoters, enhancers and cell types for the expression of proteins, see, eg, Sambrook et al. (2012). The promoter utilized may be a constitutive promoter, a tissue specific promoter, an inducible promoter, and / or conditions suitable for directing high level expression of the DNA segment to be introduced, eg, large recombinant proteins and / or peptides. It may be a useful promoter under conditions that favor scale production. The promoter may be heterologous or endogenous.

  The promoter sequence exemplified in the experimental examples presented herein is the sequence of the cytomegalovirus (CMV) earliest promoter. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively 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 early 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. By using an inducible promoter in the present invention, 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 promoter, glucocorticoid promoter, progesterone promoter and tetracycline promoter. Furthermore, the invention also includes the use of tissue specific promoters, which are only active in the desired tissue.

  In order to evaluate the expression of the nucleotide sequence encoding the peptide of the present invention, the expression vector to be introduced into cells also contains either a selectable marker gene or a reporter gene or both, and can be transfected by a viral vector. It can also facilitate identifying and selecting cells that are expressed from cell populations targeted for infection or infection. In other embodiments, selectable markers can be placed on separate pieces of DNA and used in co-transfection procedures. Both the selectable marker gene and the reporter gene may be flanked by appropriate regulatory sequences to allow expression in a 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 encoding easily assayable proteins are well known in the art. In general, the reporter gene is neither present in the recipient organism or tissue nor expressed by the recipient organism or tissue, and some readily detectable property, eg, enzyme activity, reveals expression It is a gene encoding a protein. Expression of the reporter gene is assayed at an appropriate time after introducing the DNA into recipient cells.

  Suitable reporter genes can include luciferase, beta-galactosidase, chloramphenicol acetyltransferase, a gene encoding secreted alkaline phosphatase, or a green fluorescent protein gene (eg, Ui-Tei et al., 2000, FEBS). Lett. 479: 79-82). Suitable expression systems are well known and they may be prepared using 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 showing the highest levels of expression of the reporter gene are identified as promoters. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

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

  Vaccines of the invention can induce antigen-specific T cell responses and / or high titer antibody responses, thereby being directed against or reactive to an antigen expressing cancer or tumor. An immune response is induced or elicited. In some embodiments, the induced or elicited immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or 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. The vaccine can include one or more cancer antigens. Vaccines can block tumor growth. Vaccines can reduce tumor growth. Vaccines can block metastasis of tumor cells. Depending on the cancer antigen, target the vaccine, breast cancer, liver cancer, prostate cancer, melanoma, hematologic 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, tumor cells It can mediate growth clearance or arrest. Vaccines, tumor free survival, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, It can be increased by 44% and 45%. Vaccines, after immunization, tumor volume, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42% , 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59 % And 60% can be reduced.

  The vaccine is about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold over the cellular immune response in the subject receiving the vaccine, as compared to the cellular immune response in the subject not receiving the vaccine. Double-about 5000 times, about 50 times-about 4500 times, about 100 times-about 6000 times, about 150 times-about 6000 times, about 200 times-about 6000 times, about 250 times-about 6000 times, or about 300 times -It can be increased about 6000 times. In some embodiments, the vaccine is about 50-fold, 100-fold, 150-fold, 200-fold the cellular immune response in the subject receiving the vaccine as compared to the cellular immune response in the subject not receiving the vaccine. Double, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 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, 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 Double, 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, 5300 times It can be increased 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

  The vaccine is about 50-fold to about 6000-fold, about 50-fold about IFN-γ levels in subjects receiving vaccine compared to the level of interferon gamma (IFN-γ) in subjects not receiving vaccine -About 5500 times, about 50 times-about 5000 times, about 50 times-about 4500 times, about 100 times-about 6000 times, about 150 times-about 6000 times, about 200 times-about 6000 times, about 250 times-about It can be increased 6000 times, or about 300 times to about 6000 times. In some embodiments, the vaccine comprises about 50-fold, 100-fold, or more levels of IFN-γ in the subject receiving vaccine compared to the levels of IFN-γ in the subject not receiving the vaccine. 150 times, 200 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, 3700 Double, 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, 5300 times It can be increased 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

  The vaccine of the invention has the features necessary for an effective vaccine, for example, the vaccine of the invention is safe, so the vaccine itself does not cause disease or death; it provides protection against disease; neutralizing antibodies Induce a protective T cell response; easy to administer, have few side effects, are biostable, and have low cost per dose. Vaccines can be achieved by containing some or all of these features, as discussed below, with cancer antigens.

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

  Also, the APC can be "pulsed" in a manner that exposes the antigen to the antigen, and the exposure can also occur for a sufficient time 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, and these fragments are "pulsed" directly onto the exterior of the APC (Mehta-Damani et al. Or APC) can be incubated with whole proteins or protein particles, and these proteins are then taken up by the APC. All these proteins are digested by APC into small peptide fragments and finally carried 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 "pulsing" techniques described herein.

  Without intending to be bound by any particular theory, antigens in the form of foreign antigens or self antigens are processed by the APCs of the present invention to retain an 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 by immune cells, eg, T cells, to activate them. Preferably, such foreign antigens or autoantigens are proteins that are processed by APC into peptides. The relevant peptides produced by APC can be extracted, purified and used as immunogenic compositions. Also, peptides processed by APC can be used to induce tolerance to proteins processed by APC.

  Antigen-loaded APC, by another expression, is known as the "pulsed APC" of the present invention, and is generated by exposing APC to antigen either in vitro or in vivo. Ru. When APCs are pulsed in vitro, the APCs can be plated on culture dishes and exposed to the antigen for a sufficient period of time in an amount sufficient to allow the antigen to bind to the APC. . The amount and time necessary to achieve binding of 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 antigen on APC after exposure to the antigen.

  In a further embodiment of the invention, APCs can be transfected with a vector that allows expression of specific proteins by the APCs. The proteins expressed by APCs 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 to the vector encoded protein.

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

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

  As contemplated herein, various methods can be used to transfect polynucleotides into host cells. These methods include, but are not limited to, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, colloidal dispersion systems (ie, macromolecular complexes, nanocapsules, 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 it is therefore possible for those skilled in the art to carry out these methods.

  In another embodiment, by another method, a polynucleotide encoding an antigen can be cloned into an expression vector to generate a loaded APC, which can be introduced into the 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. See, eg, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY), and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York). It is readily understood that the introduction of an expression vector containing a polynucleotide encoding an antigen results in pulsed cells.

  The invention includes various methods for pulsing APCs, including, but not limited to, loading APCs with whole antigens in the form of proteins, cDNA or mRNA. However, the present invention should not be construed as limited to the particular form of antigen used to pulse APC. Rather, the present invention encompasses other methods known in the art for generating antigen-loaded APCs. Preferably, APCs are transfected with mRNA encoding the defined antigen. Use of appropriate primers and reverse transcriptase-polymerase chain reaction (RT-PCR) coupled with a transcription reaction to rapidly generate in vitro mRNA corresponding to a gene product whose sequence is known it can. When generating pulsed APCs, transfection with APC mRNA offers advantages over other antigen-loaded techniques. For example, being able 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 to the antigen in cells, tissues or mammals (eg, humans). As used herein, an "immunological composition" may express or display an antigen (eg, a peptide or polypeptide), a nucleic acid encoding an antigen (eg, an antigen expression vector), or an antigen or cell component. Cells can be included. In particular embodiments, the antigen composition comprises or encodes all or part of any of the antigens described herein, or an immunological functional equivalent thereof. In other embodiments, the antigen composition is a mixture comprising an additional immunostimulatory agent or nucleic acid encoding such an agent. 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 linked to the antigen or immunostimulatory agent in any combination. In certain embodiments, the antigen composition is conjugated to or comprises amino acids of an HLA anchor motif.

  Vaccines, as contemplated herein, may vary in their composition of nucleic acids and / or cell components. In a non-limiting example, nucleic acids encoding the antigen could also be formulated with an adjuvant. Of course, it will be understood that the various compositions described herein can further include 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 of the methods disclosed herein, or this will be understood by one of ordinary skill in the art in light of the present disclosure. Will.

  It is understood that the antigen composition of the present invention can be produced by methods well known in the art, and such methods include, but are not limited to, chemical synthesis by solid phase synthesis and chemical reaction by HPLC Other methods of purification removal from products, or expression of nucleic acid sequences (eg, DNA sequences) encoding a peptide or polypeptide comprising an antigen of the invention in an in vitro translation system or in a living cell There is a way to generate by. Additionally, the antigen composition can also include cell components isolated from the biological sample. Antigen compositions can be isolated and extensively dialyzed to remove one or more unwanted low molecular weight molecules, and / or lyophilize in the desired vehicle to enhance the immediate use of the formulation . It should be further understood that if there are additional amino acids, mutations, chemical modifications, etc. to be introduced into the vaccine components, they preferably do not substantially prevent the antibody from recognizing the epitope sequences.

Antigen Presenting Cell Therapy The present invention encompasses methods of generating a population of APCs (eg, dendritic cells; DCs) presenting the peptides of the present invention on the 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 generated form 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 originally obtained from the cells, thus forming an embodiment of the treatment of the 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 DCs capture an antigen, they process the antigen into small peptides and migrate toward secondary lymphoid organs. It is within the lymphoid organs that DC presents antigenic peptides to naive T cells, and presentation of the peptide initiates a signal cascade that biases T cell differentiation. When exposed to DC, they 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; Ginhoux et al., 2007, J. Exp. Med. 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. Im 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 the adaptive immune response, and also serve to integrate the communication between the natural and adaptive arms of the effectors of the immune system. These characteristics make DCs powerful candidates for immunotherapy. DCs have a unique ability to identify their environment by macropinocytosis and receptor-mediated endocytosis (Gerner et al., 2008, J. Immunol. 181: 155-164; Stoitzner et al., 2008, Cancer Immunol. Immunoother 57: 1665-1673; Lanzevecchia A., 1996, Curr. Opin. Immunol. 8: 348-354; Delamarre et al., 2005, Science, 307 (5715): 1630-1634).

  DCs also require maturation signals to enhance their antigen presentation capabilities. By providing additional maturation signals, such as TNF-α, CD40L or calcium signaling agents, DCs are surface molecules, such as CD80 and CD86 (also known as second signal molecules). Upregulation of expression (Czerniecki et al., 1997, J. Immunol. 159: 3823-3837; Bedrosian et al., 2000, J. Immunother. 23: 311-320; Mailliard 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). Alternatively, DCs can be matured with a calcium ionophore prior to pulsing the antigen.

  DCs are receptors that recognize pathogens, for example, PKR and MDA-5 (Kalali et al., 2008, J. Immunol. 181: 2694-2704; Nallagatla et al., 2008, RNA Biol. 5 (3): In addition to pages 140-144), it also contains a series of receptors known as Toll-like receptors (TLRs), which are also capable of sensing the risk from pathogens. . Stimulation of these TLRs induces a series of activity changes in DC that lead to 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 the cell-mediated response, such as natural killer γ-δ T cells and α-β T cells, which, once activated, Retain their immunizing 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; Bykovskaia et al., 1999, J. Leukoc. Biol. 66: 659-666; Clark et al., 2000, Microbes Infect. 2: 257-272).

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

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

  The present invention provides compositions and methods for activating APCs, preferably DCs, in the context of immunotherapy for activating an immune response in a mammal. By activating DCs with the peptide of the present invention or a combination of peptides of the present invention so that DCs activate anti-tumor immunity in a mammal in need thereof, DCs are induced by causing DC maturation. It can be operated.

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

  In one embodiment, the invention relates, inter alia, to expand desired T cells, to activate T cells, to generate new APCs for the purpose of expanding specific T cells, and to use them for such purposes. The invention relates to methods for using APCs, and also to a number of therapeutic uses related to T cell expansion and activation using the peptide-loaded APCs and peptides of the invention. In some cases, DCs activated by OCT4 can also be used to expand T cells specific for the peptide.

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

Methods for Treating Diseases The invention also encompasses methods of 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 for activating DC), which itself has utility in the treatment of diseases. Exemplary diseases which can be treated and / or prevented using the pharmaceutical composition of the present invention include, but are not limited to, infections caused by viruses, such as HIV, influenza, herpes, viral hepatitis , Epstein-Barr, polio, viral encephalitis, measles, chicken pox, papilloma virus etc .; or infections caused by bacteria, eg, pneumonia, tuberculosis, syphilis etc .; or infections caused by parasites, eg, malaria, Trypanosomiasis, leishmaniasis, trichomoniasis, amebiasis etc. may be mentioned.

  Non-limiting examples of preneoplastic or hyperplastic states that can be treated or prevented using the pharmaceutical compositions of the invention (transduced DCs of the invention, expression vectors, expression constructs, etc.) These include preneoplastic or hyperplastic states 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's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, gastrointestinal cancer, brain cancer, bladder cancer , Cervical cancer and the like.

  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 vessels Tumor, fibroma, vascular occlusion, restenosis, atherosclerosis, preneoplastic condition lesions (eg, adenomatous hyperplasia and neoplasia in prostate epithelium), carcinoma in situ, cholecystitis alveolar or psoriasis It can be mentioned.

  Autoimmune disorders which can be treated using the compositions of this invention include, but are not limited to, AIDS, Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis , Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes, emphysema, nodular erythema, 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; infections by viruses, bacteria, fungi, parasites, protozoa and helminths; and trauma.

  In methods of treatment, the administration of the compositions of the present invention may either be for "prevention" or for "treatment". The compositions of the invention, when provided for prophylaxis, are provided prior to any symptom, but in certain embodiments, the vaccine is provided after one or more symptoms have developed. , Prevent the onset of additional symptoms, or prevent the aggravation of the emerging symptoms. The 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 the symptoms of the infection or disease. Thus, the present invention can be provided before exposure to the substance causing the disease is expected or before the disease state occurs or after the infection or disease has started.

  An effective amount of the composition is that which enhances the immune response and is such an amount to achieve these selected results, such amount will be determined by one of ordinary skill in the art as a matter of routine. For example, an effective amount to treat the immune system's failure to cancer or pathogens would be the amount necessary to cause activation of the immune system to generate an antigen-specific immune response upon exposure to the antigen. . This term is also synonymous with "sufficient amount".

  The effective amount for any particular application may 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 of ordinary skill in the art can empirically determine the effective amount of a particular composition of the present invention without requiring undue experimentation.

Vaccine Formulations The invention further comprises vaccine formulations suitable for use in immunotherapy. In certain embodiments, vaccine formulations are used to prevent and / or treat diseases, eg, cancer and infectious diseases. In one embodiment, administration to a patient of a vaccine according to the invention for the prevention and / or treatment of a cancer, prior to 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 the cancer vaccine prepared according to the present invention is broadly applicable to the prevention or treatment of cancer which is determined to some extent by the choice of the part that forms the antigen of the cancer vaccine. Cancers that can be suitably treated according to the practice of the invention include, but are not limited to, lung, breast, ovary, cervix, colon, head and neck, pancreas, prostate, stomach, bladder, kidney, bone, Includes cancers 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 cells 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, etc. are also produced as immunotherapeutic agents and prevent and / or prevent the tumor or cancer cells from which the vaccine is generated. It is used according to the implementation procedure for treatment.

  In another embodiment, as also described, a vaccine according to the invention is obtained by collecting appropriate antigens excreted into the culture medium by the pathogen in order 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, preparing a multivalent vaccine by preparing a vaccine from a pool of organisms expressing different important antigens It 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 also be administered intradermally or subcutaneously to the limbs, arms and legs of the patient being treated. While this approach is generally satisfactory for melanoma as well as other cancers, other routes of administration, including the prevention or treatment of infectious diseases, such as, for example, the intramuscular or in the bloodstream route. It can also be used.

  Additionally, the vaccine can be administered with an adjuvant and / or an immunomodulator to boost the activity of the vaccine and the patient's response. Such adjuvants and / or immunomodulators are understood by those skilled in the art and are easily described in the available published literature.

  As contemplated herein, depending on the type of vaccine produced, if desired, culturing the cells in a bioreactor or fermentor or other such vessel or device suitable for growing the cells in bulk By doing this, the vaccine production can be scaled up. In such devices, the culture medium is periodically, frequently or continuously collected to recover any material or antigen from the culture medium prior to degradation of such material or antigen in the culture medium. Will be done.

  The device or composition containing the vaccine or antigen, which device is suitable for sustained or intermittent release, produced according to the invention and recovered, is optionally implanted in the body or It can be applied externally to the body to release such materials into the body relatively slowly or at defined times.

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

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

  These methods described herein are by no means all-inclusive and additional methods suitable for particular applications will be apparent to those skilled in the art. In addition, the effective amount of the composition can also be determined to a more accurate approximation by analogy with compounds that are known to exert the desired effect.

<Example of experiment>
The invention is further described in detail by reference to the following experimental examples. These examples are provided for the purpose of illustration only and are not intended to limit the invention unless otherwise specified. Accordingly, the present invention should not be construed as being limited in any way to the following examples, but rather is to be construed as encompassing any modifications that may become apparent as a result of the teachings provided herein. It should.

  Without further elaboration, it is believed that one skilled in the art can, using the preceding description and the following illustrative examples, make and use the present invention and practice the claimed method. Conceivable. Thus, the following examples specifically point out the preferred embodiments of the present invention and should not be construed as limiting the remainder of the present disclosure in any way.

Creation of peptide vaccines against other receptor tyrosine kinases that cause breast cancer and other solid cancers Experiments were designed to develop alternative therapies for patients designated as carriers of BRCA mutations. That is, they are looking for an alternative to bilateral mastectomy, and the need for younger patients at genetic risk for breast cancer has not been met.

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. To develop vaccines for this group and to evaluate their safety in immune induction trials, experiments were designed; this is the first ever vaccination for primary prevention of breast cancer It is an attempt. We also observe whether estrogen receptor- ^positive breast cancer can be prevented using the multivalent vaccine of the invention, including carriers of BRCA2 mutations.

  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 c-MET oncogene and HER-3 prematurely, while tumors from non-mutant or sporadic patients show HER-2 and HER- It was observed that 3 was expressed. This is important; as disclosed herein the targets for tumor immunotherapy that can be used to develop vaccines for carriers of sporadic and BRCA mutations. Based on the fact that it is now known. This is an initial and striking feature and can be targeted to use an immune response for prevention. Thus, the invention includes compositions and methods for developing a vaccine, and their use for prophylaxis as an alternative to bilateral mastectomy.

Creation of peptide vaccines The HER family consists of four related signaling molecules, HER1, HER2, HER3 and HER4, which are involved in a variety of cancers. It is known that overexpression of HER2 is found in 20% to 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, HER1 is expressed on a minority of breast cancers, which are generally triple negative. c-MET is a growth factor receptor involved in the relapse of many cancers, which activates HER3. HER3 is overexpressed in colon, prostate, breast and melanoma. HER3 is expressed in a number of DCIS lesions and breast cancer. In some patients receiving the HER2 vaccine, HER3 may be detected in the residual DCIS at the time of surgery. As a result of these findings, in breast cancer, the potential to target these molecules in addition to HER2 would be beneficial.

Immunogenic peptides derived from HER3 have been identified (Figures 1 and 2) and are shown below:
p11-13 (peptides 51-75): KLYERCEVVMGNLEIVLTGHNADLSFLQW (SEQ ID NO: 1);
p81-83 (peptides 401-425): SWPPHMHNFSVFSNLTTIGGRSLYN (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): SWPPHM HNFS VFSNL (SEQ ID NO: 5);
p84 (peptides 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6); and p91 (peptides 451-465): AGRIYISANRQLCYH (SEQ ID NO: 7).

The results presented herein indicate that these peptides can activate CD4 T cells across many patients. The peptide can be pulsed on dendritic cells and T cells can be educated to recognize HER3. HER3 is expressed in triple negative breast cancer and may lead to resistance to anti-estrogen in ER ^-positive breast cancer. HER3 is also expressed in other cancers, including melanoma, lung cancer, colon cancer, prostate cancer and metastatic brain tumors. Without 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 provided herein, immunogenic peptides for the receptor tyrosine kinase molecules HER1 and c-MET were screened and identified based on the procedure that identified immunogenic peptides for HER3. can do. The immunogenic peptides of the invention can be used to prepare multivalent prophylactic vaccines for breast and other cancers.

  The results presented herein demonstrate the identification of the role of HER2 sister protein in breast cancer. These sister proteins can be effective targets and vaccines can be developed for other solid tumors. We have developed peptides that can be used to target HER1 and HER3. Specifically, DCIS leads to the identification of specific anti-HER1, anti-HER2 and anti-HER3 responses in patients before and after vaccination, which prevents cancer early or DCIS Assist in the development of multivalent vaccines that can be used to treat women with. The compositions of the invention are useful for treating other cancers including, but not limited to, colon cancer, melanoma, brain cancer, 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 standard dendritic cell vaccines and engineered dendritic cells to exhibit a mutated protein (BRAF) that causes approximately 70% of melanomas . Vaccination with these dendritic cells protects mice from challenge with melanoma cells, indicating that it is possible to develop a vaccine for melanoma. While not intending to be bound by any particular theory, targeting combined with BRAF and HER3 is not limited to melanoma, but not limited to solid cancers such as colon cancer, pancreatic cancer and lung cancer and others. It may be useful to treat other cancers, including gastrointestinal tumors.

  In addition, melanoma tumors have also been shown to use B cells to evade immune surveillance, and it is therefore thought that therapy can be improved by eliminating certain B cells. Experiments can be designed to assess whether altering the tumor's microenvironment to a Th1-type response can help prevent avoidance.

  In some cases, the vaccines of the invention can also be used to treat spread melanomas. In some cases, the present invention also provides a therapy to eliminate residual cells, often becoming resistant to drug therapy.

Novel strategies to identify MHC class II-promiscuous CD4 ⁺ peptides from tumor antigens for use in vaccination. Cytotoxic CD8 + T lymphocytes (CTLs) have historically been the mainstay of anti-tumor immunity Although considered to be an effector, just boosting the CTL response with the CD8 + vaccine in various tumor types has produced unpredictable clinical results, which is probably due to the CTL being appropriate Without the help of these CD4 + T lymphocytes, it would only function suboptimally. CD4 + T helper type 1 (Th1) cells secrete IFN-γ / TNF-α to induce tumor senescence and apoptosis. However, the successful integration of CD4 + epitopes in constructing cancer vaccines and the development of durable, antigen-specific CD4 + immunity remains a challenge. An immunogenic HER3 CD4 + that exhibits class II promiscuity and generates anti-HER3 CD4 + immunity, with the aim of including it in a vaccine construct, using the extracellular domain (ECD) of HER3 as a candidate "oncogenic driver" tumor antigen Experiments were performed to identify the peptides.

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

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 long peptides, overlapping by 5 amino acids, was generated from the HER3 ECD. These peptides were pulsed onto mononuclear cell-derived DC obtained from a donor and allowed to mature into a type 1 polarized (DC1; secreting IL-12) phenotype. DC1 was collected, co-cultured with purified CD4 + T cells obtained from subjects belonging to our DCIS vaccine study and known to have an anti-HER3 Th1 response. A large pool of 10 peptides was used to refine the process of identification, as measured by the secretion of interferon gamma (IFN-γ) of CD4 + T cells, to obtain a single reactive epitope. 4 to 6 subjects are screened to appear to react across most donors, ie, HER3 _56-70 (SEQ ID NO: 4), HER3 _401-415 (SEQ ID NO: 5), HER3 _416-430 (SEQ ID NO: 6), and HER3 _51-465 (SEQ ID NO: 7) were identified. Identify subjects that do not show evidence of responsiveness to HER3 extracellular domain CD4 + T cell recognition, pulsing the four HER3 peptides into their DC1s, and culturing pulsed DC1 with CD4 T cells for 1 week Then, it was tested for reactivity to HER2 peptide and response to extracellular HER3 protein. In each case, at least one peptide resulted in the recognition of both the peptide pulsed on mononuclear cells and the whole protein of HER3, suggesting that primary sensitization occurred ex vivo . Healthy donors were also able to respond to these peptides, and in triple-negative breast cancer patients, it was also shown that the anti-HER3 Th1 response is lost. Also, Gala, K. et al. , Clin. Cancer Res 2014; 20: 1410-1416, and Datta, J. et al. See also, "Progressive Loss of Anti-HER2 CD4 + T-helper Type 1 Response in Breast Tumorigenesis and the Potential for Immune Restoration", Onco Immunology (nearly published).

Key points of the protocol further illustrated in FIG. 19:
A library containing 123 15 amino acid long peptide fragments overlapping by 5 amino acids was generated from the extracellular domain (ECD) of HER3.
-Autologous mononuclear cell-derived dendritic cells (DC) obtained from a donor are rapidly transformed into type 1 (secreting DC1 → IL-12) phenotype by GM-CSF, IFN-γ and LPS. It was matured and pulsed with related peptides (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 for 8-10 days with purified CD4 + T cells.
-Sensitized CD4 + T cells (most of them are antigens against immature DCs (iDCs) pulsed with a specific CD4 + peptide of interest (eg a cluster of HER3 library peptides) or a control of an irrelevant class II peptide Was expected to be specific) was reactivated.
-The supernatant was then collected from these co-cultures. Th1 responses measured by IFN-γ ELISA were considered to be antigen specific if the production of IFN-γ was at least twice that of an unrelated control.
In order to assess MHC class II promiscuity of CD4 ⁺ Th1 responses, HLA-DR, DP, DQ typing on donors was performed by the Clinical Immunology laboratory at the Hospital of the University of Pennsylvania.

  A library containing 123 15 amino acid peptide fragments with overlap was generated from HER3-ECD. Autologous mononuclear cell-derived DC obtained from a donor 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 reactivated against immature DCs (iDCs) pulsed with clusters of HER3 library peptide or irrelevant CD4 control peptide 1. Th1 responses measured by IFN-γ ELISA were considered to be antigen specific if the production of IFN-γ was at least twice that of an unrelated control.

  1) Obtain a breast cancer patient known to show anti-HER3 ECD reactivity after DC1 vaccine pulsed with HER2 to identify immunogenic CD4 + peptides; 2) Immunogenicity of these peptides Were confirmed by the process of "reverse" sensitization in the same patient; 3) Obtained a patient known to show anti-HER3 ECD non-reactivity after vaccination and used to identify CD4 + peptides The experiments were carried out in a three-step process, in which cells were sensitized to the native HER3 ECD, thus observing whether tolerance to self antigens (ie, HER3) was overcome / nulled.

  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, anti-HER3 ECD to identify a single immunogenic HER3 CD4 + epitope Th1 sensitization was performed in 5 breast cancer patients known to be reactive. To achieve this, starting with 10 peptide clusters (1 to 10, 11 to 20, etc.), 3 peptide clusters (1 to 3, 3 to 6, 7 to 10,...) Etc., and finally, focused on single immunogenic HER3 peptides to reactivate HER3 ECD sensitized CD4 + Th1 against them sequentially. Representative screens are shown in FIGS. 2, 13 and 14. Four immunogenic peptides, namely, 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 reproducibly identified and they were promiscuous across HLA-DR, DP and DQ subtypes. Th1 cells obtained from 4 non-HER3-reactive donors are sensitized using DC1 pulsed with 4 identified HER3 peptides, and then these cells are iDC pulsed with HER3 ECD Not only did all donors show good sensitization to the individual immunogenic HER3 peptides, but also recognized native HER3-ECD when challenged to recognize.

  The results presented herein demonstrate that DC1 pulsed with a library of overlapping tumor antigen-derived peptides can identify promiscuous class II peptides for the development of CD4 T cell vaccines There is. In this study, the immunogenic HER3 CD4 peptide effectively overcomes immune tolerance to self tumor antigens. Vaccines utilizing 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 from any tumor antigen for cancer immunotherapy using the DC1-Th1 platform New strategies for 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 the immunogenicity of the identified CD4 + HER3 ECD epitope by “inverse” sensitization, ie confirmation of the ability of the individual epitope-sensitized CD4 + Th1 to recognize the native HER3 ECD FIG. 15 shows that it has HER3 ECD reactivity In living donors, CD4 + T cells are sensitized by DC1 pulsed with their respective donor-specific, immunogenic HER3 epitopes and reactivated against iDCs pulsed with their respective HER3 epitopes and native HER3 ECD. Show that.

  Figures 20 and 21 show the additional results of "reverse" sensitization.

CD4 + Th1 sensitized with DC1 pulsed with an immunogenic HER3 epitope were obtained from 4 HER3 ECD non-responsive donors, as seen in Figure 16 which appears to abolish anti-HER3 immunoself tolerance All CD4 + Th1 cells were sensitized using DC1 pulsed with 4 identified HER3 peptides, and all these cells were subsequently challenged to recognize HER3 ECD pulsed iDC. Not only did donors not only show good sensitization to individual HER3 epitopes, but also recognized native HER3 ECD.

CD4 + HER3 epitopes show MHC class II promiscuity As seen in Figure 17, experiments were performed using the extracellular domain (ECD) of HER3 as a candidate "oncogenic driver" tumor antigen Immunogenic HER3 CD4 + peptides have been identified that display differential and generate anti-HER3 CD4 + immunity; these peptides can be used in vaccine constructs.

Peptides Derived from Tumor Antigens The results presented herein are:
-To be able to identify promiscuous MHC class II peptides by DC1 pulsed with a library of overlapping peptides obtained from tumor antigens to develop a CD4 + T cell vaccine,
-Immunogenic HER3 CD4 + peptides effectively overcome immune tolerance to self tumor antigens,
-From these results, in order to rapidly and reproducibly identify class II promiscuous immunogenic CD4 + epitopes obtained from any tumor antigen, for cancer immunotherapy using the DC1-CD4 + Th1 platform That a new strategy for
-Vaccines constructed utilizing these HER3 CD4 + peptides have been shown to be worthwhile to study in patients with cancers that overexpress HER3.

HER3 expression is a marker of tumor progression in premalignant lesions of the gastroesophageal junction Overexpression of RTKs including members of the HER family has prognostic and therapeutic significance in invasive esophageal gastric cancer. RTK expression in premalignant gastroesophageal lesions has not been extensively investigated before.

  The presence of Barrett's esophagus, or metaplastic columnar epithelium of the distal esophagus, predisposes to the development of esophageal adenocarcinoma. While histologic metastasis from dysplasia to invasive malignancy is well characterized, carcinogenesis in metaplastic cells is associated with poorly understood genetic alterations (Cameron, A. J. Et al., 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 esophagus lesions with dysplasia. (Almhanna, K. et al., Appl. Immunohistochem. Mol. Morphol. July 16 2015) (Almhanna, et al.); Fassan, M. et al. , Et al. , Histopathology 61 (5): 769-76 (2012) (Fassan, et al.); And Rossi, E. et al. Et al. Cell. Mol. Med. 13 (9 B): 3826-33 (2009) (Rossi et al.). Furthermore, the rate of HER2 expression correlates with the degree of dysplasia, suggesting a relevant pathway in tumorigenesis.

  Overexpression of RTK molecules including members of the mesenchymal-epidermal transition factors HER family (HER1, HER2, and HER3) and cMET has been demonstrated in many of the more common malignancies including breast cancer, lung cancer and gastrointestinal cancer (Yokata, J., et al., Lancet 1: 765-767 (1986)) and the identification of HER2 overexpression in a subset of breast cancer with esophageal cancer, overexpression of HER2 and more aggressive biology and efficacy Inhibition of HER2 by targeted monoclonal antibodies was a central event in the evolution of targeted therapies for the treatment of solid tumors (Joensuu, H. et al., N. Eng. J. Med. 354 (8): 809). -20 (2006)) This experience targets RTK molecules in the treatment of other malignancies It provides a basis for further efforts to.

  Overexpression of HER2 has been demonstrated in a small number of gastric cancers and has a minor impact on the outcome and is a target for trastuzumab in metastatic settings (Bang, Y. J. et al., Lancet 376 (9742) 687). -97 (2010) (Bang et al.)). Overexpression of HER2 is more frequently expressed at the proximal gastric and gastroesophageal junction as compared to the more distal gastric adenocarcinoma (Rajagopal et al., I., et al., J. Clin. Diagn. Res. 9 (3): EC 06-10 (2015)) Trastuzumab was targeted for translocation and had a modest impact on 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 is a poor prognosis in most relevant 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 modulates the activity of HER1 and HER3. These data provide the basis for further efforts to characterize RTK expression in esophagogastric 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 Clinical approval of 73 patients with Barrett's esophagus with dysplasia (low grade dysplasia (LGD), n = 32, high grade (HGD), n = 59) after approval of the University of Pennsylvania Institute Review Committee Records and histological specimens were reviewed retrospectively. Formalin-fixed paraffin-embedded tissue blocks from stored endoscopic biopsy and mucosal resection specimens from 2003-2012 are sectioned at 5 μm on plus slides (Fisher Scientific, Waltham, Mass.) And subsequently deparaffinized And allowed to rehydrate. All biopsies were taken 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 apparatus), evaluated by a single pathologist (Leica Bond-III) under a microscope. Membrane 3+ HER staining was considered as positive as membrane 2+ HER2 staining in 10% or more of the tumor cells as seen in FIG. CMET immunohistochemistry was performed in 42 cases where sufficient tissue was available. Moderate or scleral staining of 50% or more of tumor cells was considered positive. RTK overexpression was correlated with clinical data to assess its relevance to either diagnosing invasive carcinoma, adenocarcinoma of the paired aplastic adenocarcinoma biopsy specimen or subsequent biopsy specimens.

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

Results Seventy-three Barrett's esophagus patients with low grade dysplasia (n = 32) or high grade formation (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). 81.9% were male and 87.5% were white. Alcohol use in the cohort was 14.3% and active tobacco use was 6.3%, compared to 55.6% for previous smokers. 26.4% had a family history of malignancy. As shown in Table 3 below, there were no significant differences between the LGD and HGD cohorts in the 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).

  Foci of invasive esophageal adenocarcinoma are associated with dysplastic lesions in 6 cases, all in association with HGD (HGD: 10.2% vs. LGD: 0.0%, p <0.001) Occurred. An additional nine patients were diagnosed with invasive esophageal adenocarcinoma in subsequent biopsy specimens (HGD: 17.0% vs. LGD: 0.0%, p = 0.017). 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 , Patients with significant association of HER3, as seen in Figures 23A and 23B, patients without lesions of invasive cancer (71.4% vs 38.6%, p = 0.032).

  Overexpression of cMET was observed in 42 (42.9%) specimens with HGD compared to LGD specimens (58.3% vs 36.7%, p = 0.200), most frequently with HER3 It was 38.2% of the HER3 positive sample expressed versus the HER3 negative sample (p = 0.212). The same tendency was not observed in HER1 positive (p = 0.729) or HER2 positive (p = NA) samples. In 1 of 42 patients (5.6%), invasive cancer was identified. CMET was overexpressed in this patient (p = 0.243).

Discussion This analysis of RTK expression in dysplastic lesions of the gastroesophageal junction (1) HER family proteins upregulate dysplasia in Barrett's esophagus, (2) the frequency of HER family and cMET overexpression differ There is a positive correlation with the degree of formation; (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 potentially invasive disease in Barrett's esophagus and HGD patients. Furthermore, therapeutics 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 was limited to assessment of over-expressed HER2 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 in particular frequent overexpression of HER3 are unexpected new findings. Homodimerization and heterodimerization of HER receptors drive signal activation. Clustered overexpression of multiple members of the HER family has been observed in other tumor types. Together, activated c-MET positively regulates the activity of HER1 and HER311. In fact, the interaction between these receptors provides the rationale for multivalent therapies that target multiple RTKs. (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 ductal carcinoma of the breast (DCIS) has targeted promising results. U.S. Patent Application Publication No. 2015/0323547; U.S. Patent Application Serial No. 14 / 985,303, filed December 30, 2019, Datta, J. et al. Et al, Onco Immunology 4: 8 e 102 2301 (2015) DOI: 10. 1080/2162402 X. Datta, J. 2015. 102 2301; Et al, Breast Cancer Res. 17 (1): 71 (2015). Such an approach remains a distant goal for gastrointestinal malignancies. Nevertheless, all of the current treatment options of the Barrett's esophagus, including endoscopic resection and removal modes and radical surgery, have serious limitations. Alternative strategies to rescue morbidity and reduce the risk of invasive cancer are needed. This analysis of RTK expression in dysplastic lesions of the gastroesophageal junction (1) HER family proteins upregulate dysplasia in Barrett's esophagus, (2) frequency of HER family and cMET overexpression, dysplasia There is a positive correlation with the degree of (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 potentially invasive disease in Barrett's esophagus and HGD patients. Furthermore, therapeutics that target HER3 or CMET can provide secondary prevention of gastroesophageal cancer in a subset of patients.

  In summary, the present data show that over-expression of HER3 in high-grade dysplastic lesions of the gastroesophageal junction, especially those with latent invasive cancer and malignant transformation, and these findings indicate that HER3-expressing dysplasia It may justify a more aggressive management approach of the lesions and provides a basis for the future application of HER3 targeted therapeutics in early disease setting that would be readily understood by those skilled in the art.

  We previously showed progressive loss of native anti-HER-2 CD4 Th1 during HER-2 pos breast tumorigenesis. This loss of response was associated with the absence of a pathological complete response ("pCR") to neoadjuvant treatment, correlated with an increased risk of recurrence of breast cancer and could be reversed with vaccination. Example 4 below examines whether there is a similar loss of anti-HER3 Th1 responses during breast cancer development.

Loss of anti-HER3 CD4 Th1 occurs in breast tumorigenesis and is negatively associated with outcome
Summary of Overall Example 4 We previously showed progressive loss in native anti-HER2 CD4 Th1 during HER2 ^pos breast tumorigenesis. This loss of response was associated with the absence of a pathological complete response ("pCR") to neoadjuvant treatment, correlated with an increased risk of recurrence of breast cancer and could be reversed with vaccination. This example examines whether there is a similar loss of anti-HER3 Th1 responses during breast tumor development.

  Peripheral blood was collected from 131 subjects including healthy donor ("HD"), benign breast disease ("BD"), in situ ductal adenocarcinoma ("DCIS") and invasive breast cancer ("IBC") patients . The immune response to the four different HER3 immunogenic peptides identified in Examples 1 and 2 above was tested via an enzyme-linked immunosorbent (ELISpot) assay to analyze all metrics of immune response.

There was a significant reduction in the anti-HER3 response from HD to IBC. Triple negative ("TN") IBCs showed the lowest response across all three immune parameters. HD had significantly higher immune responses than ER ^pos IBC and TN IBC patients across all three immune parameters. Interestingly, HER2 ^pos IBC showed similar immune responses as HD and BD. Patients with recurrent breast cancer and lack of pCR for neoadjuvant therapy had significantly lower anti-HER3 CD4 Th1 responses than patients without subsequent relapse or patients with neoadjuvant therapy from pCR.

  Thus, CD4 Th1 anti-HER3 was found to be lost during breast tumorigenesis, especially in the group with limited treatment options, prominent in TN IBC, a group with a significant prognosis of HER3 overexpression It was found to be. These findings imply an attempt to restore this response to prevent recurrence.

Background: About 1 in 8 women develop breast cancer in their lifetime. Among these, cancers overexpressing HER2 have a high rate of distant metastasis and a poor prognosis overall. Introduction of trastuzumab, a monoclonal antibody to HER2, dramatically improves progression free survival and overall survival in HER2-positive cancer patients and points out an important role of HER2 in regulating breast cancer progression. Giordano S. H. Et al. Clin. Oncol. (2014): JCO-2013 [May 5, 2014 published online before printing, doi: 10.1200 / JCO. 2013.54.0948].

The immune system plays an important role in the regulation of HER2-expressing tumors. It has previously been shown that the native anti-HER2 CD4 T cell response transitioning from healthy subjects to HER2 ^pos DCIS, HER2 ^pos IBC, HER2 ^neg IBC is progressively reduced. Furthermore, lower anti-HER2 immune response correlates with subsequent breast cancer recurrence, while higher anti-HER2 immune response correlates with pathological complete response to preoperative chemotherapy and the role of immune system in HER2 ^pos tumorigenesis Suggested. Datta, J. See, Onco Immunology 4 (10): e1027474. DOI: 10.1080 / 2162402X. 2015. 102. 2301 (2015) and U.S. Patent Application Publication No. 2015/023547 A1 (collectively "Datta et al." Hereinafter). Our group has developed a HER2 pulsed dendritic cell vaccine that ameliorates anti-HER2 CD4 and CD8 T cell responses in both DCIS and IBC patients. Sharma, A. Et al., Cancer 118 (17): 4354-4362 (2012). Koski, G. K. Et al. Immunother. 35 (1): 54-65 (2102); and U.S. Published Application No. 2015/0323547 A1.

  HER2 is a member of the EGFR family, an RTK group that also includes HER1 and HER3. While it is well known that HER2 self-dimerizes, the role of HER3 in signal transduction is less clear and may act to dimerize both HER2 itself and HER2. HER3 dimerization by HER2 has been proposed as an escape mechanism in trastuzumab-treated breast cancer patients. Czopek, J.M. Et al., Contemp. Oncol. 17 (5): 446-9 (2013) ("Czopek et al.") And Bae, S. et al. Y. Et al, Breast Cancer Res. Treat. 139 (3): 741-50 (2013) ("Bae, et al."). Pertuzumab is the latest addition to the market and is the first tumor treatment to receive accelerated FDA approval as neoadjuvant treatment, inhibits HER2 / HER3 dimerization and is combined with trastuzumab for breast cancer patients Use has been shown to have an overall survival benefit. Jhaveri, K. Et al. Natl. Compr. Canc. Netw. 12 (4): 591-8 (2014) and Harbeck, N. et al. Et al., Breast Care 8 (1): 49-55 (2013).

Although there is over-expression seen in ER positive, HER2 positive and triple negative ("TN") subtypes, HER3 expression is less well delineated among breast cancer subtypes. Moeder, C.I. Et al., Cancer 115 (11): 2400-9 (2009). It is interesting to note that while HER3 overexpression may be more common in HER2 ^pos IBC, its prognostic value is more important in TN IBC. HER3 expression in ER ^pos / HER2 ^pos IBC did not affect disease free survival ("DFS") or overall survival ("OS"), whereas HER3 expression in TN IBC was 5 years of DFS and 10 years of OS Correlated with the deterioration of the Bae et al., And Czopek et al. Notably, a significant subset of TN IBC patients with HER3 overexpression, a group that by definition does not have any of the classical treatment options, can benefit from recognizable new targets. It is unknown whether an anti-HER3 CD4 Th1 response is present in healthy donors and whether this response changes during breast tumorigenesis. This study seeks answers to these questions.

Method
Subject Registration A total of 131 subjects met the study criteria and were consecutively enrolled at the University of Pennsylvania with informed consent. The study was conducted prior to being approved and approved by the Institutional Review Board of the University of Pennsylvania and the Abramson Cancer Center. Of the 131 subjects, 9 performed the assay in the absence of peripheral blood monocytes, and 122 subjects left immune response data for review. (HD, n = 30), benign breast disease (BD, n = 11), DCIS (n = 13), HER2 ^pos IBC (n = 21), and CD4 against 4 different HER3 immunogenic peptides against HER2 polypeptide T cell responses were compared. ER ^pos invasive breast cancer (ER ^pos IBC, n = 20) and triple negative IBC (TN IBC, n = 27) are included.

Peripheral Blood Monocyte Collection Peripheral blood was collected by venipuncture. Blood was diluted in Hank's buffer or PBS at a ratio of 1: 1, and lymphocyte separation medium was overlaid under diluted blood in conical tubes. The blood was then separated by density centrifugation at 1200 rpm for 30 minutes. The mononuclear cell layer was collected and washed twice with Hanks buffer or PBS. The cells were counted and resuspended at 10 million cells per milliliter and frozen at minus 80 ° C. for 24-48 hours before transferring to minus 200 ° C. Here, cells remained stored until experimental assay.

Measurement of Anti -HER3 CD4 Th1 Response The anti-HER3 CD4 Th1 cell response was measured by ELISpot assay according to the manufacturer's protocol. In brief, 96 well PVDF membrane plates were activated with 70% ethanol, washed with PBS, coated with anti-IFN-γ (anti-IFN-γ) antibody and incubated overnight at 4 ° C. After 24 hours, the plates were again washed with PBS and blocked for 1 hour with Iscove's medium containing 10% human serum. Peripheral blood monocytes are thawed at 37 ° C., washed with PBS or Hanks buffer, resuspended at 1 million cells per milliliter, four immunogenic HER3 peptides (p12 (peptide 56-70): CEVVMGNLEIVLTGH (sequence No. 4); p81 (peptide 401-415): SWPPHMHNFFSVFSNL (SEQ ID NO: 5); p 84 (peptide 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6); Anti-CD3 / CD28 (polyclonal stimulation, positive control) Tetanus toxoid (Santa Cruz Biotechnology, Dallas, TX) or nothing (non-stimulation control) assays were performed in triplicate. The plate was washed with PBS, biotinylated anti-IFN-γ antibody was added, and the plate was incubated for 2 hours at 38 ° C. The plate was washed again with PBS and the streptavidin-HRP was incubated for 1 hour at 38 ° C. Finally, the plate was washed with PBS and then TMB substrate solution was added Color development was stopped after 5 minutes by thorough washing with tap water The plate was dried overnight at 4 ° C.

Immune response analysis spots were counted by immunospot software. (1) Cumulative response, or total response to all four HER3 immunogenic peptides in spots per million cells, (2) repertoire, or number of peptides per subject with 20 or more spots, (3) responses Three parameters or metrics were quantified: gender, or percent of subjects responding to at least one peptide (defined as a threshold of 20 or more thresholds). Tetanus responses at spots per 200,000 cells and anti-CD3 / CD28 responses at spots per 200,000 cells were also quantified as controls. All immune response metrics were analyzed with GraphPad Prism software.

result
A total of 131 subjects under study met the test criteria and were enrolled consecutively with informed consent at the University of Pennsylvania hospital. Nine subjects had insufficient cells for analysis, leaving 122 subjects. Among them, the average age was 50 years, between 25 and 83 years. Caucasians were 72.1%, African Americans 18.0%, and another race 9.8%. HD (n = 30), BD (n = 11), DCIS (n = 13), HER2 ^pos IBC (n = 21), ER ^pos IBC (n = 20) or TN IBC (n = 27). Of the 68 IBC subjects, 35 (51.5%) in stage I, 22 (32.4%) in stage II, 9 (13.2%) in stage IV and 2 in stage IV 2.9%). There were 52 (76.5%) patients who received chemotherapy and / or herceptin and / or tamoxifen and 16 (23.5%) who had no treatment. Three DCIS patients and three HER2 ^pos IBC patients received HER2 pulsed dendritic cell vaccination. Other properties are reported in Table 4 below.

Decreased anti-HER3 immune response of CD4 Th1 cells from healthy donors to subsets of invasive breast cancer There is a reduction in all three immune parameters when comparing HD, BD, DCIS, HER2pos IBC, ERpos IBC, and TN IBC And reached the lowest point of the TN IBC: cumulative response (90 vs 80 vs 66 vs 79 vs 48 vs 40, p = 0.01 respectively, as shown in FIG. 24A) repertoire (as shown in FIG. 24B) 1.0 vs. 0.6 vs. 0.8 vs. 0.8 vs. 0.5 vs 0.3, p = 0.003) and responsiveness (respectively as shown in FIG. 24C, 76.7% vs. 63. 6% vs. 53.8% vs. 66.7% vs. 45.0% vs. 33.3%, p = 0.002). In particular, these differences are cumulative response (90 vs. 40, p = 0.002), repertoire (1.0 vs. 0.3, p = 0.004), responsiveness (76.7% vs. 33.3%) Not only statistically significant higher in HD but also more than doubled compared to TN IBC patients across all three immune parameters, p = 0.001). BD has a significantly higher cumulative response (40 vs. 80, p = 0.007, respectively) and DCI patients show a significantly higher repertoire compared to TN IBC patients (0.8 vs. 0.3 respectively) , P = 0.04), HER2pos IBC showed significantly higher repertoire (0.3 vs. 0.8, p = 0.01) and responsiveness (33.3% vs. 66.7%, respectively p = 0.04). ERpos IBC patients are second to HD over all three immune parameters: cumulative response (48 vs. 90, p = 0.03 respectively), repertoire (0.5 vs. 1.0, p = 0.008) Have a low anti-HER3 CD4 T cell response and are responsive (45.0% vs. 76.7% respectively, p = 0.03 respectively). Of note is the fact that HER2 ^pos IBC anti-HER3 responses did not change significantly from HD, BD or DCIS subjects.

Decreased anti-HER3 immune response of CD4 Th1 cells in patients with invasive breast cancer is specific for HER3 and analyzes polyclonal stimulation with tetanus response and anti-CD3 / CD28 response that are not attributable to a broad loss of immune response The overall immune responsiveness was compared and controlled. There was no difference in the anti-tetanus response of CD4 Th1 cells as measured via ELISpot assay among patients with HDIS, BD, DCIS, HER2 ^pos IBC, ER ^pos IBC or TN IBC (37 vs. 30 vs. 19 vs. 34) Vs. 24 29 p = 0.65). Importantly, the anti-tetanus response (the most divergent anti-HER3 CD4 Th1 cell response group) between HD and TN IBC patients was similar (37 vs. 29, p = 0.37). Similarly, there was no difference in polyclonal stimulation with anti-CD3 / CD28, and the majority of subjects in each group had astonishing spot development, which was uncountable. Among computable patients, HD, BD, DCIS, HER2 ^pos IBC, ER ^pos IBC or TN IBC patients (688 vs 549 vs 804 vs 699 vs 629 vs 675, p = 0.68, respectively, as shown in FIG. 25. There was no statistically significant difference between

Anti-HER3 CD4 Th1 Cell Responses in Patients with Invasive Breast Cancer Correlate with Prognosis and Features of Tumor Invasion Initial surgery (node positive) to determine whether anti-HER3 CD4 T cell responses correlate with features of tumor infiltration Compare immune response of IBC patients with ("LN ^pos ") versus lymph node negative ("LN ^neg ") lymph node status ("pCR") vs. residual disease ("pCR") Diagnosis and response for at least 1 year from diagnosis and response LN ^pos IBC patients (n = 28) have an overall immune response across all three parameters, compared to LN ^neg patients (n = 31). But not statistically significant: cumulative response (40 vs. 56, p = 0.12 respectively), repertoire (as shown in FIG. 26A). Are each, 0.4 vs. 0.6, p = 0.08) and responsive (respectively 35.7% vs. 54.8%, p = 0.19). ^Remarkably, the ^{LN pos} subjects According to the analysis, subjects with lymph node metastasis after preoperative chemotherapy were included, because it was unclear whether subjects after LN ^neg postoperative chemotherapy had a positive node before treatment. Of patients with at least 1 year or more since diagnosis, recurrent breast cancer (n = 7 for either local or distant metastasis) remained disease free (n = 36) for all three immune parameters ): As shown in FIG. 26B, cumulative response (17 vs. 66, p = 0.04 respectively), repertoire (0.0 vs. 0.6 respectively, p <0.05) and responsiveness (0% vs. 55 respectively) 6%, p = 0.01) Finally, FIG. As shown in C, among patients who received neoadjuvant chemotherapy (n = 16), pCR (n = 5) had a significantly higher cumulative response (144 vs. 32, respectively) compared to pCR (n = 11). There was a statistically significant difference between pCR and <pCR, with p = 0.004) and repertoire (each 0.8 vs 0.4, p = 0.05) (80.0% vs. 27.3% respectively, p = 0.10) There was no difference in tetanus response between LN ^pos and LN ^neg patients (22 vs. 29, p = 0.35 respectively) There was no difference between relapsed vs non-relapsed patients (27 vs. 35, p = 0.65 respectively) and pCR vs <pCR (17 vs. 59 respectively, p = 0.15), thus more aggressive tumor characteristics The reduction of CD4 Th1 cell responses in IBC patients with It is specific to R3.

Anti-HER3 CD4 Th1 cell response under age 50 (n = 25) or over 50 (n = 16) according to characteristics of healthy donors , race (Caucasian (n = 29), African American (n = 12)) Anti-HER3 CD4 Th1 responses were compared in HD and BD up to. Pregnancy status (0 (n = 17) or one or more pregnancy (n = 24)) and menopausal status (pre-menopausal (n = 30) or post-menopausal (n = 11)). There was no difference in cumulative peptide response, repertoire or responsiveness by age (FIG. 27A), race (FIG. 27B) or past pregnancy history (FIG. 27C). However, compared to postmenopausal women, postmenopausal women showed significantly higher cumulative responses compared to premenopausal women, as shown in FIG. Spot pairs p = 0.005) (upper panel) and repertoire (1.4 vs 0.8 peptides, respectively p = 0.03) (second panel). There was no statistically significant difference in responsiveness (90.9% vs. 66.7%, p = 0.23, respectively) (third panel). There was no statistically significant difference in tetanus response between premenopausal women (lower panel), indicating that menopausal differences in immune response were specific for HER3.

ELISpot Assay is Accurate as Demonstrated by Linearity Precision Assay ELISpot assay has been previously validated in our laboratory. Linearity accuracy assays were performed with serial dilutions from known high anti-HER3 CD4 T cell responders to confirm the accuracy of this assay under the operator who performed all experiments for this study. Peripheral blood monocytes were serially diluted in the medium at a concentration of 1.0-0.1-0.01-0.001 and the cumulative anti-HER3 immune response was measured in spots per million cells. FIG. 28 shows that there was a linear decrease in points where the cumulative value was 230-35-12 (p <0.0001, r = 0.88).

Discussion The knowledge of the role of the immune system in the development, progression and prognosis of cancer is rapidly expanding. Immunodeficiency states are well established to increase the risk of developing cancer from tumors of viral as well as tumors of non-viral origin. Boshoff, C.I. , Et al. Nature Rev. Cancer 2: 373-82 (2002). Sheil, A. G. , World J. Surg. 10: 389-96 (1986); Transplantation 61: 274-78 (1996); and Penn, I., et al. Transplantation 60: 1485-91 (1995). It is also known that certain immunophenotypes are associated with breast cancer. The circulating inflammatory cytokines TNF-α and IL-6 are higher in breast cancer patients and low CD4 ^pos / CD8 ^pos T cell ratios are associated with a more aggressive breast cancer phenotype, but tumor infiltrating lymphocytes are more selective in some breast cancers It is associated with a better prognosis. Alokai, M .; S. , Et al. Med. Oncol. 31 (8): 38 (2014) doi: 10.1007 / s 12032-014-0038-0; Et al, Med. Oncol. 31: 981 (2014); and Matsumoto, H. et al. Et al. Clin. Pathol. doi: 10.1136 / jclinpath-2015-202944. However, the evidence associated with the loss of immune recognition for specific molecules Oncodria in other immunocompetent hosts is relatively new. Recently, a decrease in native anti-HER2 CD4 Th1 cell responses to transition from healthy donors to HER2 ^pos DCIS, HER2 ^pos IBC has been shown, the first to show a loss of immune response to this particular oncodriver in breast cancer development It is one of the research. Those skilled in the art will readily understand that the identification and understanding of such specific losses have many of the potentials of a particular immunotargeting therapy.

This study shows that (1) anti-HER3 CD4 Th1 cell response decline from HD to ER ^pos and TN IBC, (2) anti-HER3 response correlates with prognosis, specifically lower response is relapse Related (3) post-menopausal HD has been shown to have a significantly higher anti-HER3 immune response. All of these findings will have diagnostic and clinical applications of anti-HET3 CD4 Th1 cell responses.

The anti-HER3 CD4 Th1 cell response was highest in HD and lowest in TN IBC whose prognosis is more severely affected by HER3 overexpression than other types of IBC. Bae et al., And Czopek, J.A. Et al. While HER3 expression is unknown in the current cohort of IBC patients, the TN IBC and ER ^pos IBC groups have higher levels of HER3 expression compared to the HER2 ^pos IBC cohort and have a response similar to that of HD. Indicated. In fact, our previous studies showed that the anti-HER2 CD4 Th1 cell response was directly correlated with HER2 expression; there was a significant reduction of HER2 ^pos IBC but no reduction of HER2 ^neg IBC. HER3 not only has a major impact on the prognosis of TN IBC compared to receptor-expressing breast cancer, but even on TN IBC may have a major impact on the prognosis of HER2 (0) compared to HER2 (1+) tumors There is sex. Schmidt, G., et al. Et al, Arch. Gynecol. Obstet. 290: 1221-29 (2014). This may explain in part the similarity in the immune response between HER2 ^pos IBC and HD. Tumor progression can not circumvent immune surveillance if the tumor is already infiltrated due to overexpression of HER2. However, if the immune system already recognizes and targets HER2, tumors can be adapted by HER3 overexpression, where immune evasion becomes an evolutionary advantage for tumor cell survival.

Interestingly, ER ^pos IBC showed anti-HER3 CD4 T cell responses similar to TN IBC and was significantly lower than HD or HER2 ^pos IBC. Although HER3 expression is not of prognostic significance in ER ^pos IBC compared to TNF-IBC, there is evidence to show that HER3 mRNA expression is positively correlated with ER expression. Fujiwara, S. Et al., Breast Cancer 21: 472-81 (2014) which may explain the lower immune response seen with this subgroup of IBCs.

  Breast cancer patients with recurrent disease have lower anti-HER3 CD4 Th1 responses compared to patients who remain disease free, indicating that immune surveillance may be an important mechanism for successful long-term treatment ing. Patients with relapse are more likely to have HER3-expressing tumors and may be correlated with a higher risk of recurrence, metastasis, and overall survival. Li, Q. Et al., Oncology Reports 30: 2563-70 (2013); Smirnova, T. et al. Et al, Oncogene 31: 706-15 (2012); and Ocana, A. Et al. N. C. I. 105 (No. 4): 266-73 (2013). Furthermore, immunoediting has been proposed as an escape mechanism by which the neoplastic cells are selectively eliminated by the immune system until the neoplastic cells have evolved to express the molecule Oncodria like HER3 which escapes immune recognition. Dunn, G. P. Et al., Nature Immunology 3 (11): 991-8 (2008). Thus, expression of HER3 is possible due to the lack of immune surveillance, which increases the risk of relapse. Interestingly, recent evidence suggests that recurrent tumors may not be pathologically identical to primary tumors. The mismatch rate between primary and secondary tumors is particularly high for progesterone receptors, and any type point mismatch worsens the prognosis of recurrent tumors. Idriisinghe, P .; K. A. , Et al. , Am. J. Clin. Pathol. 133: 416-29 (2010); Broom, R., et al. J. , Et al. , Anticancer Research 29: 1557-62 (2009); and Liedtke, C.I. , Et al. , Annals of Oncology 20: 1953-58 (2009). Studies to date have not been considered to compare HER3 expression between primary and secondary tumors. It is unclear whether HER3 expression is inconsistent between primary and recurrent tumors, and whether HER3 expression represents a relapse avoidance mechanism to occur. If so, targeting patients with low anti-HER3 CD4 T cell responses may help improve immune surveillance and prevent long-term recurrence.

  Also, suggesting a role for the immune system in prognosis, patients with pCR and preoperative chemotherapy had significantly higher anti-HER3 CD4 T cell responses than those with <pCR. HER3 signaling has been shown to mediate acquired resistance to targeted therapies. Sergina, N .; V. Et al., Nature 445: 437-41 (2007); and Frogne, T. et al. Et al, Breast Cancer Res. Treat. 114: 263-75 (2009). Here, it is not an acquired resistance but rather an early resistance, making the anti-HER3 immune response a potential prognostic marker for patients most likely to benefit from neoadjuvant treatment. Further studies should reveal whether anti-HER3 immune responses can be intervened to promote not only prognosis but also response to treatment.

In current studies, subsets of HD, especially postmenopausal women, showed higher anti-HER3 responses. However, unlike previous findings of anti-HER2 responses, there was no difference based on pregnancy history. Biologically, this higher anti-HER2 response may be due to breast regression and subsequent exposure of cellular proteins to immune surveillance by pregnancy, but such changes in chest parenchyma are menopausal Hard to happen in Press, M. F. Et al, Oncogene 5: 953-62 (1990). In imaging that mimics breast regression during pregnancy and also exposes cellular proteins normally expressed in breast tissue to the immune system, changes in mammary gland density with a hormonal change (menopausal) have been observed. Clendenen, T .; V. Et al, Magnetic Resonance Imaging 31: 1-9 (2013). Alternative explanations may indicate that higher anti-HER3 immune responses in postmenopausal women are attributable to differences in risk. Expression in various breast cancer recipients is clearly age-dependent: HER2 ^pos IBC is less likely to decline with age, and ER ^pos IBC becomes more likely. Clark, G .; M. Et al. Clin. Oncol. 2: 1102-09 (1984); Eppenberger-Castori, S. et al. , Et al. , Int. J. Biochem. And Cell Biol. 34: 1318-30 (2002). Furthermore, these two receptors are not independent of one another due to age-dependent HER2 dependent ER / PR expression and vice versa. Neven, P .; Et al, Breast Cancer Res. Treat. 110: 153-59 (2008). TN IBC patients, the group with the lowest anti-HER3 immune response and the highest prognosis for HER3 overexpression, occur more frequently in premenopausal women. Bae et al., And Howlander, N .; Et al. N. C. I. 106 (5): 1-8 (2014). Thus, a subset of premenopausal HD is a group at high risk of developing TN IBC, and postmenopausal HD has already exceeded this higher risk period, and a low risk group that actually has both HER2 and HER3 Over-express breast cancer. It is also worth noting that while TNIBC is more common in young women, it has shown that in older people the cause is a better prognosis for unknown reasons. This partially explains the good prognosis of TN IBC in postmenopausal women, if the higher anti-HER3 immune response is in fact due to biological mechanisms occurring in menopausal rather than risk aversion groups be able to.

Conclusions This example demonstrated a reduction in anti-HER3 CD4 T cell responses from HD, BD and DCIS towards ER ^pos and TN IBC. Furthermore, lower anti-HER3 responses are correlated with relapse and <pCR and neoadjuvant treatment, indicating that this immune response may play a prognostic role in invasive breast cancer. Most importantly, these results are consistent with previous studies that the reduction of the HER2 immune response from HD to HER2 ^pos DCIS is migrating to HER2 ^pos IBC. Such similar results are promising not only to confirm previous findings but also to point out the larger role of the immune system in molecular oncodriver patrol. Interestingly, postmenopausal HD is generally at higher risk of developing HER3-overexpressing breast cancer and is significantly more immune response than premenopausal HD, potentially pointing to a mechanism that mediates this risk. Had. It is important to determine whether HER3 pulsed DC1 vaccination may have a therapeutic and / or risk modifying effect on the development of HER3 overexpressing breast cancer. It will be equally important to continue to investigate the role of the immune system in other oncodriver-specific cancers.

  Conclusion: The anti-HER-3 immune response of CD4 Th1 cells is lost from healthy donors in invasive breast cancer, in particular TN IBC, a group with limited treatment options and a significant prognosis of HER-3 overexpression. The anti-HER3 immune response also moderates the response to treatment and prognosis and indicates a potential immunotherapeutic target. The addition of the HER3 immunogenic peptide to the DC1 vaccine may increase the population of IBC patients who benefit from vaccination. Most importantly, these results reflect the previous findings, pointing out that the role of the immune system in the patrol of the molecule Oncodria is significant.

  The findings of this example, namely the significant loss of anti-HER3 CD4 + Th1 in breast tumorigenesis from HDS to IBC, will be understood by those skilled in the art as useful for the diagnosis and treatment of HER3. In particular it expresses breast cancer, in particular triple negative IBC. It is believed that blood tests can be developed to detect circulating anti-cancer CD4 + Th1 responses in subjects to take advantage of these findings. Preferably, such blood tests are of the four HER3 immunogenic peptides used herein or any other MHC class II immunogenic peptide based on the type of cancer a patient is suffering from. Patients using such HER3 immunogenic peptides, and by way of non-limiting example, patients with recurrent breast cancer and pCR for neoadjuvant therapy are monitored by such blood tests to determine their anti-HER3 CD4 Th1 Responses can be determined and treated accordingly.

  Low anti-HER3 responses detected by blood tests or other means of the patient, eg recovery methods such as vaccines and preferably monocyte-derived dendritic cell based vaccines of patients pulsed / incubated with HER3 immunogenic peptides , Such as, for example, the 4HER3 immunogenic peptide used in the examples herein. Those skilled in the art will readily understand that there are other ways to restore the patient's immune response. In particular, for patients with TN IBC, a group with essentially limited treatment options, methods to measure HER3 responses, and, if necessary, methods to recover such responses via DC1 vaccine are very valuable. is there. Anti-HER3 immune response can also be used as a potential prognostic biomarker for patients in need of neoadjuvant treatment. The HER3 pulsed DC1 vaccine or other suitable vaccine may have a therapeutic and / or risk modifying effect on the development of HER3 overexpressing breast cancer as well as other HER3 expressing cancers. The findings herein can be evaluated as useful in the development of array blood tests and tests contemplated herein for diagnosis and / or treatment.

  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 particular embodiments, it is understood that other embodiments and modifications of the invention can be devised without departing from the true spirit and scope of the invention. It is clear to the trader. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

(Cross-reference to related applications)
This application is a continuation-in-part of PCT / US16 / 21042 filed on March 4, 2016, which application is a continuation-in-part of PCT / US15 / 41034 on July 17, 2015 . The application claims priority to US Provisional Patent Application No. 62 / 076,789, filed Nov. 7, 2014, and US Provisional Patent Application No. 62/025, 681, filed Jul. 17, 2014. Do. The entire content of each patent application is incorporated herein by reference.

(Sequence listing)
This application contains a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. This ASCII copy, created on 14 June 2016, is 319-003_PCT_CIP2_SL. It is named txt and its size is 2,666 bytes.

  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) enhances tumor aggressiveness and relapse and It is 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 ("RTK"). As one of the four members of the human epidermal growth factor receptor ("EGFR") family, HER2 stands out from others in several aspects. First, HER2 is an orphan receptor. High affinity ligands have not been identified. Second, HER2 is a preferred partner for other EGFR family members (HER1 / EGFR, HER3 and HER4) to form heterodimers, which have high ligand affinity and superiority. Signal transduction activity. Third, full-length HER2 undergoes proteolytic cleavage to release soluble extracellular domain ("ECD"). Shedding of ECD has been shown to be an alternative activation mechanism for full-length HER2 both in vitro and in vivo, as membrane anchor fragments with kinase activity remain. The central role of HER2 in EGFR family signaling correlates with its involvement in carcinogenesis of several types of cancer, for example 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 cause tumor cells to be resistant to certain chemotherapies (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 RTKs regulate a wide 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). At the moment, epidermal growth factor ("EGF"), heparin-binding EGF-like growth factor ("HB-EGF"), transforming growth factor alpha (TGFα), amphiregulin (AR), epiregulin, betacellulin and heregulin Eleven ligands have been reported, including. These ligands bind directly to their cognate receptors, which leads to the formation of homodimers or heterodimers of the receptor that cause activation of multiple signal transduction pathways. Dysregulation of the HER-family members either by mutational activation, 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. When HER3 is activated by heregulin, it 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 proliferation and survival. To date, HER3 is inactive as a kinase due to the apparently unusual sequence features in its kinase domain, and the kinase activity of the HER-family is impaired to initiate signal transduction events It has been speculated that heterodimerization with members not required is required. Consistent with this, it has been shown that HER2 requires HER3 to drive cell growth of mammary tumors. However, recent findings have also shown that HER3 is also able to phosphorylate Pyk2, resulting in activation of 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 evade HER-family inhibitor therapy by up-regulating HER3 signaling, and HER3 inhibition abrogates HER2-driven tamoxifen resistance in breast cancer cells Indicated. Furthermore, resistance to the EGFR small molecule inhibitor gefitinib (Iressa) therapy has also been shown to be linked to HER3 signal activation.

  HER3 is a receptor protein and plays an important role in regulating normal cell growth. HER3 lacks its intrinsic kinase activity and, depending on the presence of HER2, transduces signals across cell membranes. Upon transcription, HER3 pre-mRNA contains 28 exons and 27 introns. The intron has been spliced out and the fully spliced HER3 mRNA is composed of 28 exons.

  In the past decade, targeted therapies have emerged as the basis for cancer therapeutics. Members of the EGF receptor family, namely EGFR (or HER1) and ErbB2 (or HER2 / neu), are becoming regarded as particularly intriguing targets, since these RTKs It is because it is deregulated in cancer. The oncogenic function of another member of the EGF receptor family, ErbB3 or HER3, has only recently become controversial due to its ability to mediate resistance to HER2 and its major role in therapies directed against the PI3K pathway It came to be done. Mutation activation 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 precursor.

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

  Despite advances in the art, it remains uncertain whether an effective immune response can be generated in humans using cell or protein based vaccine strategies that target HER3. Thus, there is a need in the art to have additional immunotherapeutic approaches to treat or prevent breast cancer and other malignancies associated with overexpression of HER3 protein. The present embodiment meets 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. Presently preferred embodiments are illustrated in the drawings in order to illustrate the present invention. However, it should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 5 is a graph showing an immunogenic peptide derived from HER3 showing the ability to activate CD4 T cells across many patients (SEQ ID NO: 1-3 , respectively, in the order shown ). FIG. 10 is a graph showing the overall screen of HER3 using a group of 10 peptide fragments. Also shown is a screen plot of HER3 using a single peptide ( each in SEQ ID NO: 4-7 in the order shown ). FIG. 10 is a graph showing the overall screen of HER3 using a group of 10 peptide fragments. Also shown are graphs of the screen of HER3 using a single peptide (SEQ ID NOS: 4 and 7 respectively in the order shown ) . FIG. 10 is a graph showing the overall screen of HER3 using a group of 10 peptide fragments. Also shown is a screen plot of HER3 using a single peptide (each in the order shown, SEQ ID NO: 1-3) . It is a graph which shows the production amount of IFN-gamma from different HER3 peptide. It is a graph which shows the production amount of IFN-gamma from different HER3 peptide. FIG. 16 is a graph showing the amount of IFN-γ produced from the “reverse” screen that sensitized the peptide and the HER3 extracellular domain after previously identifying the peptide. FIG. 16 is a graph showing the amount of IFN-γ produced from the “reverse” screen that sensitized the peptide and the HER3 extracellular domain after previously identifying the peptide. FIG. 16 is a graph showing IFN-γ production from a “reverse” screen initiated with peptide and sensitized to the peptide and HER3 extracellular domain in patients not previously sensitized to the HER extracellular domain . Demonstrates IFN-γ production from a "reverse" screen starting with the entire peptide library and sensitizing to the peptide and HER3 extracellular domain in patients not previously sensitized to the HER extracellular domain It is a graph. Demonstrates IFN-γ production from a "reverse" screen starting with the entire peptide library and sensitizing to the peptide and HER3 extracellular domain in patients not previously sensitized to the HER extracellular domain It is a graph. FIG. 16 is a graph showing IFN-γ production from a “reverse” screen initiated with peptide and sensitized to the peptide and HER3 extracellular domain in patients not previously sensitized to the HER extracellular domain . FIG. 16 is a graph showing a screen of consecutive peptides in the UPCC 15107-24 donor. FIG. 16 is a graph showing a screen of consecutive peptides in the UPCC 15107-38 donor. FIG. 17 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 to show no anti-HER3 reactivity to the identified HER3 peptide and / or native HER3 ECD) FIG. 6 is a graph showing that DC1 pulsed with HER3 epitope sensitized CD4 + Th1 and overcome anti-HER3 immune tolerance. FIG. 16 is a graph showing that CD4 + HER3 epitopes show MHC class II promiscuity. When activated HER-3 CD4 + cells are placed alongside cells expressing HER-3 in a chamber, HER-3 CD4 + cells cause apoptosis or death of breast cancer cells expressing HER-3 FIG. FIG. 1 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. 16 is a graph showing that the immunogenicity of the identified CD4 + HER3 ECD epitope was confirmed by “reverse” sensitization. A screen of HER3 ECD was performed using the single peptide shown. FIG. 7 is a graph showing additional results where the immunogenicity of the identified CD4 + HER3 ECD epitope was confirmed by “reverse” sensitization. The photograph of immunohistochemistry scoring of HER staining is shown. Histogram showing the overexpression rate of HER family in Barrett's esophagus with low grade dysmorphism (LGD) or high grade dysmorphism (HGD) (Figure 23A), and high dysplastic Barrett lesion with associated invasive cancer (with carcinoma FIG. 23 is a histogram (FIG. 23B) showing overexpression rates of HGD) or highly dysplastic Barrett's lesion (HGD) without associated invasive cancer . Figure 24A-24C show a decrease in anti-HER3 CD4 Th1 cell responses to HD (healthy donors) from ER IBC / ^ER-positive IBC (estrogen receptor positive invasive breast cancer (IBC)) and TN IBC (triple negative IBC) . The figure shows histograms (left panel) of IFN-γ ELISpot analysis of systemic CD4 ⁺ Th1 cell responses. Patients studied were HD ; B D (benign breast biopsy); DCIS (HER2 positive (“HER2 ^positive ”) non-invasive ductal carcinoma); HER2 IBC / HER2 ^positive IBC ; ER IBC / ER ^positive IBC a and TN IBC (triple negative IBC); (estrogen receptor positive IBC). The corresponding table to the right of each histogram is an individual comparison by Student 's t-test between the two groups performed at one time. One-way ANOVA tests were performed in all groups. 24A is cumulative anti HER3 CD4 T cell response measured by IFN-gamma spots per million cells via ELISpot assay, the HDS to BD, the DCIS, to HER2 ^positive IBC, the ^ER-positive IBC, Finally, it shows a significant decrease to TN IBC (90 vs 80 vs 66 vs 79 vs 48 vs 40 , p = 0.01, respectively). Figure 24B, BD from H D, the DCIS, to HER2 ^positive IBC, the ^ER-positive IBC, and ultimately the number is significantly reduced to TN IBC (respectively, 1.0 to 0.6: 0. 8 vs. 0.8 vs. 0.5 vs. 0.3, p = 0.003), indicating the repertoire is the number of HER3 peptide having cationic properties CD4 Th1 response. FIG. 24C shows a significant reduction from HD to BD, DCIS, HER2 ^positive IBC, ER ^positive IBC, and finally to TN IBC ( 76.7% vs. 63.6% vs. 53. 8% vs. 66.7% vs. 45.0% vs. 33.3% , p = 0.02) Responsiveness (percent of subjects responding to at least one peptide) is shown. 25A and 25B because there is no difference in tetanus or anti-CD3 / CD28 stimulated between the test group of patients, indicating that the loss of CD4T cell response is specific to HER3. The figure shows histograms (left panel) of IFN-γ ELISpot analysis of systemic CD4 + Th1 cell responses. Studied group of patients, HD; a and TN IBC; BD; DCIS; HER2 IBC / HER2 ^-positive IBC; ER IBC / ^ER-positive IBC. The corresponding table to the right of each histogram is an individual comparison by Student 's t-test between the two groups performed at one time. One-way ANOVA tests were performed in all groups. FIG. 25A shows the disruption measured by IFN-γ spots per 200,000 cells via ELISpot assay between HD, BD, DCIS, HER2 IBC / HER2 ^positive IBC, ER IBC / ER ^positive IBC or TN IBC. indicating that there is no statistically significant difference in wound wind response (respectively 37 versus 30 versus 19 versus 34 versus 24 versus 29, p = 0.65). FIG. 25B shows anti-CD3 / anti-CD28 polyclonal as measured by IFN-γ spots per 200,000 cells via ELISpot assay between HD, BD, DCIS, HER2 IBC / HER2 ^positive IBC, ER IBC / ER ^positive IBC It indicates no statistically significant difference in stimulation (respectively 688 versus 549 vs. 804 vs. 699 vs. 629 vs. 675, p = 0.68). Figure 26A-26C, the anti-HER3 CD4 T cell responses, but correlated with relapse and response to neoadjuvant chemotherapy, indicating that not correlate with lymph node metastasis. Figure 26A compares the immune response of the IBC patients with lymph node state definitive the initial surgery (node-positive ( "LN +" or "LN ^positive" versus node-negative ( "LN-" or "LN ^negative")) Four histograms , cumulative response (upper panel) (40 vs. 56 each , p = 0.12 respectively ), repertoire (second panel) (respectively, between LN ^positive and LBC ^negative IBC patients ) 0.4 vs. 0.6, p = 0.08) Responsiveness (third panel) (35.7% vs. 54.8% respectively, p = 0.19) or tetanus response (lower panel) ( each ) , 22 vs. 29 , p = 0.35 ) Figure 26B shows immune response due to relapse vs non-relapse (no disease) in IBC patients at least one year after diagnosis . Compare The cumulative response, repertoire and responsiveness were significantly lower: cumulative response (upper panel) (17 vs. 66, respectively p = 0.04), repertoire (second panel ) ( 0. 0 vs. 0.6, p <0.05) and responsive (third panel) (respectively, 0% vs. 55.6%, p = 0.01). relapsed IBC patients and non-recurrent I BC patients difference in tetanus responses between did (lower panel) (35, respectively 27 versus, p = 0.65). FIG. 26C, neoadjuvant chemotherapy (pathologic complete response ( "pCR")) versus disease remains ( has four histograms comparing the "<pCR"). of the patients receiving neoadjuvant chemotherapy, <compared to patients with pCR, patients with pCR is significantly higher cumulative response and Repa Showed Lee (cumulative response (upper panel) (144 vs. 32, respectively, p = 0.004) and repertoire (the second panel) (respectively, 0.8 vs. 0.4, p = 0.05)) Between the immune response of pCR and <pCR patients, either responsive (third panel) (80.0% vs. 27.3% , respectively p = 0.10) or tetanus response (lower panel) (lower panel) There was no difference of 17 vs. 59 , p = 0.15) , respectively . Figures 27A-27D show that anti-HER3 CD4 T cell responses are significantly higher in postmenopausal HD / BD but do not change with age, race or pregnancy history. Figure 27A has four histograms comparing the immune response of HD patients and age (50 Toshihitsuji Mitsurutai 50 years of age). There was no statistically significant difference by age in cumulative response, repertoire, responsiveness or tetanus response: cumulative response (upper panel) (respectively, 77 vs. 103, p = 0.25), repertoire ( second panel ) (respectively, 0.8 vs 1.1 , p = 0.38), responsive (third panel) ( 72.0% vs 75.0% respectively , p = 1.0) or tetanus response (lower panel) (lower panel) 39:30, p = 0.40, respectively. Figure 27B has four histograms comparing the immune response of HD patients with race (white people vs. African American vs. others). Cumulative response repertoire, there was no statistically significant difference due to racial responsiveness or tetanus responses ((cumulative response) (respectively 87 versus 83 versus 95, p = 0.96), repertoires (second panel) (respectively , 0.9 vs. 0.7 vs. 1.4, p = 0.31), responsiveness ( third panel ) (respectively 69.0% vs. 71.4% vs. 100% , p = 0.35) Or tetanus response (bottom panel) ( 33, 51 vs. 26 , p = 0.30 , respectively ) ) . FIG. 27C has 4 histograms comparing the immune response of HD patients by pregnancy history / pregnancy (0 pregnancy vs 1 or more pregnancy) . Cumulative response by pregnancy history, there were no statistically significant differences repertoire or responsiveness: Cumulative response (upper panel) (respectively 82 versus 91, p = 0.71), repertoires (second panel) (respectively 1. 0 vs 0.9 , p = 0.62) or responsiveness (third panel) (76.5% vs 70.8% respectively , p = 0.74). Interestingly, tetanus reaction (lower panel), as compared to patients with pregnancy history of at least one, was significantly higher in non-parous women (respectively 47 versus 27, p = 0.04). Figure 27D is the immune response of HD patients menopausal status - with four histograms were compared by (premenopausal postmenopausal). Compared to premenopausal HD / BD, HD / BD postmenopausal showed significantly higher cumulative response and repertoire: Cumulative response (upper panel) (136 spot pairs 70 spots for each, one million cells , P = 0.005), repertoire (second panel) ( each, 1.4 vs 0.8 peptides , p = 0.03) ) . There was no difference between postmenopausal and premenopausal HD / sBD by responsiveness or tetanus response: responsiveness (third panel) ( 90.9% vs 66.7% , p = 0.23 respectively) or tetanus Response (lower panel) ( respectively 38 vs. 28, p = 0.37) ). FIG. 28 is a graph of the determination of the linearity of ELISpot. The ELISpot assay was determined to be linear and accurate under the operator who performed all the assays for this study by serial dilution of known anti-HER3 CD4 T cell responders into media. Cumulative responses are from 1.0 fold dilution to 0.1 fold dilution, 0.01 fold dilution, and 0.001 fold dilution ( 230 to 35 , 12 and 5 spots per million cells, respectively ) The linear regression line was followed ( p <0.0001, r ² = 0.88) .

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

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

One embodiment includes a composition comprising a multivalent peptide. Multivalent peptides include two or more peptides of the invention.

  Further provided are methods of activating an immune response and methods of treating cancer in a subject. Also provided are vaccines for therapeutic and prophylactic use. The peptides of this example 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 for tumor progression in precancerous lesions of the gastroesophageal junction.

In another embodiment, HER3 MHC class II immunogenic peptides are used to determine anti-HER3CD4 Th1 loss.

Definitions Unless otherwise defined, 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 pertains. 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, the nomenclature used herein as well as laboratory procedures in cell culture, molecular genetics, organic chemistry and chemistry and hybridization of nucleic acids are well known and commonly employed in the art .

  Standard techniques are used for nucleic acid and peptide synthesis. Techniques and procedures generally refer to conventional methods in the art and various general references (e.g., Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY, And according to Ausubel et al., 2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout the present description.

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

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

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

  As used herein, “about” when referring to a measurable value, such as amount, time period, etc., is ± 20%, or ± 10%, or ± from a particular value. It is intended to encompass variations of 5%, or ± 1%, or ± 0.1%, such variations being appropriate to practice the disclosed method.

  When used in the context of an organism, a tissue, a cell or components thereof, the term "abnormal" means that at least one observable or detectable feature (eg, age, treatment, time of day, etc.) An organism, tissue, cells or components thereof that differ from the organisms, tissues, cells or components thereof that exhibit the "normal" (expected) respective characteristics. For one cell or tissue type, normal or expected characteristics may be abnormal for different cell or tissue types.

As used herein, "adjuvant therapy" for breast cancer refers to any therapy given after the primary therapy (ie, surgery) to increase the chance of long-term survival. "Neoadjuvant therapy " is a treatment given prior to 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 antibody production or activation of specific 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. Furthermore, antigens can also be obtained from recombinant or genomic DNA. It is understood by those skilled in the art that any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes the term "antigen" as used herein Will. Furthermore, one skilled in the art will also 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 various combinations of these nucleotide sequences can be easily arranged to elicit the desired immune response. It becomes clear. Furthermore, one skilled in the art will also understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that the antigen may be produced synthetically or obtained from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

  An "antigen presenting cell" ("APC") is a cell capable of activating T cells, including, but not limited to, mononuclear cells / macrophages, B cells and dendritic cells (DCs).

An "antigen- loaded APC" or an "antigen-pulsed APC" includes APCs that have been exposed to antigen and have been activated by the antigen. For example, while culturing APC in vitro, for example, in the presence of the antigen, it may be in a state of taking up the antigen . It can also be incorporated into APCs by exposure to antigen in vivo. The "antigen- loaded APC" is conventionally either of two methods: (1) "pulsing" the small peptide fragment known as an antigenic peptide directly onto the exterior of the APC, or (2) the APC, Incubation with total protein or protein particles is then prepared by either method of allowing the whole protein or protein particles to be consumed by APC. These proteins are digested by APC into small peptide fragments and finally transported to the APC surface where they are presented. Furthermore, APCs that have taken up antigens can also be generated by introducing polynucleotides encoding the antigens into cells.

  "Anti-HER3 response", "anti-HER3 CD4 Th1 response", "anti-HER3 CD4 T cell response" and the like mean an immune response specific to HER3 protein.

  The term "anti-tumor effect" as used herein, refers to a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, a prolongation of life expectancy, or various physiological conditions associated with a cancerous condition Refers to biological effects that can be manifested by amelioration of In the first place, the "anti-tumor effect" can also be revealed by the ability of the peptides, polynucleotides, cells and antibodies of the invention in preventing the development of tumors.

  The term "autoimmune disease" as used herein is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of inappropriate and excessive responses to self antigens. Notably, examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotiditis, Crohn's disease, diabetes (type I), nutrition Failure 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, Sjögren's syndrome, spondyloarthritis, thyroiditis, vasculitis, vitiligo, myxedema, malignant anemia, ulcerative colitis.

  As used herein, the term "autologous" is intended to refer to any material obtained from an individual that is later intended to be reintroduced into the same individual.

  The term "B cells" as used herein is defined as cells 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 hyperproliferation of cells and results from the abandonment of normal control, which is a unique trait of cancer, resulting in unregulated growth, lack of differentiation, invasion of local tissue. 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, renal cancer, liver cancer, brain cancer, brain cancer, lymphoma, leukemia, lung cancer, germ cell tumor, etc. It can be mentioned.

“ CD4 + Th1 cells”, “Th1 cells”, “CD4 + T helper type 1 cells”, “CD4 + T cells” and the like express the surface protein CD4 and produce high levels of the cytokine IFN-γ T Defined as a helper cell subtype (see also "T helper cells" ) .

"Cumulative response" is expressed as the sum of the reactive spots (cells 10 per ⁶ spot forming cells from IFN-gamma ELISpot analysis "SFC") from all the MHC class II binding peptides from a given patient group that, that means the overall immune response of the patient group.

"DC vaccination", "DC immunization", etc. "DC1 immunization", by using the immune system recognize specific molecules using autologous dendritic cells to acquire a specific response to their Point to a strategy.

The terms "dendritic cells" or "DCs" are antigen presenting cells which may be present in vivo, in vitro, ex vivo, or in a host or subject, or derived from hematopoietic stem cells or monocytes. Dendritic cells and their precursors can be isolated from various lymphoid organs such as spleen, lymph nodes, and bone marrow and peripheral blood. DC have a characteristic morphology with thin sheets (lamellipodia) extending in multiple directions from dendritic cell bodies. Typically, dendritic cells express high levels of MHC and costimulatory (e.g., B7-1 and B7-2) molecules. Dendritic cells can induce antigen specific differentiation of T cells in vitro and can initiate primary T cell responses in vitro and in vivo. In the context of vaccine production, "activated DC" is DC exposed to a Toll-like receptor agonist such as lipopolysaccharide "LPS". Activated DC may or may not Itemoi a antigen.

  A "disease" is a state of health of an animal where the animal can not maintain homeostasis and if the disease does not ameliorate the health of the animal continues to deteriorate.

  A "disorder" in an animal refers to the case where the animal is capable of maintaining homeostasis, but where the animal's health is less favorable than in the absence of the disorder. Left untreated, a disorder does 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.

  An "effective amount" or "therapeutically effective amount" is used interchangeably herein and, as described herein, a compound, formulation, material or composition effective to achieve a particular biological result. It refers to the amount of goods. Such results may 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 introduced from or produced outside of an organism, cell, tissue or system.

  "HER receptor" is a receptor protein tyrosine kinase belonging to the HER receptor family, and is an EGFR (ErbB1, HER1) receptor, a HER2 (ErbB2) receptor, a HER3 (ErbB3) receptor and a HER4 (ErbB4) receptor including. The HER receptor is generally capable of binding to a HER ligand and / or dimerizing with another HER receptor molecule; an extracellular domain; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain As well as the carboxyl terminal signal transduction domain with several tyrosine residues which 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, HER activation results in signal transduction (eg, signal transduction triggered by the intracellular kinase domain of the HER receptor which phosphorylates a tyrosine residue or substrate polypeptide in the HER receptor). HER activation can be mediated by binding of the 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 causing one or more HER receptors to Phosphorylation of tyrosine residues and / or phosphorylation of additional substrate polypeptides, such as, for example, phosphorylation of tyrosine residues in Akt intracellular kinase or MAPK intracellular kinases occur.

  "HER2" is a member of the human epidermal growth factor receptor ("EGFR") family. HER2 is overexpressed in about 20-25% of human breast cancers and is expressed in many other cancers.

"HER2- ^positive " is a classification or molecular subtype of certain types of breast cancer as well as many other cancers. HER2 positive is currently defined by gene amplification by FISH (fluorescent in situ hybridization) assay and 2+ or 3+ of pathological staining intensity.

"HER2- ^negative " is defined by the lack of gene amplification by FISH and can almost always include the range of pathological staining forms 0 to 2+.

  “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). Receptor polypeptide.

  "The extracellular domain of HER3" or "HER3 ECD" refers to a domain of HER3 that is external to the cell and that is anchored or circulating to 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 signal peptide). In one embodiment, HER3 domain III comprises amino acids 328-532 (numbering including signal peptide).

As used herein, " HER3 immunogenic peptides " , "HER3 binding peptides", "HER3 epitopes" etc. are MHC class II derived from or based on the sequences of HER3 proteins, in particular HER3 ECD Refers to peptides and their equivalents. HER3 peptides can activate CD4 T cells across many patients. This peptide can be used to pulse dendritic cells and educate T cells to recognize HER3. HER3 is expressed in triple negative breast cancer and can confer resistance to anti-estrogen in ER ^positive breast cancer. HER3 is also expressed in other cancers, including melanoma, lung, colon, prostate cancer, and metastatic brain tumors. According to a preferred embodiment, four HER3 immunogenic peptides (epitopes) or binding peptides are identified as follows:
p12 (peptide 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4).
p81 (peptides 401-415): SWPPHMNHFSVFSNL (SEQ ID NO: 5).
p84 (peptides 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6). And p91 (peptides 451-465): AGRIYISANRQLCYH (SEQ ID NO: 7).

  “Homology” as used herein refers to two polymeric molecules, eg, two nucleic acid molecules, eg, a subunit between two DNA molecules or two RNA molecules, or two polypeptide molecules. Refers to sequence similarity. When the same monomer subunit occupies a certain subunit position in both of two molecules, for example, if adenine occupies a certain position in each of two DNA molecules, these molecules Are completely homologous or 100% homologous. The percent homology between two sequences is a direct function of the number of matching or homologous positions, eg, half of the positions (eg, a length of 10 subunits in the two compound sequences) These two sequences are 50% identical if five positions in the molecule are homologous, and these two if 90% of the positions, eg 9 out of 10, are matched or homologous. The sequences share 90% homology. By way of example, the DNA sequences 5 'ATTGCC 3' and 5 'TATTGGC 3' share 50% homology.

  Furthermore, when the terms "homology" or "identity" are used herein to refer to nucleic acids and proteins, it is understood to apply to homology or identity at both the nucleic acid and amino acid sequence levels. Should.

  The term "hyperproliferative disease" is defined as a disease resulting from hyperproliferation of cells. 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 materials" includes publications, records, diagrams or any other presentation media that can be used to convey the utility of the compositions and methods of the present invention. Instructions for use of the kit of the present invention are attached, for example, to a container containing the nucleic acid, peptide and / or composition of the present invention, or shipped together with the container containing the nucleic acid, peptide and / or composition be able to. Alternatively, the instructional material can be shipped separately from the container, with the intention that the instructional material and the compound be used in combination by the recipient.

  By "immune response" as used herein is meant activation of the host's immune system, eg, the mammalian immune system, in response to the introduction of an antigen. The immune response may take the form of a cellular or humoral response, or both.

  "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not in the "isolated state", but the same nucleic acid or peptide partially or completely separated from coexisting materials in its natural state 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, when compared to the level of response in a subject in the absence of a therapeutic or compound, and / or otherwise identical but not identical Means of mediating a detectable increase or decrease in the level of response in the subject when compared to the level of response in the subject of treatment. The term encompasses perturbing and / or affecting the native signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

For each group of subjects analyzed for anti-HER3 CD4 + Th1 immune responses , the following "assessment indices" for CD4 + Th1 responses or "immune response evaluation indices" are defined: (a) overall anti-HER3 responses sex (expressed as a percentage of the target (%) responsive to one or more immunogenicity peptide); (b) response repertoire (average number of immunogenic peptide recognized by each group of subjects (n) represented) as; and, (c) the cumulative response (reactive spot (spot forming cells per 10 ⁶ cells from IFN-gamma ELISpot analysis from four MHC class II HER3 immunogenic peptides from each group of subjects Expressed as the sum of the cells "SFC" ).

  "Peptide", "protein" or "polypeptide" as used herein can mean a sequence of linked amino acids and can be natural, synthetic, or a modified form or combination of natural and synthetic .

  As used herein, "population" includes reference to cultures in isolated form, including homogeneous, substantially homogeneous, or xenogeneic cell cultures. In general, "population" can also be considered as "isolated" cell culture.

Receptor tyrosine kinases ("RTKs") are high affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. The human EGF receptor ("HER") family of RTKs regulate a wide variety of biological processes including cell proliferation, migration, invasion and survival. This family consists of four members: HER1 (ErbB1), HER2 (neu or ErbB2), HER3 (ErbB3), and HER4 (ErbB4).

  As used herein, a "recombinant cell" is a host cell that comprises a recombinant polynucleotide.

  "Responsive" or "anti-HER3 responsive", as used interchangeably herein, means the percentage of subjects that respond to at least one of the four HER3 immunogenic peptides.

  "Response repertoire" is defined as the average number ("n") of HER3 immunogenic peptides recognized by each subject group.

  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 bodily fluids. Means biological material. The sample may be a 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 DC and is sensed by T cells through T cell receptors.

  "Signal 2" as used herein generally refers to the second signal provided by DC to T cells. Signal 2 is provided by "co-stimulatory" molecules, usually CD80 and / or CD86 (but other co-stimulatory molecules are also known) on activated DCs, sensing by T cells through surface receptor CD28 Be done.

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

  By the term "specifically bind", as used herein, to recognize and bind to another molecule or feature in a sample but to substantially recognize the other molecule or feature And molecules that do not bind to them, such as antibodies.

  The terms "subject", "patient", "individual" etc are used interchangeably herein and any animal or those suitable for the methods described herein, whether in vitro or in situ Point to the cell of In certain non-limiting embodiments, the patient, subject or individual is a human.

  As used herein, the term "targeted therapy" usually refers to drugs or other agents that interfere with specific target molecules involved in the growth of cancer cells, with little damage to normal cells to achieve an anti-tumor effect. Refers to cancer treatment using the substance of In contrast, traditional cytotoxic chemotherapeutic drugs act on all actively dividing cells. Specifically for breast cancer therapeutic monoclonal antibodies, trastuzumab / HERCEPTIN® targets the HER2 / neu receptor.

  The term "T cell" as used herein is defined as a thymus derived cell involved in a variety of cell mediated immune responses.

  The term "T-helper" as used herein with reference to cells refers to a subgroup of lymphocytes (type of white blood cells or white blood cells), including 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 stimulate or interact with other leukocytes.

As used herein, the terms " T helper cells ", "helper T cells", "Th cells" and the like refer to cells and to lymphocytes (including those of various cell types distinguishable by one skilled in the art ) It shows the sub-group of a type of white blood cell). In particular, T-helper cells are effector T cells is that primary function is to promote the activation and functions of other B and T lymphocytes and / or macrophages. Helper T cells differentiate into two major subtypes of cells known as "Th1" or "type 1" and "Th2" or "type 2" phenotypes. These Th cells secrete cytokines, proteins or peptides that stimulate or interact with other leukocytes.

As used herein, "Th1 cells,""CD4 + Th1 cells,""CD4 + T helper type 1 cells,""CD4 + T cells," etc. refer to mature T cells that express the surface glycoprotein CD4. . CD4 + T helper cells are activated upon presentation of peptide antigens by MHC class II molecules expressed on the surface of antigen presenting peptides ("APCs") such as dendritic cells. C D4 + T helper cells, once more activated MHC antigen complexes secrete cytokines, such as high levels of interferon-gamma ( "IFN-gamma"). Such cells are believed to be highly effective against the specific disease causing microbes present inside the host cell, and against the microbes and cancers causing the specific disease within the host cell. Is also important for anti-tumor response in human cancer.

  "Th17 T cells" as used herein are considered to be highly effective against disease causing microorganisms that produce high levels of cytokines IL-17 and IL-22 and survive on mucosal surfaces Refers to T cells that are

  A "therapeutically effective amount" is that amount of a compound of the invention that ameliorates the symptoms of the disease when administered to a patient. The amount of a compound of the invention which constitutes a "therapeutically effective amount" will vary depending upon the compound, the condition of the disease and its severity, the age of the patient to be treated, etc. A therapeutically effective amount can be determined routinely by one of ordinary skill in the art, given the knowledge of one of ordinary skill in the art and the present disclosure.

  The terms "treat", "treating" and "treatment" refer to the therapeutic or prophylactic strategies described herein. The method of “treatment” may be for the prevention, cure, delay, reduction in severity or amelioration of one or more symptoms of the disorder or relapse disorder, or in excess of the expected survival if not receiving such treatment. In order to prolong the survival of a subject, the composition of the present invention is ultimately obtained from a subject in need of such treatment, eg, a subject suffering from a disease or disorder or such disease or disorder Take advantage of administering to a subject at risk.

"Triple negative" and "TN" breast cancer refers to any breast cancer cell that is negative in the test for estrogen receptor ("ER"), progesterone receptor ("PR") and HER2.

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

  "Variant" with respect to a peptide or polypeptide differs in amino acid sequence by insertion, deletion or conservative substitution of amino acids but retains at least one biological activity. 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 substitutions of amino acids, ie, replacing an amino acid with an amino acid that differs in similar properties (eg, hydrophilicity, degree and distribution of charged regions), typically produces minor changes in the art. Are recognized. These minor changes can be identified, in part, by examining the hydropathic index of amino acids, as understood in the art. Kyte et al. Mol. Biol. 157: 105-132 (1982). The hydrophobicity index of amino acids is based on examination of their hydrophobicity and charge. It is known in the art that amino acids of similar hydrophobicity index can be substituted and still retain the function of the protein. In one aspect, amino acids with a hydrophobicity index of ± 2 are substituted. Also, the hydrophilicity of amino acids can be used to indicate substitutions that would result in a protein retaining biological function. In the context of peptides, examining the hydrophilicity of amino acids 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 strategy that has been reported. US Patent No. 4,554,101, which is incorporated herein by reference in its entirety. As understood in the art, substitutions of amino acids with similar hydrophilicity values can result in peptides that retain biological activity, eg, immunogenicity. Substitutions can be performed with amino acids having hydrophilicity values within ± 2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. In line with such observations, amino acid substitutions corresponding to biological functions are related to the relative similarity of amino acids, in particular their amino acids, as indicated by their hydrophobicity, hydrophilicity, charge, size and other properties. It is understood to depend on the side chain.

  Scope: Throughout the disclosure, various aspects of the invention can be presented as a scope 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 rigid limitation on the scope of the present invention. Therefore, the description of a range should be considered as specifically disclosing all possible partial ranges and individual numerical values within the range. For example, the description of the 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 the range Individual numbers of, for example, 1, 2, 2.7, 3, 4, 5, 5.3 and 6, should be considered as being specifically disclosed. This applies regardless of the width of the range.

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

  The invention also provides methods for treating or preventing cancer. Such methods comprise the step of administering a peptide of the invention or a combination of peptides of the invention to a subject in need thereof. Administration of such peptides results in the induction of anti-tumor immunity. Thus, the invention provides a method for inducing anti-tumor immunity in a subject, such method comprising the peptide of the invention or the combination of peptides of the invention, and pharmaceutical and cellular compositions derived therefrom Administering to the subject.

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

  In one embodiment, T cells can be cultured and expanded using APCs pulsed with a peptide of the invention. Once APCs have been used to expand T cells to obtain a sufficient number of antigen specific T cells, the antigen specific T cells thus obtained are administered to a mammal, thereby causing the antigen to be present in the mammal. Induce specific T cell responses.

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

The present invention includes mature, antigen- loaded 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 invention provides a method of pulsing and activating cells, whereby the cells maintain an active state after cryopreservation. The benefit of the DC preparation of the invention is that, from a single leucocyte apheresis (collection from a patient), cells are added to the initial vaccine and efficiency to multiple (eg 10 or more) “booster” doses They are cryopreserved and these doses can be thawed as needed at the remote treatment site, without any specialized cell processing facilities or even required quality control tests.

  The present invention also relates to the cryopreservation of these activated DCs, which, upon thawing, are cryopreserved so as to retain their potency and function in antigen presentation and the production of various cytokines and chemokines, Thus, activated DCs that are cryopreserved and then thawed are clinically effective at the same level as freshly harvested and activated DCs.

  Herein, it is contemplated that the present invention provides a method for generating and cryopreserving DCs with superior function in that they generate stronger signals to T cells, and thus more A strong, DC-based vaccine is obtained. By effectively cryopreserving such cells, the sample can be stored and thawed for later use, thereby reducing the need to repeat the process of pheresis and ertriation while producing the vaccine. Let It is an advantage that the DCs can be frozen and then later allowed to thaw and remove them, which allows the single-produced vaccine to be aliquoted and kept frozen, then several weeks This means that one can be administered to the patient one at a time, for months or years, to provide a "booster" vaccination that enhances immunity.

  This embodiment also involves the use of HER3 expression as a marker of tumor progression in premalignant lesions 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 the HER family of proteins and other receptor tyrosine kinases. In one embodiment, the 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 the corresponding HER protein or c-MET protein. In some embodiments, the epitope of the corresponding HER protein or c-MET protein is immunogenic.

  The invention provides compositions comprising one or more peptides of the 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.

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

  Included in the invention are methods of activating an immune response using the compositions of the invention and methods of treating cancer in a subject. Vaccines are also provided for therapeutic and prophylactic use. 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 HER3 epitope or other peptide of the invention is
p11-13 (peptides 51-75): KLYERCEVVMGNLEIVLTGHNADLSFLQW (SEQ ID NO: 1);
p81-83 (peptides 401-425): SWPPHMHNFSVFSNLTTIGGRSLYN (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): SWPPHM HNFS VFSNL (SEQ ID NO: 5);
p84 (peptides 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6);
p91 (peptides 451-465): AGRIYISANRQLCYH (SEQ ID NO: 7)
including.

  The HER3 peptide or any peptide of the invention may be cyclic or linear. The epitope, if circular, can be circular in any suitable manner. For example, disulfide bonds can be formed between selected pairs of cysteine (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 HER3 epitope identified by SEQ ID NO: 4 corresponds to positions 56 to 70 of the HER3 protein. The HER3 epitope identified by SEQ ID NO: 5 corresponds to positions 401 to 415 of the HER3 protein. The HER3 epitope identified by SEQ ID NO: 6 corresponds to positions 416 to 430 of the HER3 protein. The HER3 epitope identified by SEQ ID NO: 7 corresponds to positions 451-465 of the HER3 protein.

  As described herein, the epitopes of HER3 of the present invention also encompass peptides which are functional equivalents of the peptides identified by SEQ ID NO. Such functional equivalents have altered sequences, in which case one or more amino acids in the sequence of the corresponding HER3 epitope are replaced, or one or more amino acids correspond. It is deleted from or added to the reference sequence. For example, one to three amino acids can be added to the amino terminus, carboxy terminus, or both. In some instances, the HER3 epitope is glycosylated.

  In other examples, the HER3 epitope can be the retro-inverso isomer of the HER3 epitope. Retro-inverso modifications include inversion of all amide bonds in 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 form of retro-inverso isomer can retain the planarity and conformational constraints of at least some peptide bonds.

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

  In some instances, deletions and additions are located at the amino terminus, carboxy terminus, or both, of one sequence of a peptide of the invention. For example, the equivalent of the HER3 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 93%, at least 70% identical to the sequence of the corresponding HER3 epitope 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 deletion, addition or substitution, per 10 amino acids of the reference sequence. Percent identity is determined by comparing the amino acid sequence of the variant to a reference sequence using programs known or developed in the art.

  In the case of a functional equivalent which is longer than the corresponding HER3 epitope sequence, the functional equivalent is a sequence which is at least 90% identical to the HER3 epitope sequence and the sequence of the HER3 epitope in the wild-type HER3 protein May have a sequence adjacent to

  Functional equivalents of the HER3 epitope can be identified 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. Such antibodies can be found in a variety of body fluids, including serum and ascites fluid. Briefly, a fluid sample is isolated from a warm-blooded animal, such as a human, for whom it is desirable to determine whether an antibody specific for HER3 polypeptide is present. The body fluid is incubated with the HER3 polypeptide, under conditions sufficient to form an immune complex between the polypeptide and the antibody specific for the protein and for a time sufficient to allow so, and preferably then , Assay using the ELISA method.

  According to another embodiment of the present invention there is provided a composition comprising a chimeric peptide and one or more chimeric peptides. According to various embodiments, the chimeric peptide comprises a HER3 epitope, another epitope, and a linker connecting the HER3 epitope to the other epitope. In one embodiment, the other epitopes may include, but are not limited to, other HER3 epitopes, HER1 epitopes, HER2 epitopes and c-MET epitopes. It is further understood that any suitable linker may be used. For example, depending on the epitope used, the HER3 epitope can be linked to either the amino or carboxy terminus of the other epitope. The location and selection of the other epitopes depend on the structural features of the HER3 epitope, ie whether it is an alpha helix or a beta-turn or a chain.

  In one embodiment, the linker may be a peptide of about 2 to about 15 amino acids, about 2 to about 10 amino acids, or about 2 to about 6 amino acids in length. Chimeric peptides may be linear or cyclic. Furthermore, the HER3 epitope, other epitopes and / or linkers may be in the form of retro-inverso. Thus, the HER3 epitope alone may be in the form of retro-inverso. Alternatively, the HER3 epitope and other epitopes may be in the form of retro-inverso. In another example, the HER3 epitope, other epitopes and the linker may be in the form of retro-inverso.

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

  According to a further embodiment of the present invention there is provided a composition comprising a mixture of two or more of the peptides or chimeric peptides of the present invention. In some instances, the HER3 epitopes of each of the two or more chimeric peptides are different. In another example, one of the HER3 epitopes is selected from SEQ ID NOs: 1-7.

  The peptides of the invention, including chimeric peptides, can be prepared using 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 invention are preferably isolated, ie, substantially free of other naturally occurring host cell proteins and fragments thereof.

  Commercially available peptide synthesizers can be used to synthesize the peptides and chimeric peptides of the invention. For example, Kaumaya et al., "De Novo" Engineering of Peptide Immunogenics and Antigenic Determinants as Potential Vaccines, Peptides, Design, Synthesis and Biological Activity (1994), pages 133-164, specifically incorporated by reference herein. The chemical methods described in can be used. For example, HER-3 epitopes can be co-linearly synthesized with other epitopes to form chimeric peptides. Peptide synthesis can be performed using Fmoc / t-But chemistry. The peptides and chimeric peptides can be cyclic in any suitable manner. For example, methods of differentially protecting cysteine residues, performing iodine oxidation, adding water to facilitate removal of the Acm group, simultaneously forming disulfide bonds, and / or using the silyl chloride-sulfoxide method Thus, a disulfide bond can be obtained.

  Peptides and chimeric peptides can also be produced using cell-free translation systems and RNA molecules derived from DNA constructs encoding epitopes or peptides. Alternatively, the epitope or chimeric peptide is generated by transfecting the 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 It can also be done. In the case of the production of recombinants, recombinant constructs comprising one or more of the sequences encoding the epitope, the chimeric peptide or a variant thereof, can be carried out in a conventional manner, for example calcium phosphate transfection, DEAE-dextran mediated transfection Transfer into host cells by microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape lading, ballistic transfer, or infection.

  The peptides of the invention may contain modifications, such as glycosylation, oxidation of side chains, or phosphorylation, but this is limited to the case where the modifications do not destroy the biological activity of the peptide. . Other modifications include, for example, incorporation of D-amino acids or other amino acid mimetics that can be used to extend the serum half life of the peptide.

  The peptides of the invention can be prepared as a combination, which comprises two or more peptides of the invention for use as a vaccine for a disease, eg, cancer. The peptides may be made into a cocktail or may be conjugated to one another using standard techniques. For example, peptides 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 encompass “mutants”, “derivatives” and “variants” of the peptides of the present invention (or DNA encoding said peptides), mutants, derivatives thereof and A variant is a peptide in which one or more amino acids are changed (or, when referring to a nucleotide sequence encoding said amino acids, one or more base pairs are changed), and thus The peptides (or DNA) that are not identical to the sequences listed herein have the same biological properties as the peptides disclosed herein.

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

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

  The scope of the present invention includes homologs, 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 And derivatives of amino acids or nucleic acids known in the art, non-naturally occurring amino acids or nucleic acids and synthetic amino acids or nucleic acids, provided that these modifications preserve the biological activity of the original molecule Clearly understand what you have to do. Specifically, in accordance with the principles of the present invention, any active fragments of the active peptide, 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 referenced herein, provided that these homologous DNAs have the biology of the peptides disclosed herein. Should be interpreted as having a specific activity.

  The nucleic acids of the invention encompass sequences of RNA or DNA encoding the peptides of the invention, DNA making the nucleotide sequence more stable, with or without cells. Those skilled in the art will understand that it also includes any of its variants, including chemical modifications of RNA. Chemical modifications of the nucleotides can also be used to enhance the efficiency with which the cells take up the nucleotide sequence or the efficiency with which the nucleotides are expressed in the cells. The present invention contemplates any combination of nucleotide sequence modifications.

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

  The nucleic acid encoding the peptide of the invention can be incorporated into a suitable vector, for example a retroviral vector. These vectors are well known in the art. The nucleic acid, or a vector containing the nucleic acid usefully, can be transferred into the desired cells, which are preferably obtained from the patient. Advantageously, the present invention provides an off-the-shelf composition, which rapidly alters the patient's own cells (or another mammal's own cells). Thus, it becomes possible to rapidly and easily generate modified cells having excellent cancer cell killing properties.

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

  Polynucleotides can also be cloned into several types of vectors. However, it should not be construed that the present invention is limited to any particular vector. Instead, the present invention should be construed to encompass a very wide variety of vectors which are readily available and / or which are 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. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

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

  In addition, expression vectors can also be provided to cells in the form of viral vectors. Techniques for viral vectors are well known in the art and are described, for example, in Sambrook et al. (2012) and Ausubel et al. (1997) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retrovirus, adenovirus, adeno-associated virus, herpes virus and lentivirus. In general, a suitable vector contains an origin of replication functional 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 sequences of the invention, at least one module in each promoter is functioned to position the initiation site for RNA synthesis. The best known example of this is the TATA box, but some promoters lacking the TATA box, such as in the promoter of the mammalian terminal deoxynucleotidyl transferase gene and in the promoter of the SV40 gene, distinctly overlapping the start site The element itself helps to fix the start location.

  Additional promoter elements, ie, enhancers, regulate the frequency of transcription initiation. Typically, they 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 promoter function is preserved, even when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can work together or independently to activate transcription.

  The promoter may be one that is naturally associated with the gene or polynucleotide sequence, which may be obtained by isolating the 5 'non-coding sequence located upstream of the coding segment and / or the exon. Such promoters can be termed "endogenous." Similarly, an enhancer can also be an enhancer that is naturally associated with a polynucleotide sequence, which is either downstream or upstream of that sequence. Alternatively, placing the coding segment of the polynucleotide under the control of a recombinant or heterologous promoter provides certain advantages, in which case 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 the polynucleotide sequence in its natural environment. Such promoters or enhancers can be promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell, and non-naturally occurring promoters or enhancers, 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, sequences may also be generated using recombinant cloning and / or nucleic acid amplification techniques, which can be used in the compositions disclosed herein. The relevant PCRTM is included (US Pat. No. 4,683,202, US Pat. No. 5,928,906). Furthermore, it is also contemplated that regulatory sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, etc., may be utilized as well.

  Of course, it is important to utilize promoters and / or enhancers which effectively direct the expression of the DNA segment in the cell type, organelle and organism chosen for expression. Those skilled in molecular biology are generally aware of how to use combinations of promoters, enhancers and cell types for the expression of proteins, see, eg, Sambrook et al. (2012). The promoter utilized may be a constitutive promoter, a tissue specific promoter, an inducible promoter, and / or conditions suitable for directing high level expression of the DNA segment to be introduced, eg, large recombinant proteins and / or peptides. It may be a useful promoter under conditions that favor scale production. The promoter may be heterologous or endogenous.

  The promoter sequence exemplified in the experimental examples presented herein is the sequence of the cytomegalovirus (CMV) earliest promoter. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively 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 early 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. By using an inducible promoter in the present invention, 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 promoter, glucocorticoid promoter, progesterone promoter and tetracycline promoter. Furthermore, the invention also includes the use of tissue specific promoters, which are only active in the desired tissue.

  In order to evaluate the expression of the nucleotide sequence encoding the peptide of the present invention, the expression vector to be introduced into cells also contains either a selectable marker gene or a reporter gene or both, and can be transfected by a viral vector. It can also facilitate identifying and selecting cells that are expressed from cell populations targeted for infection or infection. In other embodiments, selectable markers can be placed on separate pieces of DNA and used in co-transfection procedures. Both the selectable marker gene and the reporter gene may be flanked by appropriate regulatory sequences to allow expression in a 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 encoding easily assayable proteins are well known in the art. In general, the reporter gene is neither present in the recipient organism or tissue nor expressed by the recipient organism or tissue, and some readily detectable property, eg, enzyme activity, reveals expression It is a gene encoding a protein. Expression of the reporter gene is assayed at an appropriate time after introducing the DNA into recipient cells.

  Suitable reporter genes can include luciferase, beta-galactosidase, chloramphenicol acetyltransferase, a gene encoding secreted alkaline phosphatase, or a green fluorescent protein gene (eg, Ui-Tei et al., 2000, FEBS). Lett. 479: 79-82). Suitable expression systems are well known and they may be prepared using 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 showing the highest levels of expression of the reporter gene are identified as promoters. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

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

  Vaccines of the invention can induce antigen-specific T cell responses and / or high titer antibody responses, thereby being directed against or reactive to an antigen expressing cancer or tumor. An immune response is induced or elicited. In some embodiments, the induced or elicited immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or 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. The vaccine can include one or more cancer antigens. Vaccines can block tumor growth. Vaccines can reduce tumor growth. Vaccines can block metastasis of tumor cells. Depending on the cancer antigen, target the vaccine, breast cancer, liver cancer, prostate cancer, melanoma, hematologic 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, tumor cells It can mediate growth clearance or arrest. Vaccines, tumor free survival, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, It can be increased by 44% and 45%. Vaccines, after immunization, tumor volume, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42% , 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59 % And 60% can be reduced.

  The vaccine is about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold over the cellular immune response in the subject receiving the vaccine, as compared to the cellular immune response in the subject not receiving the vaccine. Times-about 5000 times, about 50 times-about 4500 times, about 100 times-about 6000 times, about 150 times-about 6000 times, about 200 times-about 6000 times, about 250 times-about 6000 times, or about 300 times -It can be increased about 6000 times. In some embodiments, the vaccine is about 50-fold, 100-fold, 150-fold, 200-fold the cellular immune response in the subject receiving the vaccine as compared to the cellular immune response in the subject not receiving the vaccine. Double, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 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, 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 Double, 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, 5300 times It can be increased 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

  The vaccine is about 50-fold to about 6000-fold, about 50-fold about IFN-γ levels in subjects receiving vaccine compared to the level of interferon gamma (IFN-γ) in subjects not receiving vaccine -About 5500 times, about 50 times-about 5000 times, about 50 times-about 4500 times, about 100 times-about 6000 times, about 150 times-about 6000 times, about 200 times-about 6000 times, about 250 times-about- It can be increased 6000 times, or about 300 times to about 6000 times. In some embodiments, the vaccine comprises about 50-fold, 100-fold, or more levels of IFN-γ in the subject receiving vaccine compared to the levels of IFN-γ in the subject not receiving the vaccine. 150 times, 200 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, 3700 Double, 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, 5300 times It can be increased 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

  The vaccine of the invention has the features necessary for an effective vaccine, for example, the vaccine of the invention is safe, so the vaccine itself does not cause disease or death; it provides protection against disease; neutralizing antibodies Induce a protective T cell response; easy to administer, have few side effects, are biostable, and have low cost per dose. Vaccines can be achieved by containing some or all of these features, as discussed below, with cancer antigens.

Antigen uptake (antigen pulsing) generation of immune cells The invention includes cells that have been exposed to or otherwise "pulsed" an antigen of the invention or other peptides of the invention. For example, APCs, such as DCs, can be in the state of antigen taken up in vitro, eg, by culturing ex vivo in the presence of the antigen, or by exposing the antigen in vivo.

  Also, the APC can be "pulsed" in a manner that exposes the antigen to the antigen, and the exposure can also occur for a sufficient time 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, and these fragments are "pulsed" directly onto the exterior of the APC (Mehta-Damani et al. Or APC) can be incubated with whole proteins or protein particles, and these proteins are then taken up by the APC. All these proteins are digested by APC into small peptide fragments and finally carried 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 "pulsing" techniques described herein.

  Without intending to be bound by any particular theory, antigens in the form of foreign antigens or self antigens are processed by the APCs of the present invention to retain an 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 by immune cells, eg, T cells, to activate them. Preferably, such foreign antigens or autoantigens are proteins that are processed by APC into peptides. The relevant peptides produced by APC can be extracted, purified and used as immunogenic compositions. Also, peptides processed by APC can be used to induce tolerance to proteins processed by APC.

APCs that have taken up antigens are , according to another expression, known as "pulsed APCs" according to the invention, which are produced by exposing APCs to antigens either in vitro or in vivo . When APCs are pulsed in vitro, the APCs can be plated on culture dishes and exposed to the antigen for a sufficient period of time in an amount sufficient to allow the antigen to bind to the APC. . The amount and time necessary to achieve binding of 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 antigen on APC after exposure to the antigen.

  In a further embodiment of the invention, APCs can be transfected with a vector that allows expression of specific proteins by the APCs. The proteins expressed by APCs 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 to the vector encoded protein.

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

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

  As contemplated herein, various methods can be used to transfect polynucleotides into host cells. These methods include, but are not limited to, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, colloidal dispersion systems (ie, macromolecular complexes, nanocapsules, 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 it is therefore possible for those skilled in the art to carry out these methods.

In another embodiment, the polynucleotide encoding the antigen can be cloned into an expression vector to generate the APC incorporating the antigen by another method, and the vector is introduced into the APC It will be 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. See, eg, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY), and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York). It is readily understood that the introduction of an expression vector containing a polynucleotide encoding an antigen results in pulsed cells.

The present invention includes various methods for pulsing APCs, including, but not limited to, incorporating whole antigens in the form of proteins, cDNA or mRNA into APCs. However, the present invention should not be construed as limited to the particular form of antigen used to pulse APC. Rather, the present invention encompasses other methods known in the art for generating APCs that have taken up antigen. Preferably, APCs are transfected with mRNA encoding the defined antigen. Use of appropriate primers and reverse transcriptase-polymerase chain reaction (RT-PCR) coupled with a transcription reaction to rapidly generate in vitro mRNA corresponding to a gene product whose sequence is known it can. When producing pulsed APCs, transfection with APC mRNA offers advantages over other techniques that incorporate antigens. For example, being able 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 to the antigen in cells, tissues or mammals (eg, humans). As used herein, an "immunological composition" may express or display an antigen (eg, a peptide or polypeptide), a nucleic acid encoding an antigen (eg, an antigen expression vector), or an antigen or cell component. Cells can be included. In particular embodiments, the antigen composition comprises or encodes all or part of any of the antigens described herein, or an immunological functional equivalent thereof. In other embodiments, the antigen composition is a mixture comprising an additional immunostimulatory agent or nucleic acid encoding such an agent. 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 linked to the antigen or immunostimulatory agent in any combination. In certain embodiments, the antigen composition is conjugated to or comprises amino acids of an HLA anchor motif.

  Vaccines, as contemplated herein, may vary in their composition of nucleic acids and / or cell components. In a non-limiting example, nucleic acids encoding the antigen could also be formulated with an adjuvant. Of course, it will be understood that the various compositions described herein can further include 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 of the methods disclosed herein, or this will be understood by one of ordinary skill in the art in light of the present disclosure. Will.

  It is understood that the antigen composition of the present invention can be produced by methods well known in the art, and such methods include, but are not limited to, chemical synthesis by solid phase synthesis and chemical reaction by HPLC Other methods of purification removal from products, or expression of nucleic acid sequences (eg, DNA sequences) encoding a peptide or polypeptide comprising an antigen of the invention in an in vitro translation system or in a living cell There is a way to generate by. Additionally, the antigen composition can also include cell components isolated from the biological sample. Antigen compositions can be isolated and extensively dialyzed to remove one or more unwanted low molecular weight molecules, and / or lyophilize in the desired vehicle to enhance the immediate use of the formulation . It should be further understood that if there are additional amino acids, mutations, chemical modifications, etc. to be introduced into the vaccine components, they preferably do not substantially prevent the antibody from recognizing the epitope sequences.

Antigen Presenting Cell Therapy The present invention encompasses methods of generating a population of APCs (eg, dendritic cells; DCs) presenting the peptides of the present invention on the 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 generated form 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 originally obtained from the cells, thus forming an embodiment of the treatment of the 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 DCs capture an antigen, they process the antigen into small peptides and migrate toward secondary lymphoid organs. It is within the lymphoid organs that DC presents antigenic peptides to naive T cells, and presentation of the peptide initiates a signal cascade that biases T cell differentiation. When exposed to DC, they 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; Ginhoux et al., 2007, J. Exp. Med. 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. Im 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 the adaptive immune response, and also serve to integrate the communication between the natural and adaptive arms of the effectors of the immune system. These characteristics make DCs powerful candidates for immunotherapy. DCs have a unique ability to identify their environment by macropinocytosis and receptor-mediated endocytosis (Gerner et al., 2008, J. Immunol. 181: 155-164; Stoitzner et al., 2008, Cancer Immunol. Immunoother 57: 1665-1673; Lanzevecchia A., 1996, Curr. Opin. Immunol. 8: 348-354; Delamarre et al., 2005, Science, 307 (5715): 1630-1634).

  DCs also require maturation signals to enhance their antigen presentation capabilities. By providing additional maturation signals, such as TNF-α, CD40L or calcium signaling agents, DCs are surface molecules, such as CD80 and CD86 (also known as second signal molecules). Upregulation of expression (Czerniecki et al., 1997, J. Immunol. 159: 3823-3837; Bedrosian et al., 2000, J. Immunother. 23: 311-320; Mailliard 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). Alternatively, DCs can be matured with a calcium ionophore prior to pulsing the antigen.

  DCs are receptors that recognize pathogens, for example, PKR and MDA-5 (Kalali et al., 2008, J. Immunol. 181: 2694-2704; Nallagatla et al., 2008, RNA Biol. 5 (3): In addition to pages 140-144), it also contains a series of receptors known as Toll-like receptors (TLRs), which are also capable of sensing the risk from pathogens. . Stimulation of these TLRs induces a series of activity changes in DC that lead to 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 the cell-mediated response, such as natural killer γ-δ T cells and α-β T cells, which, once activated, Retain their immunizing 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; Bykovskaia et al., 1999, J. Leukoc. Biol. 66: 659-666; Clark et al., 2000, Microbes Infect. 2: 257-272).

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

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

  The present invention provides compositions and methods for activating APCs, preferably DCs, in the context of immunotherapy for activating an immune response in a mammal. By activating DCs with the peptide of the present invention or a combination of peptides of the present invention so that DCs activate anti-tumor immunity in a mammal in need thereof, DCs are induced by causing DC maturation. It can be operated.

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

In one embodiment, the invention relates, inter alia, to expand desired T cells, to activate T cells, to generate new APCs for the purpose of expanding specific T cells, and to use them for such purposes. The invention relates to methods for using APCs, and also to a number of therapeutic uses , involving APCs incorporating the peptides of the invention and T-cell expansion and activation using the peptides. In some cases, DCs activated by OCT4 can also be used to expand T cells specific for the peptide.

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

Methods for Treating Diseases The invention also encompasses methods of 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 for activating DC), which itself has utility in the treatment of diseases. Exemplary diseases which can be treated and / or prevented using the pharmaceutical composition of the present invention include, but are not limited to, infections caused by viruses, such as HIV, influenza, herpes, viral hepatitis , Epstein-Barr, polio, viral encephalitis, measles, chicken pox, papilloma virus etc .; or infections caused by bacteria, eg, pneumonia, tuberculosis, syphilis etc .; or infections caused by parasites, eg, malaria, Trypanosomiasis, leishmaniasis, trichomoniasis, amebiasis etc. may be mentioned.

  Non-limiting examples of preneoplastic or hyperplastic states that can be treated or prevented using the pharmaceutical compositions of the invention (transduced DCs of the invention, expression vectors, expression constructs, etc.) These include preneoplastic or hyperplastic states 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's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, gastrointestinal cancer, brain cancer, bladder cancer , Cervical cancer and the like.

  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 vessels Tumor, fibroma, vascular occlusion, restenosis, atherosclerosis, preneoplastic condition lesions (eg, adenomatous hyperplasia and neoplasia in prostate epithelium), carcinoma in situ, cholecystitis alveolar or psoriasis It can be mentioned.

  Autoimmune disorders which can be treated using the compositions of this invention include, but are not limited to, AIDS, Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis , Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes, emphysema, nodular erythema, 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; infections by viruses, bacteria, fungi, parasites, protozoa and helminths; and trauma.

  In methods of treatment, the administration of the compositions of the present invention may either be for "prevention" or for "treatment". The compositions of the invention, when provided for prophylaxis, are provided prior to any symptom, but in certain embodiments, the vaccine is provided after one or more symptoms have developed. , Prevent the onset of additional symptoms, or prevent the aggravation of the emerging symptoms. The 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 the symptoms of the infection or disease. Thus, the present invention can be provided before exposure to the substance causing the disease is expected or before the disease state occurs or after the infection or disease has started.

  An effective amount of the composition is that which enhances the immune response and is such an amount to achieve these selected results, such amount will be determined by one of ordinary skill in the art as a matter of routine. For example, an effective amount to treat the immune system's failure to cancer or pathogens would be the amount necessary to cause activation of the immune system to generate an antigen-specific immune response upon exposure to the antigen. . This term is also synonymous with "sufficient amount".

  The effective amount for any particular application may 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 of ordinary skill in the art can empirically determine the effective amount of a particular composition of the present invention without requiring undue experimentation.

Vaccine Formulations The invention further comprises vaccine formulations suitable for use in immunotherapy. In certain embodiments, vaccine formulations are used to prevent and / or treat diseases, eg, cancer and infectious diseases. In one embodiment, administration to a patient of a vaccine according to the invention for the prevention and / or treatment of a cancer, prior to 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 the cancer vaccine prepared according to the present invention is broadly applicable to the prevention or treatment of cancer which is determined to some extent by the choice of the part that forms the antigen of the cancer vaccine. Cancers that can be suitably treated according to the practice of the invention include, but are not limited to, lung, breast, ovary, cervix, colon, head and neck, pancreas, prostate, stomach, bladder, kidney, bone, Includes cancers 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 cells 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, etc. are also produced as immunotherapeutic agents and prevent and / or prevent the tumor or cancer cells from which the vaccine is generated. It is used according to the implementation procedure for treatment.

  In another embodiment, as also described, a vaccine according to the invention is obtained by collecting appropriate antigens excreted into the culture medium by the pathogen in order 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, preparing a multivalent vaccine by preparing a vaccine from a pool of organisms expressing different important antigens It 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 also be administered intradermally or subcutaneously to the limbs, arms and legs of the patient being treated. While this approach is generally satisfactory for melanoma as well as other cancers, other routes of administration, including the prevention or treatment of infectious diseases, such as, for example, the intramuscular or in the bloodstream route. It can also be used.

  Additionally, the vaccine can be administered with an adjuvant and / or an immunomodulator to boost the activity of the vaccine and the patient's response. Such adjuvants and / or immunomodulators are understood by those skilled in the art and are easily described in the available published literature.

  As contemplated herein, depending on the type of vaccine produced, if desired, culturing the cells in a bioreactor or fermentor or other such vessel or device suitable for growing the cells in bulk By doing this, the vaccine production can be scaled up. In such devices, the culture medium is periodically, frequently or continuously collected to recover any material or antigen from the culture medium prior to degradation of such material or antigen in the culture medium. Will be done.

  The device or composition containing the vaccine or antigen, which device is suitable for sustained or intermittent release, produced according to the invention and recovered, is optionally implanted in the body or It can be applied externally to the body to release such materials into the body relatively slowly or at defined times.

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

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

  These methods described herein are by no means all-inclusive and additional methods suitable for particular applications will be apparent to those skilled in the art. In addition, the effective amount of the composition can also be determined to a more accurate approximation by analogy with compounds that are known to exert the desired effect.

<Example of experiment>
The invention is further described in detail by reference to the following experimental examples. These examples are provided for the purpose of illustration only and are not intended to limit the invention unless otherwise specified. Accordingly, the present invention should not be construed as being limited in any way to the following examples, but rather is to be construed as encompassing any modifications that may become apparent as a result of the teachings provided herein. It should.

  Without further elaboration, it is believed that one skilled in the art can, using the preceding description and the following illustrative examples, make and use the present invention and practice the claimed method. Conceivable. Thus, the following examples specifically point out the preferred embodiments of the present invention and should not be construed as limiting the remainder of the present disclosure in any way.

Creation of peptide vaccines against other receptor tyrosine kinases that cause breast cancer and other solid cancers Experiments were designed to develop alternative therapies for patients designated as carriers of BRCA mutations. That is, they are looking for an alternative to bilateral mastectomy, and the need for younger patients at genetic risk for breast cancer has not been met.

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. To develop vaccines for this group and to evaluate their safety in immune induction trials, experiments were designed; this is the first ever vaccination for primary prevention of breast cancer It is an attempt. We also observe whether estrogen receptor- ^positive breast cancer can be prevented using the multivalent vaccine of the invention, including carriers of BRCA2 mutations.

  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 c-MET oncogene and HER-3 prematurely, while tumors from non-mutant or sporadic patients show HER-2 and HER- It was observed that 3 was expressed. This is important; as disclosed herein the targets for tumor immunotherapy that can be used to develop vaccines for carriers of sporadic and BRCA mutations. Based on the fact that it is now known. This is an initial and striking feature and can be targeted to use an immune response for prevention. Thus, the invention includes compositions and methods for developing a vaccine, and their use for prophylaxis as an alternative to bilateral mastectomy.

Creation of peptide vaccines The HER family consists of four related signaling molecules, HER1, HER2, HER3 and HER4, which are involved in a variety of cancers. It is known that overexpression of HER2 is found in 20% to 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, HER1 is expressed on a minority of breast cancers, which are generally triple negative. c-MET is a growth factor receptor involved in the relapse of many cancers, which activates HER3. HER3 is overexpressed in colon, prostate, breast and melanoma. HER3 is expressed in a number of DCIS lesions and breast cancer. In some patients receiving the HER2 vaccine, HER3 may be detected in the residual DCIS at the time of surgery. As a result of these findings, in breast cancer, the potential to target these molecules in addition to HER2 would be beneficial.

Immunogenic peptides derived from HER3 have been identified (Figures 1 and 2) and are shown below:
p11-13 (peptides 51-75): KLYERCEVVMGNLEIVLTGHNADLSFLQW (SEQ ID NO: 1);
p81-83 (peptides 401-425): SWPPHMHNFSVFSNLTTIGGRSLYN (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): SWPPHM HNFS VFSNL (SEQ ID NO: 5);
p84 (peptides 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6); and p91 (peptides 451-465): AGRIYISANRQLCYH (SEQ ID NO: 7).

The results presented herein indicate that these peptides can activate CD4 T cells across many patients. The peptide can be pulsed on dendritic cells and T cells can be educated to recognize HER3. HER3 is expressed in triple negative breast cancer and may lead to resistance to anti-estrogen in ER ^-positive breast cancer. HER3 is also expressed in other cancers, including melanoma, lung cancer, colon cancer, prostate cancer and metastatic brain tumors. Without 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 provided herein, immunogenic peptides for the receptor tyrosine kinase molecules HER1 and c-MET were screened and identified based on the procedure that identified immunogenic peptides for HER3. can do. The immunogenic peptides of the invention can be used to prepare multivalent prophylactic vaccines for breast and other cancers.

  The results presented herein demonstrate the identification of the role of HER2 sister protein in breast cancer. These sister proteins can be effective targets and vaccines can be developed for other solid tumors. We have developed peptides that can be used to target HER1 and HER3. Specifically, DCIS leads to the identification of specific anti-HER1, anti-HER2 and anti-HER3 responses in patients before and after vaccination, which prevents cancer early or DCIS Assist in the development of multivalent vaccines that can be used to treat women with. The compositions of the invention are useful for treating other cancers including, but not limited to, colon cancer, melanoma, brain cancer, 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 standard dendritic cell vaccines and engineered dendritic cells to exhibit a mutated protein (BRAF) that causes approximately 70% of melanomas . Vaccination with these dendritic cells protects mice from challenge with melanoma cells, indicating that it is possible to develop a vaccine for melanoma. While not intending to be bound by any particular theory, targeting combined with BRAF and HER3 is not limited to melanoma, but not limited to solid cancers such as colon cancer, pancreatic cancer and lung cancer and others. It may be useful to treat other cancers, including gastrointestinal tumors.

  In addition, melanoma tumors have also been shown to use B cells to evade immune surveillance, and it is therefore thought that therapy can be improved by eliminating certain B cells. Experiments can be designed to assess whether altering the tumor's microenvironment to a Th1-type response can help prevent avoidance.

  In some cases, the vaccines of the invention can also be used to treat spread melanomas. In some cases, the present invention also provides a therapy to eliminate residual cells, often becoming resistant to drug therapy.

Novel strategies to identify MHC class II-promiscuous CD4 ⁺ peptides from tumor antigens for use in vaccination. Cytotoxic CD8 + T lymphocytes (CTLs) have historically been the mainstay of anti-tumor immunity Although considered to be an effector, just boosting the CTL response with the CD8 + vaccine in various tumor types has produced unpredictable clinical results, which is probably due to the CTL being appropriate Without the help of these CD4 + T lymphocytes, it would only function suboptimally. CD4 + T helper type 1 (Th1) cells secrete IFN-γ / TNF-α to induce tumor senescence and apoptosis. However, the successful integration of CD4 + epitopes in constructing cancer vaccines and the development of durable, antigen-specific CD4 + immunity remains a challenge. An immunogenic HER3 CD4 + that exhibits class II promiscuity and generates anti-HER3 CD4 + immunity, with the aim of including it in a vaccine construct, using the extracellular domain (ECD) of HER3 as a candidate "oncogenic driver" tumor antigen Experiments were performed to identify the peptides.

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

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 long peptides, overlapping by 5 amino acids, was generated from the HER3 ECD. These peptides were pulsed onto mononuclear cell-derived DC obtained from a donor and allowed to mature into a type 1 polarized (DC1; secreting IL-12) phenotype. DC1 was collected, co-cultured with purified CD4 + T cells obtained from subjects belonging to our DCIS vaccine study and known to have an anti-HER3 Th1 response. A large pool of 10 peptides was used to refine the process of identification, as measured by the secretion of interferon gamma (IFN-γ) of CD4 + T cells, to obtain a single reactive epitope. 4 to 6 subjects are screened to appear to react across most donors, ie, HER3 _56-70 (SEQ ID NO: 4), HER3 _401-415 (SEQ ID NO: 5), HER3 _416-430 (SEQ ID NO: 6), and HER3 _51-465 (SEQ ID NO: 7) were identified. Identify subjects that do not show evidence of responsiveness to HER3 extracellular domain CD4 + T cell recognition, pulsing the four HER3 peptides into their DC1s, and culturing pulsed DC1 with CD4 T cells for 1 week Then, it was tested for reactivity to HER2 peptide and response to extracellular HER3 protein. In each case, at least one peptide resulted in the recognition of both the peptide pulsed on mononuclear cells and the whole protein of HER3, suggesting that primary sensitization occurred ex vivo . Healthy donors were also able to respond to these peptides, and in triple-negative breast cancer patients, it was also shown that the anti-HER3 Th1 response is lost. Also, Gala, K. et al. , Clin. Cancer Res 2014; 20: 1410-1416, and Datta, J. et al. See also, "Progressive Loss of Anti-HER2 CD4 + T-helper Type 1 Response in Breast Tumorigenesis and the Potential for Immune Restoration", Onco Immunology (nearly published).

Key points of the protocol further illustrated in FIG. 19:
A library containing 123 15 amino acid long peptide fragments overlapping by 5 amino acids was generated from the extracellular domain (ECD) of HER3.
-Autologous mononuclear cell-derived dendritic cells (DC) obtained from a donor are rapidly transformed into type 1 (secreting DC1 → IL-12) phenotype by GM-CSF, IFN-γ and LPS. It was matured and pulsed with related peptides (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 for 8-10 days with purified CD4 + T cells.
-Sensitized CD4 + T cells (most of them are antigens against immature DCs (iDCs) pulsed with a specific CD4 + peptide of interest (eg a cluster of HER3 library peptides) or a control of an irrelevant class II peptide Was expected to be specific) was reactivated.
-The supernatant was then collected from these co-cultures. Th1 responses measured by IFN-γ ELISA were considered to be antigen specific if the production of IFN-γ was at least twice that of an unrelated control.
In order to assess MHC class II promiscuity of CD4 ⁺ Th1 responses, HLA-DR, DP, DQ typing on donors was performed by the Clinical Immunology laboratory at the Hospital of the University of Pennsylvania.

  A library containing 123 15 amino acid peptide fragments with overlap was generated from HER3-ECD. Autologous mononuclear cell-derived DC obtained from a donor 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 reactivated against immature DCs (iDCs) pulsed with clusters of HER3 library peptide or irrelevant CD4 control peptide 1. Th1 responses measured by IFN-γ ELISA were considered to be antigen specific if the production of IFN-γ was at least twice that of an unrelated control.

  1) Obtain a breast cancer patient known to show anti-HER3 ECD reactivity after DC1 vaccine pulsed with HER2 to identify immunogenic CD4 + peptides; 2) Immunogenicity of these peptides Were confirmed by the process of "reverse" sensitization in the same patient; 3) Obtained a patient known to show anti-HER3 ECD non-reactivity after vaccination and used to identify CD4 + peptides The experiments were carried out in a three-step process, in which cells were sensitized to the native HER3 ECD, thus observing whether tolerance to self antigens (ie, HER3) was overcome / nulled.

  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, anti-HER3 ECD to identify a single immunogenic HER3 CD4 + epitope Th1 sensitization was performed in 5 breast cancer patients known to be reactive. To achieve this, starting with 10 peptide clusters (1 to 10, 11 to 20, etc.), 3 peptide clusters (1 to 3, 3 to 6, 7 to 10,...) Etc., and finally, focused on single immunogenic HER3 peptides to reactivate HER3 ECD sensitized CD4 + Th1 against them sequentially. Representative screens are shown in FIGS. 2, 13 and 14. Four immunogenic peptides, namely, 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 reproducibly identified and they were promiscuous across HLA-DR, DP and DQ subtypes. Th1 cells obtained from 4 non-HER3-reactive donors are sensitized using DC1 pulsed with 4 identified HER3 peptides, and then these cells are iDC pulsed with HER3 ECD Not only did all donors show good sensitization to the individual immunogenic HER3 peptides, but also recognized native HER3-ECD when challenged to recognize.

  The results presented herein demonstrate that DC1 pulsed with a library of overlapping tumor antigen-derived peptides can identify promiscuous class II peptides for the development of CD4 T cell vaccines There is. In this study, the immunogenic HER3 CD4 peptide effectively overcomes immune tolerance to self tumor antigens. Vaccines utilizing 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 from any tumor antigen for cancer immunotherapy using the DC1-Th1 platform New strategies for 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 the immunogenicity of the identified CD4 + HER3 ECD epitope by “inverse” sensitization, ie confirmation of the ability of the individual epitope-sensitized CD4 + Th1 to recognize the native HER3 ECD FIG. 15 shows that it has HER3 ECD reactivity In living donors, CD4 + T cells are sensitized by DC1 pulsed with their respective donor-specific, immunogenic HER3 epitopes and reactivated against iDCs pulsed with their respective HER3 epitopes and native HER3 ECD. Show that.

  Figures 20 and 21 show the additional results of "reverse" sensitization.

CD4 + Th1 sensitized with DC1 pulsed with an immunogenic HER3 epitope were obtained from 4 HER3 ECD non-responsive donors, as seen in Figure 16 which appears to abolish anti-HER3 immunoself tolerance All CD4 + Th1 cells were sensitized using DC1 pulsed with 4 identified HER3 peptides, and all these cells were subsequently challenged to recognize HER3 ECD pulsed iDC. Not only did donors not only show good sensitization to individual HER3 epitopes, but also recognized native HER3 ECD.

CD4 + HER3 epitopes show MHC class II promiscuity As seen in Figure 17, experiments were performed using the extracellular domain (ECD) of HER3 as a candidate "oncogenic driver" tumor antigen Immunogenic HER3 CD4 + peptides have been identified that display differential and generate anti-HER3 CD4 + immunity; these peptides can be used in vaccine constructs.

Peptides Derived from Tumor Antigens The results presented herein are:
-To be able to identify promiscuous MHC class II peptides by DC1 pulsed with a library of overlapping peptides obtained from tumor antigens to develop a CD4 + T cell vaccine,
-Immunogenic HER3 CD4 + peptides effectively overcome immune tolerance to self tumor antigens,
-From these results, in order to rapidly and reproducibly identify class II promiscuous immunogenic CD4 + epitopes obtained from any tumor antigen, for cancer immunotherapy using the DC1-CD4 + Th1 platform That a new strategy for
-Vaccines constructed utilizing these HER3 CD4 + peptides have been shown to be worthwhile to study in patients with cancers that overexpress HER3.

HER3 expression is a marker of tumor progression in premalignant lesions of the gastroesophageal junction Overexpression of RTKs including members of the HER family has prognostic and therapeutic significance in invasive esophageal gastric cancer. RTK expression in premalignant gastroesophageal lesions has not been extensively investigated before.

The presence of Barrett's esophagus, or metaplastic columnar epithelium of the distal esophagus, predisposes to the development of esophageal adenocarcinoma (Cameron, A. J. 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 esophagus lesions with dysplasia. (Almhanna, K. et al., Appl. Immunohistochem. Mol. Morphol. July 16 2015) (Almhanna et al. ); Fassan, M. et al . Al, Histopathology 61 (5): 769-76 (2012) (Fassan et al); and Rossi, E. Et al. Cell. Mol. Med. 13 (9 B): 3826-33 (2009) (Rossi et al.). Furthermore, the rate of HER2 expression correlates with the degree of dysplasia, suggesting a relevant pathway in tumorigenesis.

HER family while a mesenchymal epithelial transition factor (HER1, HER2, and HER3) and overexpression of RTK molecule comprising members of cMET is confirmed in many common malignancy than including breast cancer, lung cancer and gastrointestinal cancer and (Yokata, J et al., Lancet 1:.. 765-767 ( 1986) the identification of HER2 overexpression in a subset of breast cancer has also been confirmed by esophagogastric cancer, the more aggressive ecological and overexpression of HER2 Association and effective targeting of HER2 by monoclonal antibodies has been a central event in the evolution 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 identified in the treatment of other malignancies. It provides the basis for further efforts to target TK molecules.

Overexpression of HER2 has been identified in a small number of gastric cancers and the impact on outcome is a target of trastuzumab in a metastatic environment (Bang, Y. J. et al . , Lancet 376 (9742) 687). -97 (2010) (Bang et al.)). Overexpression of HER2 is more frequently expressed at the proximal gastric and gastroesophageal junction compared with the more distal gastric adenocarcinoma (Rajagopal , I. et al ., J. Clin. Diagn. Res. 9 (3): EC 06-10 (2015), the impact on outcome is slight and is a target for 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 cancer has been associated with poor prognosis in most 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 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.Cancer Res.17 (22) : 7127-38 (2011)) . These data, that provides a basis for further effort to characterize the RTK expression in esophageal gastric and precursor lesions. The studies described below aim to characterize RTK expression in dysplastic lesions of the gastroesophageal junction in order to identify potential targets for treatment and primary prevention.

After approval of the way the University of Pennsylvania Institute Review Committee, dysplasia (low grade dysplasia (LGD), n = 32, advanced dysplasia (HGD), n = 59) clinical records and of 73 patients with Barrett esophagus Histological specimens were reviewed retrospectively. Formalin-fixed paraffin-embedded tissue blocks from stored endoscopic biopsy and mucosal resection specimens from 2003-2012 are sectioned at 5 μm on plus slides (Fisher Scientific, Waltham, Mass.) And subsequently deparaffinized And allowed to rehydrate. All biopsies were taken 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 Immunostaining for -III instrument) and evaluated by one pathologist (Leica Bond-III) under a microscope. Membranes of 3 + HER staining were considered as positive as membranes of 2 + HER2 staining in which 10% or more of the tumor cells were stained as seen in FIG. CMET immunohistological staining was performed in 42 cases where sufficient tissue was available. Moderate or scleral staining of 50% or more of tumor cells was considered positive. RTK overexpression was correlated with clinical data either in dysplasia-adenocarcinoma biopsy specimen pairs or diagnosis of adenocarcinoma in subsequent biopsy specimens to assess its relevance to invasive cancer .

Statistical analysis Two-sided tests were performed for all analyses . Descriptive statistics are shown as a frequency of categorical variables and median continuous variables (interquartile range (IQR)). Categorical and continuous variables were analyzed using Pearson's χ 2 test or Fisher's direct test and Wilcoxon's rank sum test, respectively. P values ≦ 0.05 were considered statistically significant. All tests 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 3 below, there were no significant differences between the LGD and HGD cohorts in the measured clinical and demographic variables.

Highly dysmorphic (HGD) is an over-expression of HER1 (22.4% vs. 3.1%, p = 0.016), HER2 (5.3% vs. 0 ) compared to low grade dysplasia (LGD) .0%, p = 0.187) , and HER33 overexpression (45.6% vs. 9.4%, p <0.001) .

Lesions invasive esophageal adenocarcinoma is associated with dysplastic lesions in 6 cases, all of which occurred in connection with the HGD (HGD: 10.2% vs. LGD: 0.0%, p <0 . 001 ). An additional nine patients were diagnosed with invasive esophageal adenocarcinoma in subsequent biopsy specimens (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 compared to HGD lesions without invasive cancer foci (71.4% vs. 38.6%, p = 0.032), not significantly associated with HER1 or HER2 (increasing HER1: 26.7% vs. 20.5%, p = 0.616, increasing HER2: 14.3% vs. 2. 3%, p = 0.077).

Overexpression of cMET was observed in 18 of 42 evaluation samples (42.9%) and more in HGD samples compared to LGD samples (58.3% vs 36.7%, p = 0.200) and, in most cases, coexpressed 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. Patients to invasive cancer of 42 people in one (5.6%) was confirmed. CMET was overexpressed in this patient (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) Frequency of 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 potentially invasive disease in Barrett's esophagus and HGD patients. Furthermore, therapeutics 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 was limited to assessment of HER2 overexpressing 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 cancers where HER3 is more commonly overexpressed than HER2 ( Fichter et al . ) . Increased overexpression of HER3 protein with progression from LGD to HGD and in particular frequent overexpression of HER3 is a novel finding, although not unexpected . Homodimerization and heterodimerization of the HER receptor drives signal activation. 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 a plurality of RTK provides rationale for multivalent therapies that target ((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 of the breast (DCIS) has targeted promising results ( US Patent Application Publication 2015/0323547; filed December 30, 2019) No. 14 / 985,303, Datta, J. et al., Onco Immunology 4: 8 e 102 2301 (2015) DOI: 10. 1080/2162402 X. 2015. 1022301; Datta, J. et al., Breast Cancer Res. (1): 71 (2015) ) . Such an approach remains a distant goal for gastrointestinal malignancies. Nevertheless, all of the current treatment options of the Barrett's esophagus, including endoscopic resection and removal modes and radical surgery, have serious limitations. There is a need for alternative strategies to eliminate morbidity 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 potentially invasive disease in Barrett's esophagus and HGD patients. Furthermore, therapeutics that target HER3 or CMET can provide secondary prevention of gastroesophageal cancer in a subset of patients.

In summary, the present data indicate the relationship between the frequent overexpression and malignant transformation of HER3 in high-grade dysplasia lesions, high-grade dysplasia lesions especially involving potential invasive cancer of the gastroesophageal junction. 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, future applications of HER3 targeted therapy in early disease settings Provide a basis for

We previously showed that there is progressive loss of native anti-HER-2 CD4 Th1 during HER-2 ^positive breast tumor development . This loss of response is associated with the absence of a pathological complete response ("pCR") to neoadjuvant treatment, correlates with an increased risk of recurrence of breast cancer, and could be reversed by vaccination. Example 4 below examines whether there is a similar loss of anti-HER3 Th1 responses during breast tumorigenesis .

Loss of anti-HER3 CD4 Th1 occurs in breast tumorigenesis and is negatively associated with outcome
Summary of Overall Example 4 We previously showed that, during HER2- ^positive breast tumorigenesis, native anti-HER2 CD4 Th1 is progressively lost . This loss of response is associated with the absence of a pathological complete response ("pCR") to neoadjuvant treatment, correlates with an increased risk of recurrence of breast cancer, and could be reversed by vaccination. This example examines whether there is a similar loss of anti-HER3 Th1 responses during breast tumorigenesis .

Peripheral blood from 131 subjects including healthy donor (“HD”), benign breast disease (“BD”), non-invasive ductal carcinoma (“DCIS”) and invasive breast cancer (“IBC”) patients It was collected. The immune response to the four different HER3 immunogenic peptides identified in Examples 1 and 2 above was tested via an enzyme-linked immunosorbent (ELISpot) assay to analyze all metrics of immune response.

From HD to IBC , there was a significant decrease in anti-HER3 response. Triple negative ("TN") IBCs showed the lowest response across all three immune parameters. HD had significantly higher immune responses than ER ^positive IBC and TN IBC patients across all three immune parameters. Interestingly, HER2- ^positive IBCs showed similar immune responses as HD and BD. Patients with absence of pCR for recurrent breast cancer and neoadjuvant therapy had significantly lower anti-HER3 CD4 Th1 response than patients with pCR for subsequent patient or neoadjuvant therapy without relapse.

Thus, CD4 Th1 anti HER3 has been found to be lost during the breast tumor development, in particular, treatment options are limited, remarkable in TN IBC HER3 is remarkably bad group prognosis overexpressing It was found to be. These findings influence the attempt to recover this response to prevent recurrence.

One woman in the background about 8 people will develop breast cancer during their lifetime. Among these, cancers overexpressing HER2 have a high rate of distant metastasis and a poor prognosis overall. Introduction of trastuzumab, a monoclonal antibody to HER2, dramatically improves progression free survival and overall survival in HER2-positive cancer patients and points out an important role of HER2 in regulating breast cancer progression. Giordano S. H. Et al. Clin. Oncol. (2014): JCO-2013 [May 5, 2014 published online before printing, doi: 10.1200 / JCO. 2013.54.0948].

The immune system plays an important role in the regulation of HER2-expressing tumors. It has previously been shown that native anti-HER2 CD4 T cell responses are progressively reduced from healthy subjects towards HER2- ^positive DCIS, HER2- ^positive IBC , but not in HER2- ^negative IBC . Furthermore, lower anti-HER2 immune response correlates with subsequent breast cancer recurrence, while higher anti-HER2 immune response correlates with pathological complete response to neoadjuvant chemotherapy and the role of immune system in HER2- ^positive tumorigenesis the suggested (Datta, J et al., Onco Immunology 4 (10): .. e1027474 DOI: 10.1080 / 2162402X.2015.1022301 (2015) and U.S. Patent application Publication No. 2015 / 0323547A1 (hereinafter collectively See "Datta et al.") ) . Our group has developed a HER2 pulsed dendritic cell vaccine that restores anti-HER2 CD4 and CD8 T cell responses in both DCIS and IBC patients ( Sharma, A. et al., Cancer 118 (17): 4354-4362 (2012) Koski, G. K. et al., J. Immunother. 35 (1): 54-65 (2102); and U.S. Published Application No. 2015/0323547 A1 ).

HER2 is a member of the EGFR family, an RTK group that also includes HER1 and HER3. HER2 is it is well known that self-dimerization, the role of HER3 in signal transduction is not very clear, it may act to both the dimerization of itself and HER2. The dimerization of HER3 with HER2 has been proposed as an escape mechanism in trastuzumab-treated breast cancer patients. Czopek, J.M. Et al., Contemp. Oncol. 17 (5): 446-9 (2013) ("Czopek et al.") And Bae, S. et al. Y. Et al, Breast Cancer Res. Treat. 139 (3): 741-50 (2013) ("Bae et al "). Pertuzumab is added to the recent market, is the first of a tumor therapeutic agent that has received early approval of the F DA as a neo-adjuvant therapy, inhibits the HER2 / HER3 dimerization, used in combination with trastuzumab for breast cancer patients It has then been shown to have overall survival benefits ( Jhaveri, K. et al., J. Natl. Compr. Canc. Netw. 12 (4): 591-8 (2014) and Harbeck, N. et al., Breast Care 8 (1): 49-55 (2013 ) .

Although there is overexpression seen in several ER positive, HER2 positive and triple negative ("TN") subtypes, HER3 expression is not as well delineated among breast cancer subtypes . Moeder, C.I. Et al., Cancer 115 (11): 2400-9 (2009). It is interesting to note that although HER3 overexpression may be more common in HER2- ^positive IBCs, its prognostic value is more pronounced in TN IBCs. HER3 expression in ER- ^positive / HER2- ^positive IBCs did not affect disease free survival ("DFS") or overall survival ("OS"), whereas HER3 expression in TN IBC was 5 years of DFS and 10 years of OS Correlated with the deterioration of the Bae et al., And Czopek et al. Notably, HER3 overexpression significant subset of TN IBC patients with (by definition, do not have any of the classical therapeutic option group) can benefit from recognizable new targets. It is unknown whether an anti-HER3 CD4 Th1 response is present in healthy donors and whether this response changes during breast tumorigenesis. This study seeks answers to these questions.

Method
Meet the subject test criteria of 131 people in the whole subject of registration, registered in succession to the University of Pennsylvania and received consent. The study was approved by the University of Pennsylvania Institutional Review Board and the Abramson Cancer Center prior to subject enrollment . Of the 131 subjects, 9 lacked peripheral blood monocytes to assay, resulting in 122 subjects for review of immune response data. (HD, n = 30), benign breast disease (BD, n = 11), DCIS (n = 13), HER2 ^positive IBC (n = 21), ER ^positive invasive breast cancer (ER ^positive IBC, n = 20) and Among triple negative IBCs (TN IBC, n = 27), CD4 T cell responses to four different HER3 immunogenic peptides to HER2 polypeptide were compared .

Peripheral Blood Monocytes Collection Peripheral blood was collected by venipuncture. Blood was diluted in Hank's buffer or PBS at a ratio of 1: 1, and lymphocyte separation medium was overlaid under diluted blood in conical tubes. The blood was then separated by density centrifugation at 1200 rpm for 30 minutes. The mononuclear cell layer was collected and washed twice with Hanks buffer or PBS. The cells were counted and resuspended at 10 million cells per milliliter and frozen at minus 80 ° C. for 24-48 hours before transferring to minus 200 ° C. Cells were stored until experimental assay .

Measurement of Anti -HER3 CD4 Th1 Response The anti-HER3 CD4 Th1 cell response was measured by ELISpot assay according to the manufacturer's protocol. Briefly , 96 well PVDF membrane plates were activated with 70% ethanol, washed with PBS, coated with anti-IFN-γ (anti-IFN-γ) antibody and incubated overnight at 4 ° C. After 24 hours, the plates were again washed with PBS and blocked for 1 hour with Iscove's medium containing 10% human serum. Peripheral blood monocytes are thawed at 37 ° C., washed with PBS or Hanks buffer, resuspended at 1 million cells per milliliter, four immunogenic HER3 peptides (p12 (peptide 56-70): CEVVMGNLEIVLTGH (sequence No. 4); p81 (peptide 401-415): SWPPHMHNFSVFSNL (SEQ ID NO: 5); p84 (peptide 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6); and p91 (peptide 451-465): AGRIYISAN RQLCYH (SEQ ID NO: 7) one of the anti-CD3 / CD28 (polyclonal stimulation, positive control), 200,000 tetanus toxoid (Santa Cruz Biotechnology, Dallas, TX), or with a blank (unstimulated control) Was coated plates. Assays After 48 hours of incubation was. 48 hours was carried out in triplicate. The plates at 38 ° C., plates were washed with PBS, added biotinylated anti IFN-gamma antibody, the plate at the 38 ° C. and incubated for 2 h. plates were washed again with PBS, added streptavidin-HRP, the plate was incubated for 1 hour at 38 ° C., finally the plate was washed with PBS, and then was added TMB substrate solution. color development and it was stopped after 5 minutes by extensive washing with tap water. plates were dried overnight at 4 ° C. the.

Immune response analysis spots were counted by immunospot software. (1) Cumulative responses, i.e. total response to all four HER3 immunogenic peptides in per million cells of spots, (2) the repertoire, that is, the number of peptides per person subjects with 20 or more spots, (3 ) responsiveness, i.e. to quantify the response to the at least one peptide (proportion of subjects defined) as a threshold for more than 20 spots (%), three parameters (metric). Anti CD3 / CD28 response with tetanus response and cell 200,000 per spot by cells 200,000 per spots was quantified as a control. All immune response metrics were analyzed with GraphPad Prism software.

result
Meet the subject test criteria of properties total 131 people studied, it has been registered in succession in response to a consent at the hospital of the University of Pennsylvania. Nine subjects had insufficient cells for analysis and 122 subjects . Among them, the average age was 50 years, between 25 and 83 years. Whites were 72.1%, African Americans 18.0%, and other races 9.8%. Subject, HD (n = 30), BD (n = 11), DCIS (n = 13), HER2 ^-positive IBC (n = 21), ^ER-positive IBC (n = 20), TN IBC of (n = 27) Belongs to one of six groups . Of the 68 IBC subjects, 35 (51.5%) in stage I, 22 (32.4%) in stage II, 9 (13.2%) in stage IV and 2 in stage IV 2.9%). There were 52 (76.5%) patients who received chemotherapy and / or herceptin and / or tamoxifen and 16 (23.5%) who had no treatment. Three DCIS patients and three HER2- ^positive IBC patients received HER2-pulsed dendritic cell vaccination. Other properties are reported in Table 4 below.

There is a reduction in the anti-HER3 immune response of CD4 Th1 cells from healthy donors to subsets of invasive breast cancer HD, BD, DCIS, HER2- ^positive IBCs, ER- ^positive IBCs, and TN IBCs compare all three immune parameters There decreased, reached the lowest point in the TN IBC: cumulative response (as shown in FIG. 24A, respectively, 90 vs. 80 to 66 pairs 79 pairs 48 pairs 40, p = 0.0 1), repertoire (Figure 24B As shown, respectively, 1.0 vs 0.6 vs 0.8 vs 0.8 vs 0.5 vs 0.3, p = 0.003 and responsiveness (respectively as shown in Figure 24C). .7% vs. 63.6% vs. 53.8% vs. 66.7% vs. 45.0% vs. 33.3%, p = 0.002). In particular, these differences over all three immune parameters, as compared with TN IBC patients, statistically not only significantly higher in HD, showed more than 2-fold: Cumulative response (90 vs. 40, p = 0.002), repertoire (1.0 vs 0.3, p = 0.004), responsiveness (76.7% vs 33.3%, p = 0.001). BD has a significantly higher cumulative response (40 vs. 80, p = 0.007, respectively) and DCI patients show a significantly higher repertoire compared to TN IBC patients (0.8 vs. 0.3 respectively) , P = 0.04), HER2- ^positive IBCs have significantly higher repertoire ( 0.3 vs. 0.8, p = 0.01) and responsiveness (33.3% vs. 66.7% respectively , p = 0. 04) is shown . ER- ^positive IBC patients had the second lowest anti-HER3 CD4 T cell response and showed statistically significantly lower responses compared to HD across all three immune parameters: cumulative response (48 vs 90, respectively) p = 0.03), repertoire (0.5 vs. 1.0, p = 0.008) and responsiveness (45.0% vs. 76.7%, respectively, p = 0.03) . Of note is the fact that HER2- ^positive IBC anti-HER3 responses did not change significantly from HD, BD or DCIS subjects.

Decreased anti-HER3 immune response of CD4 Th1 cells in patients with invasive breast cancer is specific for HER3 and analyzes polyclonal stimulation with tetanus response and anti-CD3 / CD28 response that are not attributable to a broad loss of immune response The overall immune responsiveness was compared and controlled. HD, BD, DCIS, ^{HER2-positive} IBC, between ^ER-positive IBC or TN IBC patient, differences in anti-tetanus response C D4 Th1 cells was measured by the spot per 200,000 cells via ELISpot assay was no ( As shown in FIG. 25 , 37: 30: 19: 34: 24 : 29 ( p = 0.65), respectively. Importantly, HD and anti-tetanus response between the TN IBC patients (group with the most divergent anti HER3 CD4 Th1 cell responses) were similar (37 vs. 29, p = 0.37). Similarly, there was no difference in polyclonal stimulation with anti-CD3 / CD28, and the majority of subjects in each group had astonishing spot development, which was uncountable. Of the computable patients, HD, BD, DCIS, HER2 ^positive IBC, ER ^positive IBC or TN IBC patients (as shown in FIG. 25B, respectively 688 vs 549 vs 804 vs 699 vs 629 vs 675, p = 0 There was no statistically significant difference between 6 and 8) .

Anti HER3 CD4 Th1 cell responses invasive breast cancer patients, for anti-HER3 CD4 T cell responses that correlate with prognosis and features of tumor invasion to determine whether correlated with characteristics of tumor invasion, the initial surgery lymphatic section state (node-positive ( "LN ^positive") to node-negative ( "LN ^negative"), recurrent vs. non recurrence in patients who have passed at least one year from diagnosis, and response (pathological for neoadjuvant chemotherapy Immune responses of IBC patients were compared by complete response ("pCR") versus disease survival ("<pCR") LN ^positive IBC patients (n = 28) compared to LN ^negative patients (n = 31) Although the immune response throughout the three parameters were generally low, there was no statistically significant ones: as shown in FIG. 26A, the cumulative response (respectively 40 to 56, = 0.22), repertoire (0.4 vs 0.6, p = 0.08 respectively) and responsiveness (35.7% vs 54.8% respectively p = 0.19). to, the ^LN-positive subjects, subjects with lymph node metastasis after neoadjuvant chemotherapy were included. ^LN-negative subjects after neoadjuvant therapy, whether had positive lymph nodes before treatment is unknown Among patients with at least 1 year or more since diagnosis, recurrent breast cancer (local metastasis or distant metastasis , n = 7) is a patient without disease (n == ) at all three immune parameters. 36) compared to, it showed significantly lower anti-HER3 response: as shown in FIG. 26B, the cumulative response (respectively 17 versus 66, p = 0.04), repertoire (respectively 0.0 vs. 0.6, <0.05) and responsive (respectively 0% vs. 55.6%, p = 0.01). Finally, as shown in FIG. 26C, of the patients (n = 16) who received neoadjuvant chemotherapy , PCR (n = 11) compared to pCR (n = 11) significantly higher cumulative response (144 vs. 32, p = 0.004 respectively) and repertoire (0.8 vs. 0.4 respectively, p = between the .pCR and <pCR which has a 0.05), statistically significant differences were not (80.0% vs. 27.3%, respectively, p = 0.10). in addition, There was no difference in tetanus response between relapsed vs non-relapsed patients and pCR vs <pCR between LN- ^positive and LN- ^negative patients: tetanus response ( 22 vs. 29 , p = 0.35 respectively ), recurrence vs non-relapse Patient (27 vs 35, p = 0.65 respectively) and pCR vs <p CR (17 vs. 59, p = 0.15 respectively). Thus, the reduction of CD4 Th1 cell responses in IBC patients with more aggressive tumor features is specific to HER3.

Anti-HER3 CD4 Th1 cell response by characteristics of healthy donors
Age ( under 50 (n = 25) or over 50 (n = 16) ) , race ( white (n = 29), African American (n = 12)) , pregnancy status (0 (n = 17) ) or by one or more pregnancy (n = 24)) and menopausal status (premenopausal (n = 30) or postmenopausal (n = 11)), were compared anti HER3 CD4 Th1 reaction in HD and BD. There was no difference in cumulative peptide response, repertoire or responsiveness by age (FIG. 27A), race (FIG. 27B) or past pregnancy history (FIG. 27C). However, women postmenopausal, as shown in FIG. 27D, showed significantly higher cumulative response and repertoire as compared to premenopausal women: Cumulative response (respectively, per million cells 136 spot pairs 70 Spot , P = 0.005) (upper panel) , repertoire (respectively, 1.4 vs 0.8 peptide , p = 0.03) (second panel) ). There was no statistically significant difference in responsiveness (respectively 90.9% vs. 66.7%, p = 0.23) (3 panel). No statistically significant difference in tetanus reaction between peri-menopausal women with (lower panels), the difference in the immune response by menopausal status showed to be specific for HER3.

ELISpot Assay is Accurate as Demonstrated by Linearity Precision Assay ELISpot assay has been previously validated in our laboratory. Linearity accuracy assays were performed with serial dilutions from known high anti-HER3 CD4 T cell responders to confirm the accuracy of this assay under the operator who performed all experiments for this study. Peripheral blood monocytes are serially diluted in the medium to concentrations of 1.0 to 0.1, 0.1 to 0.01, 0.01 to 0.001, and cumulative anti-HER3 immune response per million cells It measured by the spot of. FIG. 28 shows that there was a linear decrease in spots with cumulative values of 230 to 35 , 35 to 12, 12 to 5 (p <0.0001, r = 0.88).

Discussion The knowledge of the role of the immune system in the development, progression and prognosis of cancer is rapidly expanding. Immunodeficiency states are well established to increase the risk of developing cancer from tumors of viral as well as tumors of non-viral origin. Boshoff, C.I. Et al , Nature Rev. Cancer 2: 373-82 (2002). Sheil, A. G. , World J. Surg. 10: 389-96 (1986); Transplantation 61: 274-78 (1996); and Penn, I., et al. Transplantation 60: 1485-91 (1995). It is also known that certain immunophenotypes are associated with breast cancer. The circulating inflammatory cytokines TNF-α and IL-6 are higher in breast cancer patients and low CD4 ⁺ / CD8 ⁺ T cell ratios are associated with a more aggressive breast cancer phenotype, but tumor-infiltrating lymphocytes are associated with some breast cancers It is associated with a better prognosis. Alokai, M .; S. Et al , Med. Oncol. 31 (8): 38 (2014) doi: 10.1007 / s 12032-014-0038-0; Et al, Med. Oncol. 31: 981 (2014); and Matsumoto, H. et al. Et al. Clin. Pathol. doi: 10.1136 / jclinpath-2015-202944. However, the evidence linking the loss of immune recognition in immune response hosts with specific molecular oncogenic drivers is relatively new. Health donors from ^{HER2-positive} DCIS, the HER2 native anti HER2 CD4 Th1 decrease in cellular response to ^positive IBC is indicated are recently, the disappearance of the immune response to this specific oncogenic driver in breast tumorigenesis Is one of the first studies to show. Those skilled in the art will readily appreciate that there is likely to lead to specific immune targeting therapy to identify and understand such specific losses.

The study shows that (1) there is a reduction in the anti-HER3 CD4 Th1 cell response from HD to ER ^positive and TN IBC, and (2) that the anti-HER3 response is correlated with prognosis, specifically higher response is associated with pCR for neoadjuvant therapy, a lower response to be associated with relapse (3) postmenopausal HD are shown to have high anti-HER3 immune response significantly. All of these findings will have diagnostic and clinical applications of anti-HET3 CD4 Th1 cell responses.

The anti-HER3 CD4 Th1 cell response was highest in HD and lowest in TN IBC whose prognosis is more severely affected by HER3 overexpression than other types of IBC. Bae et al., And Czopek, J.A. Et al. Expression of HER3 is a cohort of IBC patients in this study is unknown, as compared with the ^{HER2-positive} IBC cohorts showed similar response and HD, TN IBC and ^ER-positive IBC group when a high level of HER3 expression I think . In fact, our previous studies, anti-HER2 CD4 Th1 cell responses indicated that correlates HER2 expression directly, there was a significant reduction in ^{HER2-positive} IBC, there was no significant reduction in ^{HER2-negative} IBC. HER3 not only has a major impact on the prognosis of TN IBC compared to receptor-expressing breast cancer, but even on TN IBC may have a major impact on the prognosis of HER2 (0) compared to HER2 (1+) tumors There is sex. Schmidt, G., et al. Et al, Arch. Gynecol. Obstet. 290: 1221-29 (2014) . This may partially explain the similarity in immune response between HER2- ^positive IBCs and HD. Tumor progression can not circumvent immune surveillance if the tumor is already infiltrated due to overexpression of HER2. However, if the immune system already recognizes and targets HER2, tumors can be adapted by HER3 overexpression, where immune evasion becomes an evolutionary advantage for tumor cell survival.

Interestingly, ER ^positive IBCs showed similar anti-HER3 CD4 T cell responses as TN IBCs and were significantly lower than HD or HER2 ^positive IBCs. Although HER3 expression is not of prognostic significance in ER ^positive IBC compared to TNF-IBC, there is evidence to show that HER3 mRNA expression is positively correlated with ER expression. Fujiwara, S. Et al., Breast Cancer 21: 472-81 (2014) which may explain the lower immune response seen with this subgroup of IBCs.

Breast cancer patients with recurrent disease have lower anti-HER3 CD4 Th1 responses compared to patients who remain disease free, indicating that immune surveillance may be an important mechanism for successful long-term treatment ing. Recurrent patients can potentially more have high bearing tumors with high expression of HER3, this itself, recurrence, metastasis, and you correlates with overall survival is poor high risk. Li, Q. Et al., Oncology Reports 30: 2563-70 (2013); Smirnova, T. et al. Et al, Oncogene 31: 706-15 (2012); and Ocana, A., et al . Et al. N. C. I. 105 (No. 4): 266-73 (2013). Furthermore, immune editing has been proposed as an escape mechanism by which neoplastic cells are selectively eliminated by the immune system until neoplastic cells have evolved to express molecular oncogenic drivers such as HER3 that evade immune recognition. . Dunn, G. P. Et al., Nature Immunology 3 (11): 991-8 (2008). Thus, expression of HER3 is possible due to the lack of immune surveillance, which increases the risk of relapse. Interestingly, recent evidence suggests that recurrent tumors may not be pathologically identical to primary tumors. Discrepancy rate between the primary tumor and the secondary tumors, especially high in the case of the progesterone receptor, of any type mismatch, indicating a trend of bad prognosis of recurrent tumors. Idriisinghe, P .; K. A. Et al ., Am. J. Clin. Pathol. 133: 416-29 (2010); Broom, R., et al. J. Et al. , Anticancer Research 29: 1557-62 (2009); and Liedtke, C.I. Et al , Annals of Oncology 20: 1953-58 (2009) . Studies comparing the HER3 expression between the nuclear power tumor and the secondary tumor is considered that there is no until today. It is unclear whether HER3 expression is inconsistent between primary and recurrent tumors, and whether HER3 expression represents a relapse avoidance mechanism to occur. If so, targeting patients with low anti-HER3 CD4 T cell responses may help improve immune surveillance and prevent long-term recurrence.

Also, suggesting a role for the immune system in prognosis , patients who were pCR to neoadjuvant chemotherapy had significantly higher anti-HER3 CD4 T cell responses than those with <pCR. HER3 signaling has been shown to mediate acquired resistance to targeted therapies. Sergina, N .; V. Et al., Nature 445: 437-41 (2007); and Frogne, T. et al. Et al, Breast Cancer Res. Treat. 114: 263-75 (2009). Here, it is not an acquired resistance but rather an early resistance, making the anti-HER3 immune response a potential prognostic marker for patients who will most benefit from neoadjuvant treatment. Further studies should reveal whether anti-HER3 immune responses can be intervened to promote not only prognosis but also response to treatment.

In this study, a subset of HD, especially postmenopausal women, showed higher anti-HER3 responses. However, unlike previous findings of anti-HER2 responses, there was no difference based on pregnancy history. Biologically, this higher anti-HER2 response may be due to regression of the breast and subsequent exposure of cellular proteins to immune surveillance by pregnancy, but such changes in breast parenchyma are menopausal Difficult to occur in Press, M. F. Et al, Oncogene 5: 953-62 (1990). Mimic breast involution during pregnancy, the imaging exposing the cell protein that is normally expressed at similarly breast tissue to the immune system, and is (as in menopause) changes in breast density due to hormonal changes observed. Clendenen, T .; V. Et al, Magnetic Resonance Imaging 31: 1-9 (2013). Alternatively, high anti-HER3 immune response than in post-menopausal women can be explained to be due to differences in risk. The expression of various oncogenic drivers is clearly age-dependent: HER2- ^positive IBCs are less likely to decline with age, and ER- ^positive IBCs are more likely to occur. Clark, G .; M. Et al. Clin. Oncol. 2: 1102-09 (1984); Eppenberger-Castori, S. et al. Et al ., Int. J. Biochem. And Cell Biol. 34: 1318-30 (2002). Furthermore, these two receptors are not independent of one another. That is because ER / PR expression by age is HER2-dependent or vice versa. Neven, P .; Et al, Breast Cancer Res. Treat. 110: 153-59 (2008). TN IBC patients, the group with the lowest anti-HER3 immune response and the highest prognostic sensitivity to HER3 overexpression, occur more frequently in premenopausal women. Bae et al., And Howlander, N .; Et al. N. C. I. 106 (5): 1-8 (2014). Thus, a subset of the premenopausal HD, while the risk of developing TN IBC is Ru high group der postmenopausal HD has exceeded the higher risk period already, in fact over-expressing both HER2 and HER3 low group der risk of contracting breast cancer Apply predicates. It is also worth noting that although TNIBC is more common in young women, the incidence in the elderly predicts a good prognosis for unknown reasons. Aapro, M. , Et al, annals of Oncology 23 (6) koronvi 52-55 (2012). This partially explains the good prognosis of TN IBC in postmenopausal women, if the higher anti-HER3 immune response is in fact due to biological mechanisms occurring in menopausal rather than risk aversion groups be able to.

Conclusions This example demonstrated a reduction in anti-HER3 CD4 T cell responses from HD, BD and DCIS towards ER ^positive and TN IBC. Furthermore, lower anti-HER3 responses are correlated with relapse and <pCR for neoadjuvant treatment , indicating that this immune response may play a prognostic role in invasive breast cancer. Most importantly, these results are consistent with the results of previous studies showing that the native anti-HER2 immune response decreases from HD to HER2- ^positive DCIS to HER2- ^positive IBC. Such similar results are promising not only to confirm previous findings but also to show a greater role of the immune system in the patrol of molecular carcinogenesis drivers . Interestingly, postmenopausal HD is generally at a higher risk of developing HER3-overexpressing breast cancer and is significantly more immune than premenopausal HD , a group that potentially exhibits a mechanism that mediates this risk. I had a response. It is important to determine whether HER3 pulsed DC1 vaccination may have a therapeutic and / or risk modifying effect on the development of HER3 overexpressing breast cancer. It will be equally important to continue investigating the role of the immune system in other carcinogenic driver specific cancers.

Conclusion: The anti-HER-3 immune response of CD4 Th1 cells is lost from healthy donors in invasive breast cancer, in particular TN IBC, a group with limited treatment options and a significant prognosis of HER-3 overexpression. The anti-HER3 immune response also moderates the response to treatment and prognosis and indicates a potential immunotherapeutic target. The addition of the HER3 immunogenic peptide to the DC1 vaccine may increase the population of IBC patients who benefit from vaccination. Most importantly, these results reflect previous findings and point out that the role of the immune system in the patrol of molecular carcinogenesis drivers is significant.

Findings of this embodiment, i.e., that there is a significant loss of anti-HER3 CD4 + Th1 in countercurrent selfish mammary tumorigenesis IBC from HDS is a cancer expressing HER3, particularly breast cancer, in particular diagnosis and treatment of triple negative IBC be useful is understood by those skilled in the art. Would be able to develop a blood test in order to use these findings to detect circulating anticancer CD4 + Th1 response in test subjects. Preferably, such blood tests are based on the type of cancer the patient is suffering from , as used herein four HER3 immunogenic peptides or any other MHC class II immunogenicity A HER3 immunogenic peptide such as a peptide is used which can elicit an immune response in the patient. As a non-limiting example, monitoring patients with recurrent breast cancer and pCR for neoadjuvant therapy with such blood tests to determine their anti-HER3 CD4 Th1 response and treat accordingly Can.

The low anti-HER3 response detected by blood test or other means of the patient may for example be a vaccine, preferably a HER3 immunogenic peptide (eg four HER3 immunogenic peptides used in the examples herein) The patient can be countered with a recovery method such as a vaccine based on monocyte-derived dendritic cells of the patient pulsed / incubated. Those skilled in the art will readily understand that there are other ways to restore the patient's immune response. In particular, for TN IBC patients, who are a group with essentially limited treatment options , methods of measuring HER3 responses, and, if necessary, methods of recovering such responses via DC1 vaccines are very valuable. Seems to be . Anti-HER3 immune response can also be used as a potential prognostic biomarker for patients in need of neoadjuvant treatment. The HER3 pulsed DC1 vaccine or other suitable vaccine may have a therapeutic and / or risk modifying effect on the development of HER3 overexpressing breast cancer as well as other HER3 expressing cancers. The findings herein can be evaluated as useful for the development of array blood tests and assays contemplated herein for diagnosis and / or treatment.

  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 particular embodiments, it is understood that other embodiments and modifications of the invention can be devised without departing from the true spirit and scope of the invention. It is clear to the trader. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims (28)

  p11 to 13 (peptides 51 to 75): KLYERCEVVMGNLEIVLTGHNADLS FLQW (SEQ ID NO: 1), p81 to 83 (peptides 401 to 425): SWPPHMHNFFSVFSNLTTIGGRSLYN (SEQ ID NO: 2), p84 to 86 (peptides 416 to 440): TTIGGRSLYNRGFSLLIMKNLNVTS (SEQ ID NO: 3) , P12 (peptides 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4), p 81 (peptides 401 to 415): SWPPHM HNFFS VFSNL (SEQ ID NO 5), p 84 (peptides 416 to 430): TTIGGRSLYNRGFSL (SEQ ID NO 6), and p91 (peptides 451-465): selected from the group consisting of AGRIYISANRQLCYH (SEQ ID NO: 7) Away peptide.   An immunomodulator comprising one or more of the peptides of claim 1.   A vaccine comprising one or more of the peptides of claim 1 and a pharmaceutically acceptable salt.   The vaccine of claim 3 further comprising an adjuvant.   A cell contacted with one or more peptides according to claim 1.   6. The cell of claim 5, which is an antigen presenting cell.   The cell according to claim 5, which is a T cell.   A method of 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 one or more of the peptides of claim 1 to the subject.   10. 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 cancer and any combination thereof.   A method of activating a cell, comprising contacting the cell with one or more peptides according to claim 1.   13. The method of claim 12, wherein the cell is an antigen presenting cell.   13. The method of claim 12, wherein the cell is a T cell. A method of generating activated dendritic cells (DCs) loaded with peptides for use in immunotherapy, comprising
Pulsing the DC with one or more peptides according to claim 1;
Activating the DC with at least one TLR agonist.   16. The method of claim 15, comprising contacting the DC with an agent that raises intracellular calcium concentration in the DC.   16. The method of claim 15, wherein the agent comprises a calcium ionophore.   16. The method of claim 15, further comprising the step of cryopreserving the DC, wherein the DC produces an effective amount of at least one cytokine to generate a T cell response when the DC is thawed.   A cell generated from the method of claim 15.   A vaccine comprising cells produced from the method of claim 15.   21. The vaccine of claim 20, wherein the vaccine is in the form of an injectable multiple dose vaccine.   21. A method of raising an immune response in a mammal comprising administering the cell population generated from the method of claim 20 to a mammal in need thereof.   21. A method of treating a disease or disorder in a mammal comprising administering the cell population generated from the method of claim 20 to a mammal in need thereof.   A biomarker for detecting tumor progression in a premalignant lesion of a gastroesophageal junction in a subject having Barrett's esophagus, comprising detecting overexpression of HER3 in said subject.   A method of treating a patient who has lost anti-HER3 CD4 + Th1, at least one dose of antigen-pulsed DC1 vaccine derived from monocytic dendritic cell (DC) precursors of said patient pulsed with HER3 MHC class II Administering to said patient, said peptide is an immunogenic peptide selected from the group consisting of p12 (peptide 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4), p81 (peptide 401-415) ): SWPPHM HNFFS VFSNL (SEQ ID NO: 5), p84 (peptide 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6), and p91 (peptide 451-465): AGRIYISANRQLCYH (SEQ ID NO: 7), and any combination thereof Choice It is, way.   26. The method of claim 25, wherein the patient has triple negative invasive breast cancer. A method of detecting anti-HER3 CD4 + Th1 loss in a patient, comprising:
Pulsing peripheral blood mononuclear cells from said patient with the HER3 MHC class II immunogenic peptide, said peptide being p12 (peptide 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4), p81 (peptide 401-415) Selected from the group consisting of: SWPPHM HNFFS VFSNL (SEQ ID NO: 5), p84 (peptide 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6), and p91 (peptide 451-465): AGRIYISANRQLCYH (SEQ ID NO: 7), and any combination thereof And a method of detecting an immune response generated thereby.   29. The method of claim 28, wherein detection of anti-HER3 CD4 Th1 response is measured by IFN-γ ELISpot assay.