JP2021043204A - Identification of immunogenic mhc class ii peptides for immune-based therapy - Google Patents

Abstract

To provide a biomarker for detecting tumor progression in premalignant lesions of the gastroesophageal junction in a subject having Barrett's esophagus, which involves detecting overexpression of HER3 in the subject.SOLUTION: A method of detecting anti-HER3 CD4+ Thl immune response loss is provided, comprising pulsing peripheral blood mononuclear cells with one or more HER3 MHC Class II immunogenic peptides, and measuring immune response generated thereby by means of IFN-γ ELISpot assay.SELECTED DRAWING: Figure 1

Description

(Cross-reference of related applications)
This application is a partial continuation application of PCT / US16 / 21042 filed on March 4, 2016, and the application is a partial continuation application of PCT / US15 / 41034 on July 17, 2015. .. The application claims priority over US Provisional Patent Application No. 62 / 076,789 filed November 7, 2014, and US Provisional Patent Application No. 62 / 025,681 filed July 17, 2014. To do. The entire contents of each patent application are incorporated herein by reference.

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

In 25-30% of breast cancers, amplification and overexpression of the growth factor receptor gene HER2 (also known as human epithelial growth factor receptor-2, neu / erbB2) enhances tumor invasiveness and recurrence and recurrence. 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”). HER2 stands out in several respects as one of the four members of the human epidermal growth factor receptor (“EGFR”) family. First, HER2 is an orphan receptor. High affinity ligand has not been identified. Second, HER2 has become a preferred partner for other EGFR family members (HER1 / EGFR, HER3 and HER4) to form heterodimers, which have high ligand affinity and excellent It shows signal transduction activity. Third, full-length HER2 undergoes protein cleavage to release the soluble extracellular domain (“ECD”). ECD shedding has been shown to be an alternative activation mechanism for full-length HER2 both in vitro and in vivo, as membrane anchor fragments with kinase activity remain. The central role of HER2 in EGFR family signaling, regardless of its expression level, correlates with its involvement in the carcinogenesis of several types of cancer, eg breast cancer, ovarian cancer, colon cancer and gastric cancer (Slamon, D). Et al., 1989, Science 244: 707; Hynes, N. et al., 1994, Biochem. Biophyss. Acta. 1198: 165). HER2 may also provide tumor cells with resistance to certain chemotherapy (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 RTK regulates a wide variety of biological processes, including cell proliferation, migration, infiltration and survival. The family consists of four members: EGFR (HER1), HER2 (neu or ErbB2), HER3 (ErbB3), and HER4 (ErbB4). At this time, epidermal growth factor (“EGF”), heparin-binding EGF-like growth factor (“HB-EGF”), transformative growth factor alpha (TGFα), amphiregulin (AR), epiregulin, betacellulin and heregulin Eleven ligands have been reported, including. These ligands bind directly to their homologous receptors, which leads to the formation of homodimers or heterodimers of the receptors that cause activation of multiple signaling pathways. The dysregulation of members of the HER-family, either by mutation activation, receptor overexpression, or abnormal ligand release, leads to the development of diverse human tumors. HER3 is overexpressed in breast, ovarian and lung cancers, and this genetic feature correlates with a poor prognosis. When activated by hellegrin, HER3 dimers with HER2 and EGFR to form a potent carcinogenic receptor heterodimer. Within this complex, HER3 preferentially recruits PI3 kinase to its cytoplasmic docking site, thereby regulating cell proliferation and survival. To date, HER3 has been inactive as a kinase due to apparently abnormal sequence features in its kinase domain, impairing the kinase activity of the HER-family in order to initiate signaling events. It was speculated that heterodimerization with non-members would be required. Consistent with this, it was shown that HER2 requires HER3 to drive cell growth in breast tumors. However, recent findings have shown that HER3 can also phosphorylate Pyk2, resulting in activation of the MAPK pathway in human glioma cells. In addition, HER3-specific monoclonal antibodies can also inhibit the growth and migration of cancerous cell lines. Interestingly, cancer cells have recently avoided HER-family inhibitor therapy by upregulation of HER3 signaling, and HER3 inhibition suppresses HER2-driven tamoxifen resistance in breast cancer cells. Shown. Furthermore, resistance to the EGFR small molecule inhibitor gefitinib (Iressa) therapy has been shown to be linked to signal activation of HER3.

HER3 is a receptor protein and plays an important role in regulating normal cell growth. HER3 lacks its own kinase activity and, depending on the presence of HER2, transmits signals across the cell membrane. Initially, the pre-mRNA of HER3 contains 28 exons and 27 introns. After splicing the intron, the fully spliced HER3 mRNA is composed of 28 exons.

Over the last decade, targeted therapies have emerged as the basis for cancer treatments. Members of the EGF receptor family, namely EGFR (or HER1) and ErbB2 (or HER2 / neu), have come to be regarded as particularly intriguing targets, for these RTKs are numerous. This is because it is deregulated in cancer. The carcinogenic function of ErbB3 or HER3, another member of the EGF receptor family, has only recently been examined due to its mediation of resistance to HER2 and its major role in therapy targeting the PI3K pathway. It came to be done. Activation and / or overexpression of mutations in HER3 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. It is a precursor to a typical prognosis.

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

Despite advances in the art, it remains uncertain whether a cell- or protein-based vaccine strategy targeting HER3 can be used to generate an effective immune response in humans. Therefore, 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 the HER3 protein. The present embodiment meets this need.

The following detailed description of preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. To illustrate the invention, currently preferred embodiments are shown in the drawings. However, it should be understood that the present invention is not limited to the exact arrangement and means of the embodiments shown in the drawings.

It is a graph which shows the immunogenic peptide derived from HER3 which shows the ability to activate CD4T cells over many patients (SEQ ID NOS: 1-3 in the order of illustration). FIG. 5 is a graph showing the overall screen of HER3 using a group of 10 peptide fragments. A screen graph of HER3 using a single peptide is also shown (SEQ ID NOS: 4-7, respectively, in the order shown). FIG. 5 is a graph showing the overall screen of HER3 using a group of 10 peptide fragments. Also shown are screen graphs of HER3 using a single peptide (SEQ ID NOS: 4 and 7, respectively, in the order shown). FIG. 5 is a graph showing the overall screen of HER3 using a group of 10 peptide fragments. A screen graph of HER3 using a single peptide is also shown (SEQ ID NOS: 1-3, respectively, in the order shown). It is a graph which shows the production amount of IFN-γ from a different HER3 peptide. It is a graph which shows the production amount of IFN-γ from a different HER3 peptide. It is a graph which shows the production amount of IFN-γ from the "reverse" screen which sensitizes the peptide and HER3 extracellular domain after identifying the peptide in advance. It is a graph which shows the production amount of IFN-γ from the "reverse" screen which sensitizes the peptide and the HER3 extracellular domain after identifying the peptide in advance. FIG. 6 is a graph showing the production of IFN-γ from a "reverse" screen that is initiated with a peptide and sensitized to the peptide and the HER3 extracellular domain in patients who have not been pre-sensitized to the HER extracellular domain. .. Demonstrates the production of IFN-γ from a "reverse" screen that is initiated with the entire peptide library and sensitized to peptides and the HER3 extracellular domain in patients who have not been pre-sensitized to the HER extracellular domain. It is a graph. Demonstrates the production of IFN-γ from a "reverse" screen that is initiated with the entire peptide library and sensitized to peptides and the HER3 extracellular domain in patients who have not been pre-sensitized to the HER extracellular domain. It is a graph. FIG. 6 is a graph showing the production of IFN-γ from a "reverse" screen that is initiated with a peptide and sensitized to the peptide and the HER3 extracellular domain in patients who have not been pre-sensitized to the HER extracellular domain. .. It is a graph which shows the screen of the continuous peptide in the donor of UPCC15107-24. It is a graph which shows the screen of the continuous peptide in the donor of UPCC15107-38. It is a graph which shows the "reverse" sensitization ("REVERSE" sensitivity) in the donor of UPCC15107-38 and UPCC15107-24. Immunogenicity in UPCC15107-30 and UPCC15107-32 donors (both patients are known to show no anti-HER3 responsiveness to the identified HER3 peptide and / or native HER3 ECD). FIG. 5 is a graph showing that DC1 pulsed with the HER3 epitope sensitized CD4 + Th1 and overcame anti-HER3 immune tolerance. The CD4 + HER3 epitope is a graph showing that it exhibits MHC class II indiscrimination. When activated HER-3 CD4 + cells are placed next to HER-3 expressing cells in a chamber, the HER-3 CD4 + cells cause apoptosis or death of HER-3 expressing breast cancer cells. It is a figure which shows. FIG. 5 shows a method for identifying immunogenic, class II indiscriminate HER3 CD4 + peptides using HER3 ECD as a tumor antigen to generate anti-HER3 Th1 cell-mediated immunity. It is a graph which shows that the immunogenicity of the identified CD4 + HER3 ECD epitope was confirmed by "reverse" sensitization. Screening of HER3 ECD was performed with the single peptide shown. It is a graph which shows the additional result which confirmed the immunogenicity of the identified CD4 + HER3 ECD epitope by "reverse" sensitization. A photograph of immunohistochemical scoring of HER staining is shown. A histogram showing the overexpression of the HER family in the Barrett's esophagus with low-grade dysplasia (LGD) or high-grade dysplasia (HGD) (FIG. 23A), and highly dysplastic Barrett's lesions with associated invasive cancer (with carcinoma) FIG. 23B is a histogram showing the overexpression rate of highly dysplastic Barrett's lesions (HGD) without HGD) or associated invasive carcinoma. FIGS. 24A-24C show reduced anti-HER3 CD4 Th1 cell response from HD (healthy donor) to ER IBC / ER ^positive IBC (estrogen receptor positive invasive breast cancer (IBC)) and TN IBC (triple negative IBC). .. The figure shows a histogram (left panel) of IFN-γELISpot analysis of ^{systemic CD4 + Th1 cell response.} The group of patients studied was HD; BD (beneficial chest biopsy); DCIS (HER2-positive (“HER2- ^positive ”) ductal carcinoma in situ); HER2 IBC / HER2- ^positive IBC; ER IBC / ER- ^positive 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 performed at once. One-way ANOVA tests were performed on all groups. FIG. 24A shows the cumulative anti-HER3 CD4 T cell response measured by IFN-γ spots per million cells via the ELISpot assay from HDS to BD, to DCIS, to HER2- ^positive IBC, to ER- ^positive IBC. Finally, it shows a significant decrease to TN IBC (90: 80: 66: 79: 48: 40, p = 0.01, respectively). FIG. 24B shows a significant reduction in numbers from HD to BD, DCIS, to HER2- ^positive IBC, to ER- ^positive IBC, and finally to TN IBC (1.0 vs. 0.6 vs. 0.8, respectively). The repertoire is the number of HER3 peptides with a positive CD4 Th1 response vs. 0.8 vs. 0.5 vs. 0.3, p = 0.003). FIG. 24C shows a significant decrease from HD to BD, to DCIS, to HER2- ^positive IBC, to ER- ^positive IBC, and finally to TN IBC (76.7% vs. 63.6% vs. 53, respectively). 8% vs. 66.7% vs. 45.0% vs. 33.3%, p = 0.02) responsiveness (percentage of subjects responding to at least one peptide (%)). Figures 25A and 25B show that loss of CD4T cell response is HER3 specific, as there is no difference in tetanus or anti-CD3 / CD28 stimulation between the group of patients tested. The figure shows a histogram (left panel) of IFN-γELISpot analysis of systemic CD4 + Th1 cell response. The patient groups studied are HD; BD; DCIS; HER2 IBC / HER2- ^positive IBC; ER IBC / ER- ^positive 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 performed at once. One-way ANOVA tests were performed on all groups. FIG. 25A shows the tetanus response measured by IFN-γ spots per 200,000 cells via the ELISpot assay between HD, BD, DCIS, HER2 IBC / HER2- ^positive IBC, ER IBC / ER- ^{positive IBC or TN IBC.} Shows that there is no statistically significant difference in (37: 30: 19: 34: 24: 29, p = 0.65, respectively). FIG. 25B shows anti-CD3 / anti-CD28 polyclonal 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 shows that there is no statistically significant difference in stimuli (688 vs. 549 vs. 804 vs. 699 vs. 629 vs. 675, p = 0.68, respectively). Figures 26A-26C show that anti-HER3 CD4 T cell response correlates with recurrence and response to neoadjuvant chemotherapy, but not with lymph node metastasis. FIG. 26A compares the immune response of IBC patients due to lymph node status (lymph node positive (“LN +” or “LN ^positive ” vs. lymph node negative (“LN-” or “LN ^{negative”)) in the first surgery 4} Two histograms, cumulative response (top panel) (40:56, p = 0.12, respectively), repertoire (second panel) (0, respectively) between ^LN-positive IBC patients and LN- ^{negative IBC patients.} .4 vs. 0.6, p = 0.08), responsiveness (third panel) (35.7% vs. 54.8%, p = 0.19, respectively) or tetanus response (lower panel) (each, respectively) It shows that there is no statistically significant difference of 22:29, p = 0.35). FIG. 26B shows the recurrence vs. non-recurrence (no disease) immune response in IBC patients at least 1 year after diagnosis. The four histograms to be compared were shown and the cumulative response, repertoire and responsiveness were significantly lower: cumulative response (upper panel) (17 vs. 66, p = 0.04, respectively), repertoire (second panel) (0 each). .0 vs. 0.6, p <0.05) and responsiveness (third panel) (0% vs. 55.6%, p = 0.01, respectively). Between recurrent IBC and non-recurrence IBC patients There was no difference in tetanus response (lower panel) (27:35, p = 0.65, respectively). Figure 26C shows neoadjuvant chemotherapy (complete pathological response (“pCR”)) vs. residual disease (“< It has four histograms comparing pCR ”). Among patients receiving neoadjuvant chemotherapy, patients with pCR showed a significantly higher cumulative response and repertoire compared to patients with <pCR (pCR”). Cumulative response (upper panel) (144 vs. 32, p = 0.004, respectively) and repertoire (second panel) (0.8 vs. 0.4, p = 0.05, respectively)). PCR and <pCR During the patient's immune response, responsiveness (third panel) (80.0% vs. 27.3%, p = 0.10, respectively) or tetanus response (lower panel) (17:59, respectively) , P = 0.15). 27A-27D show that the anti-HER3 CD4 T cell response is significantly higher in postmenopausal HD / BD but does not change with age, race or pregnancy history. FIG. 27A has four histograms comparing the immune response of HD patients to age (under 50 vs. over 50). There were no statistically significant age-related differences in cumulative response, repertoire, responsiveness or tetanus response: cumulative response (upper panel) (77 vs. 103, p = 0.25, respectively), repertoire (second panel) (each, respectively). 0.8 vs 1.1, p = 0.38), responsiveness (third panel) (72.0% vs 75.0%, p = 1.0, respectively) or tetanus response (lower panel) (lower panel) 39 to 30, p = 0.40, respectively). FIG. 27B has four histograms comparing the immune responses of HD patients by race (Caucasian vs. African American vs. Other). There were no statistically significant differences in cumulative response, repertoire, responsiveness or tetanus response by race ((cumulative response) (87:83:95, p = 0.96, respectively)), repertoire (second panel) (respectively). , 0.9 vs 0.7 vs 1.4, p = 0.31), responsiveness (third panel) (69.0% vs 71.4% vs 100%, p = 0.35, respectively) Or tetanus response (bottom panel) (33:51:26, p = 0.30, respectively). FIG. 27C has four histograms comparing the immune response of HD patients due to pregnancy history / pregnancy (0 vs. 1 or more pregnancies). There was no statistically significant difference in cumulative response, repertoire or responsiveness depending on pregnancy history: cumulative response (upper panel) (82 to 91, p = 0.71 respectively), repertoire (second panel) (1. 0 to 0.9, p = 0.62) or responsiveness (third panel) (76.5% to 70.8%, p = 0.74, respectively). Interestingly, the tetanus response (lower panel) was significantly higher in heifers compared to patients with a history of at least one pregnancy (47:27, p = 0.04, respectively). FIG. 27D has four histograms comparing the immune response of HD patients by menopausal state (premenopausal-postmenopausal). Postmenopausal HD / BD showed significantly higher cumulative response and repertoire compared to premenopausal HD / BD: cumulative response (upper panel) (136 spots vs. 70 spots per million cells, respectively) , P = 0.005), repertoire (second panel) (1.4 vs. 0.8 peptide, p = 0.03, respectively). 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 (bottom panel) (38:28, p = 0.37, respectively). FIG. 28 is a graph of determining 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 serially diluting a known anti-HER3 CD4 T cell responder into medium. Cumulative responses were 1.0-fold to 0.1-fold, 0.01-fold, and 0.001-fold (230 to 35, 12, and 5 spots per million cells, respectively). A 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, an isolated peptide from one or more of the HER-1, HER-3 and c-MET proteins is provided. In one embodiment, the peptide corresponds to an epitope of HER-1. In one embodiment, the peptide corresponds to an epitope of HER-3. In one embodiment, the peptide corresponds to an epitope of c-MET.

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

One embodiment 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 treating cancer in a subject. It also provides vaccines for use in treatment and prevention. The peptides of this example are capable of eliciting an immune response in the context of a single or chimeric peptide, as described herein. In one embodiment, the immune response is a humoral response. In another embodiment, the immune response is a cell-mediated response. According to some embodiments, the peptides of the invention confer a protective effect.

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

In another embodiment, the HER3 MHC class II immunogenic peptide is used to determine anti-HER3CD4 Th1 loss.

Definitions Unless otherwise defined, all scientific and technological terms used herein have the same meanings as those skilled in the art would normally understand. Any method and material similar to or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, but preferred methods and materials are described.

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

Standard techniques are used for the synthesis of nucleic acids and peptides. Techniques and procedures generally refer to conventional methods in the art and various general references (eg, Sambrook and Russel, 2012, Molecular Cloning, A Laboratory Application, Cold Spring Harbor Press, Cold Spring Harbor). And Ausube et al., 2012, Current Protocols in Molecular Biology, John Willey & Sons, NY), which are provided throughout this description.

The nomenclature used herein and the laboratory procedures used in the analytical chemistry and organic synthesis described below are well known and commonly used in the art. Perform chemical synthesis and analysis using standard techniques or modified forms thereof.

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

As used herein, the articles "one (a)" and "one (an)" are used to refer to one or more (ie, at least one) grammatical object of an article. As an example, "one (an) element" means one element or two or more elements.

“About” when referring to a measurable value, eg, quantity, time period, etc., as used herein, is ± 20%, or ± 10%, or ± from a particular value. It is intended to include variations of 5%, or ± 1%, or ± 0.1%, and such variations are appropriate for implementing the disclosed method.

When used in the context of living organisms, tissues, cells or their constituents, the term "abnormality" refers to at least one observable or detectable feature (eg, age, treatment, time of day, etc.). Refers to an organism, tissue, cell or component thereof that is different from the organism, tissue, cell or component thereof that exhibits each "normal" (expected) characteristic. Features that are normal or expected for one cell or tissue type may be abnormal for different cell or tissue types.

As used herein, "adjuvant therapy" for breast cancer refers to any treatment given after first-line therapy (ie, surgery) to increase the chances of long-term survival. "Neoadjuvant therapy" is a treatment given before first-line therapy.

The term "antigen" or "ag", as used herein, is defined as a molecule that provokes an immune response. This immune response can be involved in either antibody production or activation of certain immunologically competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. In addition, the antigen can be obtained from recombinant or genomic DNA. Those skilled in the art will appreciate that any DNA, including nucleotide sequences or partial nucleotide sequences that encode a protein that elicits an immune response, therefore encodes the term "antigen" as used herein. Will. Moreover, those skilled in the art will appreciate 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 a desired immune response. It becomes clear. Moreover, those skilled in the art will appreciate that the antigen does not need to 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 include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

“Antigen-presenting cells” (“APC”) are cells capable of activating T cells, including, but not limited to, mononuclear cells / macrophages, B cells and dendritic cells (DCs).

An "antigen-incorporated APC" or "antigen-pulse-added APC" comprises an APC that has been exposed to and activated by the antigen. For example, the APC may be in a state of uptake of the antigen while culturing in vitro, eg, in the presence of the antigen. It can also be incorporated into APC by exposure to the antigen in vivo. The "antigen-incorporated APC" is conventionally two methods: (1) a small peptide fragment known as an antigenic peptide is "pulse-added" directly onto the outside of the APC, or (2) the APC. It is prepared by either ingesting the whole protein or protein particles and then ingesting the whole protein or protein particles into the APC. These proteins are digested by APC into smaller peptide fragments and finally transported to the APC surface where they are presented. Furthermore, the antigen-incorporated APC can also be produced by introducing a polynucleotide encoding the antigen into the cell.

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

The term "antitumor effect", as used herein, is used herein to reduce tumor volume, reduce tumor cell count, reduce metastasis, prolong life expectancy, or various physiological conditions associated with a cancerous condition. Refers to a biological effect that can be manifested by the remission of. In the first place, the "antitumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention to prevent 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 inadequate and excessive responses to self-antigens. In particular, examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia communis, tonic spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), nutrition Disordered epidermal vesicular disease, supraclavicular inflammation, glomerulonephritis, Graves' disease, Gillan-Valley syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, severe myasthenia, pemphigus vulgaris, pemphigus vulgaris , Rheumatoid fever, rheumatoid arthritis, sarcoidosis, sclerosis, Schegren's syndrome, spondylosis, thyroiditis, vasculitis, leukoplakia, mucinous edema, pernicious anemia, ulcerative colitis.

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

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

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

"CD4 + Th1 cells", "Th1 cells", "CD4 + T helper type 1 cells", "CD4 + T cells", etc. are T helpers that express the surface protein CD4 and produce high levels of the cytokine IFN-γ. Defined as a 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 Means the overall immune response of the patient group.

"DC vaccination," "DC immunization," "DC1 immunization," etc. utilize the immune system to recognize specific molecules and use autologous dendritic cells to obtain specific responses to them. Refers to strategy.

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

A "disease" is an animal's health that continues to deteriorate if the animal is unable to maintain homeostasis and the disease does not relieve.

"Disorder" in an animal refers to the case where it is possible for the animal to maintain homeostasis, but the health of the animal is less favorable than it would be in the absence of the disorder. Disorders, if left untreated, do not necessarily cause further deterioration of the animal's health.

A disease or disorder is "alleviated" when the severity or frequency of at least one sign or symptom of the disease or disorder experienced by the patient decreases.

"Effective amount" or "therapeutically effective amount" is used interchangeably herein and, as described herein, a compound, formulation, material or composition effective in achieving a particular biological result. Refers to the quantity of things. Such results 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 that belongs to or is produced within an organism, cell, tissue or system.

As used herein, the term "extrinsic" refers to any material introduced from or produced outside an organism, cell, tissue or system.

"HER receptor" is a receptor protein tyrosine kinase belonging to the HER receptor family, EGFR (ErbB1, HER1) receptor, HER2 (ErbB2) receptor, HER3 (ErbB3) receptor and HER4 (ErbB4) receptor. including. The HER receptor is generally capable of binding to a HER ligand and / or dimerizing with another HER receptor molecule, an extracellular domain; an oily transmembrane domain; a conserved intracellular tyrosine kinase domain. Also contains a carboxyl-terminated signaling domain with several tyrosine residues that can be phosphorylated. The HER receptor may be a "native sequence" HER receptor or an "amino acid sequence variant" thereof. Preferably, the HER receptor is a native sequence human HER receptor.

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

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

"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 breast cancers as well as numerous other cancers. HER2-positive is currently defined by gene amplification by FISH (fluorescence in situ hybridization) assay and 2+ or 3+ pathological staining intensities.

"HER2 ^negative " is defined by the lack of gene amplification by FISH and can, in most cases, cover the range of pathogenic staining forms 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). Refers to a receptor polypeptide that is present.

"Extracellular domain of HER3" or "HER3ECD" refers to the domain of HER3 that is external to the cell and is anchored or circulating in the cell membrane, containing fragments thereof. In one embodiment, the extracellular domain of HER3 may include four domains: domain I, domain II, domain III and domain IV. In one embodiment, the HER3 ECD comprises amino acids 1-636, numbered with a signal peptide. In one embodiment, the HER3 domain III comprises amino acids 328-532, numbered with a signal peptide.

As used herein, "HER3 immunogenic peptide", "HER3 binding peptide", "HER3 epitope" and the like are derived from or based on the sequence of HER3 protein, specifically HER3ECD, MHC class II. Refers to peptides and their equivalents. The HER3 peptide 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-estrogens 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 (epitope) or binding peptides have been identified as follows:
p12 (Peptide 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4).
p81 (Peptide 401-415): SWPPHMHNFFSVFSNL (SEQ ID NO: 5).
p84 (Peptide 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6). And p91 (peptide 451-465): AGRIYISANRQLCYH (SEQ ID NO: 7).

As used herein, "homology" is a subunit between two polymeric molecules, eg, two nucleic acid molecules, eg, two DNA molecules or two RNA molecules, or two polypeptide molecules. Refers to sequence similarity. If the same monomeric subunit occupies the position of a subunit in both of the two molecules, for example, if adenine occupies a position in each of the two DNA molecules, then these molecules are in that position. Is completely homologous or 100% homologous. Percent homology between two sequences is a direct function of the number of matching or homologous positions, eg, in the sequences of two compounds, half the position (eg, the height of the length of 10 subunits). If the (5 positions in the molecule) are homologous, then these two sequences are 50% identical, and if 90% of the positions, eg, 9 out of 10, are matched or homologous, then these 2 sequences. The sequences share 90% homology. As an example, the DNA sequences 5'ATTGCC3'and 5'TATGGC3' share 50% homology.

In addition, the term "homology" or "identity" is used herein to refer to nucleic acids and proteins as being applied to homology or identity at both the nucleic acid and amino acid sequence levels. Should.

The term "hyperproliferative disease" is defined as a disease that results from overgrowth of cells. Exemplary hyperproliferative disorders include, but are not limited to, cancer or autoimmune disorders. Other hyperproliferative disorders include, for example, vascular occlusion, restenosis, atherosclerosis or inflammatory bowel disease.

As used herein, "Explanatory Material" includes publications, records, schematics or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the invention. Instructions for use of the kits of the invention are attached, for example, to a container containing the nucleic acids, peptides and / or compositions of the invention, or shipped with a container containing the nucleic acids, peptides and / or compositions. be able to. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound will be used together by the recipient.

As used herein, "immune response" means activation of the host's immune system, eg, the mammalian immune system, in response to the introduction of an antigen. The immune response can take the form of a cellular or humoral response, or both.

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

By the term "modulating", as used herein, when compared to response levels in a subject in the absence of treatment or compound, and / or otherwise identical but not yet. Meaning to mediate a detectable increase or decrease in response level in a subject when compared to the response level in the subject of treatment. The term includes upsetting and / or influencing a native signal or response, thereby mediating a beneficial therapeutic response in the subject, preferably human.

For each group of subjects analyzed for anti-HER3 CD4 + Th1 immune response, the following CD4 + Th1 response "evaluator" or "immune response endpoint" is defined: (a) Overall anti-HER3 response Gender (expressed as the percentage (%) of subjects responding to one or more immunogenic peptides); (b) response repertoire (as the average number (n) of immunogenic peptides recognized by each subject group) represented); and, (c) the cumulative response (four MHC class II HER3 immunoreactivity spot from the original peptide (IFN-gamma ELISpot cells 10 per ⁶ spot-forming cells from the analysis from each group of subjects Represented as the sum of "SFC").

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

As used herein, "population" includes reference to isolated cultures that include allogeneic, substantially allogeneic, or heterologous cell cultures. Generally, the "population" can also be considered as an "isolated" cell culture.

Receptor tyrosine kinase (“RTK”) is a high-affinity cell surface receptor for many polypeptide growth factors, cytokines, and hormones. The human EGF receptor (“HER”) family of RTKs regulates a wide variety of biological processes, including cell proliferation, migration, infiltration 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 contains a recombinant polynucleotide.

"Responsive" or "anti-HER3 responsive" is used interchangeably herein to mean the percentage of subjects responding to at least one of the four HER3 immunogenic peptides.

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

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

"Signal 1", as used herein, generally refers to a first biochemical signal passed from activated DC to a T cell. 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 a second signal provided by DC to T cells. Signal 2 is provided by "co-stimulation" molecules on activated DCs, usually CD80 and / or CD86 (although other co-stimulation molecules are also known) and are sensed by T cells through the surface receptor CD28. Will be done.

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

By the term "specifically binding", as used herein, recognizing and binding to another molecule or feature in a sample, but substantially recognizing another molecule or feature. Also means molecules that do not bind to them, eg, antibodies.

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

As used herein, the term "targeted therapy" is usually a drug or other drug or other that interferes with a particular target molecule involved in the growth of cancer cells with little damage to normal cells to achieve antitumor effects. Refers to cancer treatment using the substance of. In contrast, traditional cytotoxic chemotherapeutic agents act on all actively dividing cells. For breast cancer therapeutic monoclonal antibodies, specifically trastuzumab / HERCEPTIN® targets the HER2 / neu receptor.

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

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

In the present specification, terms such as "T helper cell", "helper T cell", and "Th cell" refer to cells and include lymphocytes (containing various cell types that can be identified by those skilled in the art). A subgroup of leukocytes) is shown. In particular, T helper cells are effector T cells whose primary function is to promote activation and function of other B lymphocytes and T lymphocytes and / or macrophages. Helper T cells differentiate into two major subtypes of cells known as the "Th1" or "Type 1" and "Th2" or "Type 2" phenotypes. These Th cells secrete cytokines, proteins or peptides that stimulate or interact with other white blood cells.

As used herein, "Th1 cells", "CD4 + Th1 cells", "CD4 + T helper type 1 cells", "CD4 + T cells" and the like refer to mature T cells expressing the surface glycoprotein CD4. .. CD4 + T helper cells are activated when peptide antigens are presented by MHC class II molecules expressed on the surface of antigen-presenting peptides (“APC”) such as dendritic cells. CD4 + T helper cells secrete high levels of cytokines such as interferon gamma (“IFN-γ”) when activated by the MHC antigen complex. Such cells are believed to be very effective against specific disease-causing microorganisms present inside the host cell, and against specific disease-causing microorganisms and cancers present within the host cell. Is also important for the antitumor response in human cancers.

"Th17 T cells", when used herein, are believed to be extremely effective against disease-causing microorganisms that produce high levels of cytokines IL-17 and IL-22 and live on mucosal surfaces. Refers to the T cells that are being used.

A "therapeutically effective amount" is an amount of a compound of the invention that, when administered to a patient, ameliorate the symptoms of the disease. The amount of the compound of the present invention constituting the "therapeutically effective amount" varies depending on the compound, the state and severity of the disease, the age of the patient to be treated, and the like. The therapeutically effective amount can be determined routinely by those skilled in the art in light of their own knowledge and the present disclosure.

The terms "treat," "treating," and "treatment" refer to the therapeutic or prophylactic measures described herein. Methods of "treatment" exceed the survival expected for prevention, cure, delay, reduction of severity or amelioration of one or more symptoms of the disorder or recurrent disorder or without such treatment. For the purpose of prolonging the survival of a subject, the compositions of the invention ultimately acquire a subject in need of such treatment, eg, a subject suffering from a disease or disorder or such disease or disorder. Utilize administration to subjects at risk.

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

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

A "variant" for a peptide or polypeptide retains at least one biological activity, although the amino acid sequence depends on the insertion, deletion or conservative substitution of the amino acid. Variant may also mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of amino acids, ie replacing amino acids with amino acids with similar properties (eg, hydrophilicity, degree and distribution of charged regions), typically results in minor changes in the art. It is recognized that there is. Some of these minor changes can be identified by examining the hydrophobic hydrophilicity index of amino acids, as understood in the art. Kyte et al., J. Mol. Mol. Biol. 157: 105-132 (1982). The hydrophobicity index of an amino acid is based on a study of its hydrophobicity and charge. It is known in the art that it can replace amino acids with similar hydrophobic hydrophilicity indices and still retain protein function. In one aspect, it replaces an amino acid with a hydrophobic hydrophilicity index of ± 2. The hydrophilicity of amino acids can also be used to indicate substitutions that will result in proteins that retain biological function. In the context of peptides, by examining the hydrophilicity of amino acids, it is possible to calculate the largest local average hydrophilicity of the peptide, which can be well correlated with antigenicity and immunogenicity. This is a useful measure that has been reported. U.S. Pat. No. 4,554,101, which is incorporated herein by reference in its entirety. As is understood in the art, peptides that retain biological activity, eg, immunogenicity, can be obtained from amino acid substitutions with similar hydrophilic values. Substitutions can be performed using amino acids that have hydrophilic values within ± 2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the particular side chain of that amino acid. Consistent with such observations, amino acid substitutions corresponding to biological functions are indicated by hydrophobicity, hydrophilicity, charge, size and other properties of the relative similarity of amino acids, especially of those amino acids. It is understood to depend on the side chain.

Scope: Throughout the disclosure, various aspects of the invention can be presented as range formats. It should be understood that the description as a range format is for convenience and brevity only and should not be construed as a firm limitation of the scope of the invention. Therefore, the description of a range should be regarded as specifically disclosing all possible partial ranges and individual numerical values within that range. For example, 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 within that range. It should be considered that the individual numbers of, for example, 1, 2, 2.7, 3, 4, 5, 5.3 and 6 are 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, an isolated peptide from one or more of the HER-1, HER-3 and c-MET proteins is provided. In one embodiment, the peptide is useful in eliciting an immune response. The composition containing the peptide of the present invention is also useful as a prophylactic therapeutic agent for initial defense and also as a therapeutic agent for the treatment of an ongoing condition.

The present invention also provides methods for treating or preventing cancer. Such a method comprises administering the peptide of the invention or a combination of peptides of the invention to a subject in need thereof. Administration of such peptides induces anti-tumor immunity. Accordingly, the present invention provides methods for inducing anti-tumor immunity in a subject, such methods as the peptides of the invention or combinations of peptides of the invention, as well as pharmaceutical compositions and cell compositions derived thereto. Includes the step of administering to the subject.

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

In one embodiment, T cells can be cultured and expanded using APCs pulsed with the peptides of the invention. Once the T cells have been expanded and proliferated using APC to obtain a sufficient number of antigen-specific T cells, the resulting antigen-specific T cells are administered to the mammal, thereby antigening in the mammal. Induces a specific T cell response.

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

The present invention includes mature, antigen-incorporated DCs that have been activated by any DC activation regimen. The DCs of the invention produce the desired levels of cytokines and chemokines. In one embodiment, the invention provides a method of pulsing and activating cells, thereby maintaining the cells in an active state after cryopreservation. The benefit of the DC preparation of the present invention is that from a single leukocyte apheresis (collection from a patient), cells are efficient in multiple (eg, 10 or more) "booster" doses in addition to the initial vaccine. These doses can be thawed as needed at remote treatment sites, eliminating the need for any specialized cell processing facility or additional quality control testing.

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

As used herein, the present invention contemplates providing a method for producing and cryopreserving DCs that have a superior function in generating stronger signals to T cells, and thus more. A strong, DC-based vaccine is obtained. Effective cryopreservation of such cells allows the sample to be stored, thawed and used later, thereby reducing the need to repeat the process of feresis and eltriation during vaccine production. Let me. It is an advantage that the DCs can be frozen and then thawed and removed from them later, which is to subdivide and freeze the vaccines produced in a single dose, and then for several weeks, This means that patients can be given one at a time for months or years to receive a "booster" vaccination that enhances immunity.

The present embodiment also includes 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 esophagogastric cancer.

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

The present invention provides a composition comprising one or more peptides of the invention. The present invention also provides compositions 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.

In addition, compositions having one or more multivalent peptides are also provided. These polyvalent peptides contain two or more epitopes of the invention.

Methods of activating an immune response using the compositions of the invention and methods of treating cancer in a subject are included in the invention. Vaccines are also provided for use in treatment and prevention. The epitopes of the invention are capable of eliciting an immune response in the context of a single or 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 epitopes or peptides of the invention confer a protective effect.

In one embodiment, the epitope or other peptide of HER3 of the present invention is
p11-13 (Peptides 51-75): KLYERCEVVMGNLEIVLTGHNADLSFLQW (SEQ ID NO: 1);
p81-83 (Peptides 401-425): SWPPHMHNFFSVFSNLTTIGGRSLYN (SEQ ID NO: 2);
p84-86 (Peptides 416-440): TTIGGRSLYNRGFSLLIMMKNLNVTS (SEQ ID NO: 3);
p12 (Peptides 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4);
p81 (Peptides 401-415): SWPPHMHNFFSVFSNL (SEQ ID NO: 5);
p84 (Peptides 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6);
p91 (Peptide 451-465): AGRIYISANRQLCYH (SEQ ID NO: 7)
including.

The HER3 peptide or any peptide of the present invention may be cyclic or linear. If the epitope is cyclic, it can be cyclic in any suitable manner. For example, disulfide bonds can be formed between selected cysteine (Cys) pairs to provide the desired conformation. It is believed that the formation of cyclic epitopes can result in a conformation that improves the humoral response and thus the protective effect.

The HER3 epitope identified by SEQ ID NO: 4 corresponds to positions 56-70 of the HER3 protein. The HER3 epitope identified by SEQ ID NO: 5 corresponds to positions 401-415 of the HER3 protein. The HER3 epitope identified by SEQ ID NO: 6 corresponds to positions 416-430 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 invention also include peptides that 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 have been substituted, or one or more amino acids correspond. It is deleted from the reference sequence or added to it. For example, 1-3 amino acids can be added to the amino terminus, the carboxy terminus, or both. In some examples, the HER3 epitope is glycosylated.

In another example, the HER3 epitope can be a retro-inverso isomer of the HER3 epitope. Retro-inverso modifications include reversal of all amide bonds within the peptide backbone. This reversal can be achieved by reversing the orientation of the sequence and reversing the asymmetry of each amino acid residue by using the D-amino acid instead of the L-amino acid. This retro-inverso isomer morphology is capable of retaining the planar and conformational constraints of at least some peptide bonds.

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

In some examples, deletions and additions are located at the amino terminus, carboxy terminus, or both of one sequence of the peptides of the invention. For example, an equivalent of a 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, with the sequence of the corresponding HER3 epitope. It has an amino acid sequence that is 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 per 10 amino acids in the reference sequence, i.e. any combination of deletions, additions or substitutions. Percent identity is determined by comparing the amino acid sequence of the variant with the reference sequence using a program known or developed in the art.

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

Functional equivalents of the HER3 epitope can be identified by modifying the sequence of the epitope and then assaying the resulting polypeptide for an immune response, eg, the ability to stimulate antibody production. Such antibodies can be found in a variety of body fluids, including serum and ascites. Briefly, body fluid samples are isolated from warm-blooded animals, eg, humans, for which it is desired to determine the presence of antibodies specific for the HER3 polypeptide. The body fluid is incubated with the HER3 polypeptide under conditions sufficient to form an immune complex between the polypeptide and the protein-specific antibody and for a time sufficient to do so, and then preferably. , ELSI using the ELISA method.

According to other embodiments of the present invention, there is provided a chimeric peptide and a composition comprising one or more chimeric peptides. According to various embodiments, the chimeric peptide comprises a HER3 epitope, another epitope, and a linker that connects the HER3 epitope to another epitope. In one embodiment, other epitopes can 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-terminus or the carboxy-terminus of the other epitope. The location and selection of other epitopes depends on the structural characteristics of the HER3 epitope, ie alpha helix or beta-turn or strand.

In one embodiment, the linker can be a peptide with a length of about 2 to about 15 amino acids, about 2 to about 10 amino acids, or about 2 to about 6 amino acids. The chimeric peptide may be linear or cyclic. In addition, the HER3 epitope, other epitopes and / or linkers may be in the form of retro-inverso. Therefore, 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 linkers may be in the form of retro-inverso.

In another embodiment, the peptides of the invention can be combined into a mixture instead of taking the form of a chimeric peptide. In any case, the compositions of the invention comprising peptides can be useful agents for pulse addition to antigen presenting cells (eg, dendritic cells) to generate cell vaccines. In another embodiment, the compositions of the invention comprising peptides can be useful immunogens to induce the production of antibodies. The compositions of the present invention can also be used to immunize a subject and delay or prevent the development of tumors. The compositions of the present invention can be used in vaccines to provide protective effects.

According to a further embodiment of the invention, there is provided a composition comprising a mixture of two or more of the peptides or chimeric peptides of the invention. In some examples, 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 well-known techniques. For example, peptides can be synthesized and prepared using either recombinant DNA technology or chemical synthesis. The peptides of the invention may be synthesized individually or as longer polypeptides composed of two or more peptides. The peptides of the invention are preferably isolated, i.e., substantially free of other naturally occurring host cell proteins and fragments thereof.

A commercially available peptide synthesizer can be used to synthesize the peptides and chimeric peptides of the present invention. For example, as specifically incorporated herein by reference, Kaumaya et al., “De Novo” Engineering of Peptide Immunogenic and Antigenic Determinants as Potential Vaccines, Peptide The chemical methods described in can be used. For example, the HER-3 epitope can be co-linerly synthesized with other epitopes to form a chimeric peptide. Peptide synthesis can be performed using Fmoc / t-But chemistry. Peptides and chimeric peptides can be cyclic in any suitable manner. For example, using a method of differentially protecting cysteine residues, performing iodine oxidation, adding water to facilitate the removal of Acm groups, simultaneously forming disulfide bonds, and / or the silyl chloride-sulfoxide method. Therefore, a disulfide bond can be obtained.

Peptides and chimeric peptides can also be produced using cell-free translation systems and RNA molecules derived from epitopes or DNA constructs encoding peptides. Alternatively, the epitope or chimeric peptide is made by transfecting a host cell with an expression vector containing a DNA sequence encoding each epitope or chimeric peptide and then inducing expression of the polypeptide in the host cell. You can also do it. In the case of recombinant production, recombinant constructs containing one or more of the sequences encoding an epitope, chimeric peptide or variant thereof are subjected to conventional methods, eg, calcium phosphate transfection, DEAE-dextran-mediated transfection. , Microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, or infection into host cells.

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

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

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

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

Furthermore, the present invention also includes 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", i.e. about 60% homologous, more preferably to the nucleotide sequence of the isolated nucleic acid encoding the peptide of the invention. , About 70% homology, even more preferably about 80% homology, more preferably about 90% homology, even more preferably about 95% homology, even more preferably about 99% homology.

The scope of the invention is homologues, analogs, variants, derivatives and salts, including shorter and longer peptides and polynucleotides; and analogs of peptides and polynucleotides with substitutions of one or more amino acids or nucleic acids. Includes derivatives of amino acids or nucleic acids known in the art, unnatural amino acids or nucleic acids and synthetic amino acids or nucleic acids, but these modifications preserve the biological activity of the original molecule. Please understand clearly what must be done. Specifically, according to the principles of the invention, any active fragment of an active peptide, as well as an extender, conjugate and mixture will be disclosed.

The present invention includes any isolated nucleic acid that is homologous to the nucleic acids described or referenced herein, provided that these homologous DNAs are the biology of the peptides disclosed herein. It should be interpreted as having a target activity.

The nucleic acids of the invention include sequences of RNA or DNA encoding the peptides of the invention, which make the nucleotide sequences more stable, whether without or with cells, DNA. Alternatively, those skilled in the art will understand that it also includes any form of modification thereof, including chemical modification of RNA. Chemical modifications of nucleotides can also be used to enhance the efficiency with which cells take up nucleotide sequences or the efficiency with which nucleotides are expressed in cells. The present invention contemplates any combination of nucleotide sequence modifications.

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

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

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

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

In certain embodiments, the expression vector is selected from the group consisting of viral vectors, bacterial vectors and mammalian cell vectors. There are numerous 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 utilized and used with the present invention to produce polynucleotides or their cognate polypeptides. Many such systems are commercially available and widely available.

Furthermore, the expression vector can also be provided to cells in the form of a viral vector. Viral vector techniques 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, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses and lentiviruses. In general, a suitable vector contains a replication origin, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers that function in at least one organism. (See, for example, WO 01/96584; WO 01/29058; and US Pat. No. 6,326,193.

To express the desired nucleotide sequence of the invention, at least one module in each promoter is made to function to position the starting site for RNA synthesis. The best known example of this is the TATA box, but some promoters lacking the TATA box, eg, the promoter of the mammalian terminal deoxynucleotidyltransferase gene and the promoter of the SV40 gene, have separate overlaps at the initiation site. The element itself helps to fix the starting place.

An additional promoter element, an enhancer, regulates the frequency of transcription initiation. Typically, they are located in the region 30-110 bp upstream from the initiation site, but recently it has been shown that some promoters also contain functional elements downstream of the initiation site. ing. The spacing between promoter elements is often flexible, so promoter function is preserved even when the elements are inverted or moved to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can increase up to 50 bp apart by the time activity begins to decline. Depending on the promoter, it appears that the individual elements can work together or independently to activate transcription.

The promoter can be a promoter naturally associated with a gene or polynucleotide sequence, which can be obtained by isolating a 5'non-coding sequence located upstream of a coding segment and / or exon. Such promoters can be called "endogenous". Similarly, an enhancer can be an enhancer that naturally accompanies a polynucleotide sequence, which is located either downstream or upstream of that sequence. Instead, 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 usually not associated with the polynucleotide sequence in its natural environment. Point to. Recombinant or heterologous enhancers also refer to enhancers that are not normally associated with a polynucleotide sequence in their natural environment. Such promoters or enhancers include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell, and promoters or enhancers that are not "naturally occurring". That is, it can include promoters or enhancers that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to producing promoter and enhancer nucleic acid sequences synthetically, recombinant cloning and / or nucleic acid amplification techniques can also be used to generate the sequences, which are included in the compositions disclosed herein. Includes relevant PCR ™ (US Pat. No. 4,683,202, US Pat. No. 5,928,906). It is also contemplated that non-nuclear organelles, eg, regulatory sequences that direct transcription and / or expression of sequences within mitochondria, chloroplasts, etc., can be utilized as well.

Of course, it is important to utilize promoters and / or enhancers that effectively guide the expression of DNA segments in the cell types, organelles and organisms selected for expression. Those skilled in the art of molecular biology generally know how to use a combination of promoters, enhancers and cell types for protein expression, see, for example, Sambrook et al. (2012). Promoters utilized include constitutive promoters, tissue-specific promoters, inducible promoters, and / or large amounts of recombinant proteins and / or peptides that are suitable for deriving high levels of expression of the DNA segment to be introduced. It can be a useful promoter under conditions favorable for large-scale production. Promoters may be heterologous or endogenous.

The promoter sequence exemplified in the experimental examples presented herein is the sequence of the earliest cytomegalovirus (CMV) promoter. This promoter sequence is a strongly constitutive promoter sequence and is capable of driving high levels of expression of any polynucleotide sequence operably linked to it. However, sequences of other constitutive promoters can also be used, including, but not limited to, the Salvirus 40 (SV40) Early Promoter, Mouse Breast Tumor Virus (MMTV), Human Immunodeficiency Virus (HIV). Long-Terminal Repeat (LTR) Promoter, Moloney Virus Promoter, Trileukemia Virus Promoter, Epstein-Barvirus Early Stage Promoter, Laus Sarcoma Virus Promoter, and Human Gene Promoters, eg, but not limited to, Actin Promoter, Myosin Promoter , Hemoglobin promoter and muscle creatin promoter. Furthermore, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. In the present invention, by using an inducible promoter, the expression of the polynucleotide sequence to which the inducible promoter is operably linked is turned on if such expression is desired, or if expression is not desired. Provides a molecular switch capable of turning off expression. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters and tetracycline promoters. Furthermore, the present invention also includes the use of tissue-specific promoters, which are active only in the desired tissue.

To assess the expression of a nucleotide sequence encoding a peptide of the invention, the expression vector to be introduced into a cell also contains either or both of a selectable marker gene and / or a reporter gene and is transfected by a viral vector. It can also facilitate the identification and selection of cells expressing from a cell population targeted for transfection or infection. In other embodiments, the selectable marker can be placed on a separate piece of DNA and used in the cotransfection procedure. Both the selectable marker gene and the reporter gene can be flanked by appropriate regulatory sequences to allow expression in host cells. Useful selectable markers are known in the art and include, for example, antibiotic resistance genes, such as neo.

Reporter genes are used to identify cells that may have been transfected and evaluate regulatory sequence function. Reporter genes encoding easily assayable proteins are well known in the art. In general, the reporter gene is not present in the recipient's organism or tissue, nor is it expressed in the recipient's organism or tissue, and expression is manifested by some readily detectable property, such as enzymatic activity. A gene that encodes a protein. The expression of the reporter gene is assayed at the appropriate time after introducing the DNA into the recipient cells.

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

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

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

In one embodiment, the subject of the invention is an anti-cancer vaccine. The vaccine can include one or more cancer antigens. Vaccines can block the growth of tumors. Vaccines can reduce tumor growth. Vaccines can block the metastasis of tumor cells. Targeting the vaccine according to the cancer antigen, breast cancer, liver cancer, prostate cancer, melanoma, hematological cancer, head and neck cancer, glioblastoma, recurrent respiratory papilloma, anal cancer, cervical cancer, brain tumor, etc. Can be treated.

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

Vaccines have a cell-mediated immune response of about 50-about 6000-fold, about 50-fold-about 5500-fold, and about 50-fold, compared to the cell-mediated immune response of non-vaccinated subjects. 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 -Can be increased by about 6000 times. In some embodiments, the vaccine produces a cell-mediated immune response in a vaccinated subject approximately 50-fold, 100-fold, 150-fold, 200-fold as compared to a cell-mediated immune response in a non-vaccinated subject. Double, 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 2,800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times increase Can be made to.

The vaccine has about 50-about 6000-fold, about 50-fold higher levels of IFN-γ in the vaccinated subjects compared to the levels of interferon gamma (IFN-γ) in the unvaccinated subjects. -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 by 6000 times, or about 300 times-about 6000 times. In some embodiments, the vaccine increases the level of IFN-γ in the vaccinated subject by about 50-fold, 100-fold, as compared to the level of IFN-γ in the unvaccinated subject. 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 Double, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times Or it can be increased 6000 times.

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

Generation of Antigen Uptake (Antigen Pulsed) Immune Cells The present invention includes cells that have been exposed to, or otherwise "pulsed", the antigen of the invention or other peptides of the invention. For example, APCs, eg DCs, can be in vitro uptake by, for example, by culturing ex vivo in the presence of the antigen or by exposing it to the antigen in vivo.

It can also be "pulse-added" to the APC in a manner that exposes the APC to the antigen, and this exposure can be done for a sufficient amount of time to facilitate the presentation of the antigen on the surface of the APC. Those skilled in the art will easily understand. For example, APCs can be exposed to antigens in the form of small peptide fragments known as antigenic peptides, which are "pulse-added" directly onto the outside of the APC (Mehta-Damani et al.). , 1994); or APCs can be incubated with whole proteins or protein particles, which are then ingested by APCs. All of these proteins are digested by APC into smaller peptide fragments, which are finally transported to the APC surface and presented on the APC surface (Cohen et al., 1994). Antigens in the form of peptides can be exposed to cells by standard "pulse addition" techniques described herein.

Although not intended to be bound by any particular theory, antigens in the form of foreign or self-antigens are processed by the APCs of the invention to retain the immunogenic form of the antigen. The immunogenic form of an antigen means that the antigen is processed by fragmentation to produce a form of antigen that can be recognized and activated by immune cells, such as T cells. Preferably, such a foreign antigen or self-antigen is a protein that is processed into a peptide by APC. The relevant peptides produced by APC can be extracted, purified and used as immunogenic compositions. Peptides processed by APC can also be used to induce tolerance to proteins processed by APC.

The antigen-incorporated APC is, in other words, known as the "pulse-added APC" of the invention, which is produced by exposing the APC to the antigen either in vitro or in vivo. .. For in vitro pulse addition to the APC, the APC can be sown on a culture dish and exposed to the antigen in an amount sufficient to allow the antigen to bind to the APC for a sufficient period of time. .. The amount and time required to achieve binding of the antigen to APC can be determined by using methods known in the art or other methods disclosed herein. Other methods known to those of skill in the art, such as immunoassays or binding assays, can be used to detect the presence of antigen on the APC after exposure to the antigen.

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

As discussed elsewhere herein, the vector can be prepared to contain a particular polynucleotide that encodes and expresses the protein for which an immunogenic response is desired. Preferably, a retroviral vector is used to infect cells. More preferably, an adenovirus vector is used to infect cells.

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

As defined herein, various methods can be used to transfect polynucleotides into host cells. These methods include, but are not limited to, calcium phosphate precipitation, liposomes, micelles, microinjection, electroporation, colloidal dispersions (ie, macromolecular complexes, nanocapsules, microspheres, beads, and oil-in-water types. Lipid-based systems, including emulsions, micelles, mixed micelles and liposomes). These methods are understood in the art and described in published literature, and thus those skilled in the art can be practiced.

In another embodiment, the polynucleotide encoding the antigen can be cloned into an expression vector to produce an antigen-incorporated APC by another method, which vector can be 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 publications. For example, the expression vector can be transferred into a host cell by physical, chemical or biological means. For example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, NY), and Ausube et al. (1997, Current Protocols in Molecular Biology). It is easily understood that pulsed cells can be obtained by introducing an expression vector containing a polynucleotide encoding an antigen.

The present invention includes, but is not limited to, incorporating the entire antigen in the form of a protein, cDNA or mRNA into the APC, including, but not limited to, various methods for pulsing into the APC. However, the present invention should not be construed as being limited to a particular form of antigen used for pulse addition to APC. Rather, the invention includes other methods known in the art for producing antigen-incorporated APCs. Preferably, the APC is transfected with mRNA encoding the defined antigen. The appropriate primers and reverse transcriptase-polymerase chain reaction (RT-PCR) coupled with the transcription reaction can be used to rapidly generate mRNA corresponding to a gene product of known sequence in vitro. it can. When producing pulsed APCs, transfection with APC mRNA provides advantages over other techniques of uptake of antigen. For example, the ability to amplify RNA from a small amount of tissue, i.e. tumor tissue, leads to the use of APC to vaccinate large numbers of patients.

For the antigen composition to be useful as a vaccine, the antigen composition must induce an immune response against the antigen in cells, tissues or mammals (eg, humans). As used herein, an "immunological composition" expresses or presents an antigen (eg, a peptide or polypeptide), a nucleic acid encoding an antigen (eg, an antigen expression vector), or an antigen or cell component. Can contain cells to In certain embodiments, the antigen composition comprises or encodes all or part of any of the antigens described herein, or an immunologically functional equivalent thereof. In other embodiments, the antigen composition comprises an additional immunostimulant or a mixture comprising a nucleic acid encoding such an agent. Immunostimulators include, but are not limited to, additional antigens, immunomodulators, antigen-presenting cells or adjuvants. In other embodiments, one or more of the additional agents covalently bind to the antigen or immunostimulatory agent in any combination. In certain embodiments, the antigen composition is conjugated to or comprises amino acids of the HLA anchor motif.

Vaccines can vary in their composition of nucleic acids and / or cell constituents, as herein. In a non-limiting example, the nucleic acid encoding the antigen could also be formulated with an adjuvant. Of course, it will be appreciated that the various compositions described herein can further comprise additional constituents. For example, one or more vaccine components can be included in lipids or liposomes. In another non-limiting example, the vaccine can include one or more adjuvants. The vaccines of the invention and their various constituents can be prepared and / or administered by any method disclosed herein, or those skilled in the art will understand in the light of the present disclosure. Will.

It is understood that the antigen composition of the present invention can be prepared by a method well known in the art, and such methods are not limited to these, but are chemically synthesized by solid phase synthesis and chemically reacted by HPLC. The nucleic acid sequence (eg, DNA sequence) encoding the peptide or polypeptide containing the antigen of the present invention is expressed in a method of purifying and removing from other products, or in an in vitro translation system or in living cells. There is a way to generate it. In addition, the antigen composition can also include cell constituents isolated from biological samples. The antigen composition is isolated and well dialyzed to remove one or more unwanted low molecular weight molecules and / or lyophilized in the desired vehicle to enhance the immediate usability of the formulation. .. It should be further understood that any additional amino acids, mutations, chemical modifications, etc. that result in the vaccine components preferably do not substantially prevent the antibody from recognizing the epitope sequence.

Antigen-Presenting Cell Therapy The present invention includes a method of producing a population of APCs (eg, dendritic cells; DC) that present the peptides of the invention on the surface, followed by the use of that population in therapy. Such a method can be performed ex vivo on a sample of cells obtained from a patient. Thus, the APCs thus produced form pharmaceutical agents 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 from that individual. The cells thus produced are delivered to the individual in which the cells were originally obtained, thus forming a therapeutic embodiment of the invention.

DC is derived from pluripotent mononuclear cells that act as antigen-presenting cells (APCs). DCs are ubiquitous in peripheral tissues, where they await to capture antigens. Upon capture of the antigen, the DC processes the antigen into smaller peptides and migrates towards the secondary lymphoid organs. It is within the lymphatic organs that the DC presents the antigenic peptide to naive T cells, and the presentation of the peptide initiates a signal cascade that biases T cell differentiation. When exposed, DCs present antigen molecules bound to MHC class I or class II binding peptides and ^{activate CD8 +} T cells or CD4 ⁺ T cells, respectively (Steinman, 1991, Annu. Rev. Immunol. 9: 271-296; Banchereu 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; Salulsta et al., 1999, J. Exp. Med. 189: 611-614; 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 Anti. 2: 257-272).

DCs are involved in the induction, coordination and regulation of adaptive immune responses, and also serve to integrate the communication between the natural and adaptive arms of the immune system's effectors. These characteristics make DC a strong candidate for immunotherapy. DC has a unique ability to identify the environment by macropinocytosis and receptor-mediated endocytosis (Gerner et al., 2008, J. Immunol. 181: 155-164; Stoitzner et al., 2008, Cancer Immunol. Immunother 57: 165 to 1673; Lanzeveccia A., 1996, Curr. Opin. Immunol. 8: 348 to 354; Delamarre et al., 2005, Science, 307 (5715): 163 to 1634).

DC also requires a maturation signal to enhance its antigen-presenting capacity. By providing additional maturation signals, such as TNF-α, CD40L or calcium signaling substances, DC is a surface molecule, eg, CD80 and CD86 (also known as a second signal molecule). Upregulation of expression (Czerniecki et al., 1997, J. Immunol. 159: 3823-3837; Bedrosian et al., 2000, J. Immunother. 23: 31-13-20; Mailliard et al., 2004, Cancer Res. 64. , 5934-5937; Brosart et al., 1998, Blood 92: 4238-4247; Jin et al., 2004, Hum. Immunol. 65: 93-103). Mixtures of cytokines containing TNF-α, IL-1β, IL-6 and prostaglandin E2 (PGE2) have been established to have the ability to mature DC (Jonuleit et al., 2000, Arch. Derm. Res. 292: pp. 325-332). DC can also be matured with a calcium ionophore prior to pulse addition of the antigen.

DCs are receptors that recognize pathogens, such as PKR and MDA-5 (Kalali et al., 2008, J. Immunol. 181: 2694-2704; Naralagatla et al., 2008, RNA Biol. 5 (3) :. In addition to (pages 140-144), it also contains a set of receptors known as Toll-like receptors (TLRs), which are also capable of sensing the risk of pathogen origin. .. Stimulation of these TLRs induces a series of activity changes in DC, leading to maturation and T cell signaling (Bowllart et al., 2008, Cancer Immunol. Immunother. 57 (11): 1589). Pp. 1597; Kaisho et al., 2003, Curr. Mol. Med. 3 (4): 373-385; Plendran et al., 2001, Science 293 (5528): 253 to 256; Napolitani et al., 2005, Nat. Immunol. 6 (8): pp. 769-776). DC can activate and prolong the action of various arms of the cell-mediated response, eg, natural killer γ-δ T cells and α-β T cells, and once activated, DC Retaining their immunization capacity (Steinman, 1991, Annu. Rev. Immunol. 9: 271-296; Banchereu 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 a method of inducing antigen presenting cells (APCs) using one or more peptides of the invention. In vitro, ex vivo or in vivo, induce APC by inducing dendritic cells from peripheral blood mononuclear cells and then contacting them with one or more peptides of the invention (activation). be able to. When the peptides of the invention are administered to mammals in need of them, APCs to which the peptides of the invention are immobilized are induced in the mammals. 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, administration ex vivo can include the step of collecting APCs from mammals and the step of contacting APCs with the peptides of the invention.

The invention also provides an APC that presents a complex formed between an HLA antigen and one or more peptides of the invention. APCs are obtained by contact with the peptides of the invention or nucleotides encoding such peptides, but are preferably derived from the subject to be treated and / prevented, either alone as a vaccine or the 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 mammals. By activating DC with the peptides of the invention or a combination of peptides of the invention so that DC activates anti-tumor immunity in mammals in need thereof, DC is induced to mature. 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 APC, eg, DC, in which the APC is activated by contacting the APC with a peptide of the invention or a combination of peptides of the invention, thereby. It is an APC that incorporates peptides.

In one embodiment, the invention has, among other things, novel APCs generated for the purpose of expanding and proliferating desired T cells, activating T cells, expanding and proliferating specific T cells, and for those purposes. In addition to the methods for using APCs, there are also numerous therapeutic uses related to the expansion and activation of APCs incorporating the peptides of the invention and T cells using the peptides. In some cases, OCT4 activated DCs can also be used to expand and proliferate peptide-specific T cells.

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

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

Diseases that can be treated or prevented by using the present invention include diseases caused by viruses, bacteria, yeasts, parasites, protozoa, cancer cells and the like. The pharmaceutical compositions of the present invention can be used as general immunopotentiators (compositions or systems that activate DC) and have their own utility in the treatment of diseases. Illustrative diseases that can be treated and / or prevented using the pharmaceutical compositions of the present invention include, but are not limited to, viral infections, such as HIV, influenza, herpes, viral hepatitis. , Epstein bar, polio, viral encephalitis, measles, blisters, papillomavirus, etc .; or infections caused by bacteria, such as pneumonia, tuberculosis, syphilis; or infections caused by parasites, such as malaria, Examples thereof include trypanosomatic disease, leash maniac disease, tricomonas disease, and amoeba disease.

Pretumor or hyperplastic conditions that can be treated or prevented using the pharmaceutical compositions of the invention (transfected DCs, expression vectors, expression constructs, etc.) of the invention, including, but not limited to, the large intestine. Pretumor or hyperplastic conditions such as polyps, Crohn's disease, ulcerative colitis, breast lesions and others.

Cancers that can be treated using the compositions of the invention include, but are not limited to, primary or metastatic melanomas, adenocarcinomas, squamous cell carcinomas, squamous cell carcinomas, thoracic adenomas, lymphomas, Memoroma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, gastrointestinal cancer, brain tumor, bladder cancer , Cervical cancer and the like.

Other hyperproliferative disorders 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, smooth myoma, adenoma, lipoma, vascular. Tumors, fibromas, vascular obstruction, re-stenosis, atherosclerosis, pretumorous lesions (eg, adenomatous hyperplasia and neoplasms within the prostatic epithelium), carcinoma in situ, oral folliculitis or psoriasis Can be mentioned.

Autoimmune disorders that can be treated using the compositions of the invention include, but are not limited to, AIDS, Addison's disease, adult respiratory urgency syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, dermatomyositis. , Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes, pulmonary emphysema, erythema nodosum, atrophic gastric inflammation, glomerulonephritis, gout, Graves' disease, hyperacidocytosis, hypersensitivity bowel syndrome, Erythematosus, polysclerosis, severe myasthenia, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, dermatomyositis, Sjogren's syndrome and autoimmune thyroiditis; cancer, hemodialysis And extracorporeal circulation complications; infections with viruses, bacteria, fungi, parasites, protozoa and rheumatoid arthritis; and trauma.

In the method of treatment, administration of the compositions of the invention may be either for "prevention" or for "treatment". The compositions of the present invention, when provided prophylactically, are provided prior to any symptom, but in certain embodiments, the vaccine is provided after one or more symptoms have developed. , Prevent further symptoms from developing, or prevent the emerging symptoms from getting worse. Prophylactic administration of the composition helps prevent or ameliorate any subsequent infection or disease. The pharmaceutical composition, if provided for treatment, is provided at or after the onset of symptoms of infection or disease. Accordingly, the present invention can be provided before exposure to a disease-causing substance is expected or a disease state occurs, or after an infection or disease has begun.

An effective amount of the composition is an amount that enhances the immune response and achieves these selected results, and such an amount could be determined by one of ordinary skill in the art as a matter of routine. For example, an effective amount for treating an immune system deficiency against cancer or pathogens would be the amount required to trigger activation of the immune system and generate an antigen-specific immune response upon exposure to the antigen. .. The term is also synonymous with "sufficient amount."

The effective amount for any particular application can vary depending on factors such as the disease or condition being treated, the particular composition 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 invention without the need for undue experimentation.

Vaccine Formulations The present 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 of a vaccine according to the invention for preventing and / or treating cancer to a patient, before or after a surgical procedure for removing the cancer, of chemotherapy for treating the cancer. It can be done before or after the procedure, before or after radiation therapy to treat the cancer, and in 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 who may be at risk of developing cancer.

Administration of a cancer vaccine prepared according to the present invention is widely applicable to the prevention or treatment of cancer, which is determined to some extent by the selection of antigen-forming moieties of the cancer vaccine. Cancers that can be appropriately treated according to the procedures of the present 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 can be derived from the tumor or cancer cell to be treated. For example, in the treatment of lung cancer, lung cancer cells will be treated as described above herein 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 to prevent and / or prevent tumors or cancer cells from which the vaccines are produced. It is used according to the procedure for treatment.

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

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

In addition, the vaccine can be administered with adjuvants and / or immunomodulators to boost vaccine activity and patient response. Such adjuvants and / or immunomodulators are understood by those of skill in the art and are clearly described in the available publications.

As defined herein, cells are cultured in a bioreactor or fermenter or other such tank or device suitable for growing large numbers of cells, if desired, depending on the type of vaccine produced. By doing so, vaccine production can be scaled up. In such a device, the culture medium is collected regularly, frequently or continuously in order to recover any material or antigen from the culture medium before such material or antigen is degraded in the culture medium. Will be done.

Devices or compositions containing vaccines or antigens that are produced and recovered in accordance with the present invention and are suitable for sustained or intermittent release are, if desired, actually implanted in the body, or It could be applied externally to the body to release such material into the body relatively slowly or in a fixed time.

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

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

These methods described herein are by no means inclusive and will be apparent to those skilled in the art as additional methods suitable for a particular application. Furthermore, the effective amount of the composition can be determined more accurately by analogy with a compound known to exert a desired effect.

<Experimental Example>
The present invention will be further described in detail with reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to limit the invention unless otherwise specified. Accordingly, the present invention should not be construed to be confined to the following examples under any circumstances, but rather to embrace any modification revealed as a result of the teachings provided herein. Should be.

Without further description, one of ordinary skill in the art will be able to carry out the methods of making, utilizing, and claiming the present invention using the above description and the following exemplary examples. Conceivable. Therefore, the following examples specifically point to preferred embodiments of the invention and should not be construed as limiting the rest of the disclosure under any circumstances.

Creation of Peptide Vaccines Against Breast Cancer and Other Receptor Tyrosine Kinases That Cause Breast Cancer Experiments were designed to develop alternative therapies for patients designated as carriers of BRCA mutations. That is, they are seeking an alternative to bilateral mastectomy and do not meet the needs of younger patients at genetic risk for breast cancer.

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

Experiments were designed to study the expression of receptor tyrosine kinases in breast cancer and DCIS from carriers of BRCA mutations. Tumors from carriers of BRCA mutations frequently overexpress the c-MET oncogene and HER-3 early, while tumors from non-mutated or sporadic patients are HER-2 and HER-. It was observed to express 3. This is important; because the targets for tumor immunotherapy that can be used to develop vaccines for carriers of sporadic and BRCA mutations are presented herein. This is because it has become known now. This is the first, distinctive feature and can be targeted to use the immune response for prophylaxis. Accordingly, the present invention includes compositions and methods for developing vaccines, as well as 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 cancers, as well as other cancers. For example, HER1 is expressed on a small number of breast cancers, which are generally tri-negative. c-MET is a growth factor receptor involved in the recurrence of many cancers, which activates HER3. HER3 is overexpressed in the colon, prostate, breast and melanoma. HER3 is expressed in numerous DCIS lesions and breast cancer. In some patients who receive the HER2 vaccine, HER3 may be detected in the residual DCIS during surgery. As a result of these findings, the possibility of targeting these molecules in addition to HER2 in breast cancer would be beneficial.

Immunogenic peptides derived from HER3 have been identified (FIGS. 1 and 2) and are shown below:
p11-13 (Peptides 51-75): KLYERCEVVMGNLEIVLTGHNADLSFLQW (SEQ ID NO: 1);
p81-83 (Peptides 401-425): SWPPHMHNFFSVFSNLTTIGGRSLYN (SEQ ID NO: 2);
p84-86 (Peptides 416-440): TTIGGRSLYNRGFSLLIMMKNLNVTS (SEQ ID NO: 3);
p12 (Peptides 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4);
p81 (Peptides 401-415): SWPPHMHNFFSVFSNL (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 show that these peptides are capable of activating CD4T cells across many patients. Peptides can be pulsed into dendritic cells to educate T cells to recognize HER3. HER3 is expressed in three types of negative breast cancer and may result in resistance to antiestrogens in ^{ER-positive breast cancer.} HER3 is also expressed in other cancers, including melanoma, lung cancer, colon cancer, prostate cancer and metastatic brain tumors. Although not intended to be bound by any particular theory, peptides derived from the intracellular portion of the molecule may also be advantageous.

Based on the disclosure presented herein, immunogenic peptides for the receptor tyrosine kinase molecules HER1 and c-MET are screened and identified based on the procedure for identifying immunogenic peptides for HER3. can do. The immunogenic peptides of the invention can be used to prepare multivalent prophylactic vaccines for breast cancer and other cancers.

The results presented herein show the identification of the role of the 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, in DCIS, specific anti-HER1, anti-HER2 and anti-HER3 responses in patients have been identified before and after vaccination, which leads to early prevention of cancer or DCIS. Support the development of multivalent vaccines that can be used to treat women with. The compositions of the present invention are useful for treating other cancers, including but not limited to colon cancers, melanomas, brain tumors, lung cancers, ovarian cancers 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 dendritic cells were engineered to exhibit a mutated protein (BRAF) that causes about 70% melanoma. .. When vaccinated with these dendritic cells, mice are protected from the challenge of using melanoma cells, indicating that vaccines for melanoma can be developed. Although not intended to be bound by any particular theory, the combined targeting of BRAF and HER3 is melanoma and, but not limited to, solid cancers such as colon cancer, pancreatic cancer and lung cancer and others. It may be useful in treating other cancers, including gastrointestinal tumors.

In addition, melanoma tumors have also been shown to use B cells to circumvent immune surveillance, and therefore it is believed that elimination of certain B cells can improve therapy. Experiments can be designed to assess whether altering the tumor's microenvironment for Th1 response can help prevent avoidance.

In some cases, the vaccines of the invention can also be used to treat melanoma that has become widespread. In some cases, the invention also provides therapy to eliminate residual cells, which often become resistant to drug therapy.

A novel strategy for identifying ^{MHC class II-indiscriminate CD4 +} peptides from tumor antigens for use in vaccination Cytotoxic CD8 + T lymphocytes (CTLs) have historically been the major anti-tumor immunity Though considered to be an effector, in various tumor types, simply boosting the CTL response with a CD8 + vaccine has unpredictable clinical consequences, which is probably what CTL is appropriate for. Without the help of good CD4 + T lymphocytes, it would only work below optimal. CD4 + T helper type 1 (Th1) cells secrete INF-γ / TNF-α to induce tumor aging and apoptosis. However, good incorporation of CD4 + epitopes in constructing cancer vaccines to generate durable, antigen-specific CD4 + immunity remains a challenge. An immunogenic HER3 CD4 + that exhibits class II indiscriminateness and produces anti-HER3 CD4 + immunity with the purpose of using the extracellular domain (ECD) of HER3 as a candidate "carcinogenic driver" tumor antigen for inclusion in the vaccine construct. Experiments were performed to identify the peptides.

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

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

Protocol Overview A library of 15-mer long peptides was created from HER3 ECD that overlaps only 5 amino acids. These peptides were pulsed onto mononuclear cell-derived DCs obtained from donors to mature into a type 1 biased (DC1; secreting IL-12) phenotype. DC1 was collected from subjects who belonged to our DCIS vaccine study and were known to have an anti-HER3 Th1 response and were co-cultured with purified CD4 + T cells. Using a large pool of 10 peptides, the identification process was progressively narrowed down and measured by the secretion of interferon gamma (IFN-γ) in CD4 + T cells to give a single reactive epitope. Screening of 5-6 subjects, 4 peptides that appear to react across most donors: HER3 _56-70 (SEQ ID NO: 4), HER3 _401-415 (SEQ ID NO: 5), HER3 _416-430 (SEQ ID NO: 6) and HER3 _451-465 (SEQ ID NO: 7) were identified. Subjects showing no evidence of responsiveness to CD4 + T cell recognition in the extracellular domain of HER3 were identified, four HER3 peptides were pulsed into DC1 of those subjects, and the pulsed DC1 was cultured with CD4T cells for 1 week. Then, the reactivity to the HER2 peptide and the reaction to the extracellular HER3 protein were tested. In each case, at least one peptide resulted in recognition of both the peptide pulsed onto the mononuclear cells and the total protein of HER3, suggesting that primary sensitization occurred ex vivo. It was also shown that healthy donors were able to respond to these peptides and that the anti-HER3 Th1 response was lost in patients with triple-negative breast cancer. In addition, Gala, K.K. Et al., Clin. Cancer Res 2014; 20: 141-10416, and Datta, J. et al. Et al., "Progressive Loss of Anti-HER2 CD4 + T-helper Type 1 Response in Breast Tumorigenesis and the Potential for Immune Restoration", OncoImmune.

Key points of the protocol further shown in FIG. 19:
A library containing 123 peptide fragments 15 amino acids long, overlapping only 5 amino acids, was generated from the extracellular domain (ECD) of HER3.
-Rapidly transform autologous mononuclear cell-derived dendritic cells (DCs) obtained from donors into a type 1 biased (secreting DC1 → IL-12) phenotype by GM-CSF, IFN-γ and LPS. It was matured and the relevant peptides (eg, HER3 ECD or HER3 CD4 + peptide, as specified) were pulsed. DC1 deflects the Th1 response by the production of IL-12.
-The collected DC1 was co-cultured with purified CD4 + T cells for 8 to 10 days for allosensitization.
-Sensitized CD4 + T cells (most of them antigens) to immature DCs (iDCs) pulsed with a specific CD4 + peptide of interest (eg, a cluster of HER3 library peptides) or an unrelated Class II peptide control. (Expected to be specific) was reactivated.
-The supernatant was then collected from these co-cultures. The Th1 response measured by Elisa for IFN-γ was considered antigen-specific if the production of IFN-γ was at least twice that of an irrelevant control.
-HLA-DR, DP, DQ typing on donors was performed by the Hospital of the University of Pennsylvania's Clinical Immunology Laboratory to assess MHC class II indiscriminateness of the CD4 ^{+ Th1 response.}

A library containing 123 overlapping 15 amino acid long peptide fragments was generated from HER3-ECD. The autologous monocyte-derived DC obtained from the donor was matured to DC1 and HER3-ECD was pulse-added. The collected DC1 was co-cultured with purified CD4T cells. After 10 days, sensitized CD4T cells were reactivated to immature DC (iDC) pulsed with clusters of HER3 library peptides or unrelated CD4 control peptide 1. The Th1 response measured by Elisa for IFN-γ was considered antigen-specific if the production of IFN-γ was at least twice that of an irrelevant control.

1) To identify immunogenic CD4 + peptides, we obtained breast cancer patients who were found to have shown anti-HER3 ECD reactivity after a pulsed HER2 DC1 vaccine; 2) immunogenicity of these peptides. Was confirmed by the process of "reverse" sensitization in the same patient; 3) Anti-HER3 ECD non-reactivity was obtained after vaccination and used to identify the CD4 + peptide. Experiments were performed in a three-step process of observing whether cells were sensitized to native HER3 ECD and therefore tolerance to self-antigens (ie, HER3) was overcome / abolished.

The results of the experiment are described here.

Continuous screening of the 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, start with a cluster of 10 peptides (1-10, 11-20, ..., etc.) and a cluster of 3 peptides (1, 3, 3-6, 7-10, ...). · Etc.), and finally, a single immunogenic HER3 peptide was narrowed down and HER3 ECD sensitized CD4 + Th1 was continuously reactivated against them. 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 indiscriminate across the HLA-DR, DP and DQ subtypes. Th1 cells from 4 non-HER3 reactive donors were sensitized using DC1 pulsed with 4 identified HER3 peptides, followed by these cells being pulsed with HER3 ECD iDC. When challenged to recognize, all donors not only showed good sensitization to individual immunogenic HER3 peptides, but also recognized native HER3-ECD.

The results presented herein show that pulsed DC1 with a library of overlapping tumor antigen-derived peptides can identify indiscriminate 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 autologous tumor antigens. Vaccines that utilize these HER3 CD4 peptides in their construction can be applied to patients with cancers that overexpress HER3. In addition, these results rapidly and reproducibly identify Class II indiscriminate immunogenic CD4 epitopes from any tumor antigen for cancer immunotherapy using the DC1-Th1 platform. New strategies for this are also presented. Table 1 below shows the initial identification of immunogenic CD4 + HER3 ECD peptides in patients known to have anti-HER3 reactivity. Table 2 shows the amino acid sequences of the four immunogenic HER3 CD4 + epitopes identified by continuous screening.

Confirmation of the immunogenicity of identified CD4 + HER3 ECD epitopes by "reverse" sensitization, i.e., the ability of individual epitope-sensitized CD4 + Th1 to recognize native HER3 ECD . FIG. CD4 + T cells are sensitized to each donor-specific, immunogenic HER3 epitope pulsed DC1 and reactivated to each HER3 epitope and native HER3 ECD pulsed iDC. Indicates that.

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

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

CD4 + HER3 epitopes show MHC class II indiscrimination As seen in FIG. 17, experiments were performed using the extracellular domain (ECD) of HER3 as a candidate "carcinogenic driver" tumor antigen and no class II. Immunogenic HER3 CD4 + peptides that show differentiation and generate anti-HER3 CD4 + immunity have been identified; these peptides can be used in vaccine constructs.

Peptides Derived from Tumor Antigens The results presented herein are:
-To develop a CD4 + T cell vaccine, indiscriminate MHC class II peptides can be identified by DC1 pulsed with a library of overlapping peptides obtained from tumor antigens.
-Immunogenic HER3 CD4 + peptide effectively overcomes immune tolerance to autologous tumor antigens,
-From these results, to rapidly and reproducibly identify Class II indiscriminate immunogenic CD4 + epitopes obtained from any tumor antigen for cancer immunotherapy using the DC1-CD4 + Th1 platform. New strategy is shown,
-Vaccines constructed using these HER3 CD4 + peptides have shown to be worth studying in patients with cancers that overexpress HER3.

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

The presence of metaplastic columnar epithelium in the Barrett's esophagus, or distal esophagus, predisposes to the development of esophageal adenocarcinoma (Cameron, AJ et al., Gastroenterology 109 (5): 1541-6 (1995)). Although histological metastases from dysplasia to invasive malignancies are well characterized, carcinogenesis in metaplastic cells is associated with incompletely understood genetic alterations. Several recent reports have identified human epidermal growth factor receptor 2 (HER2) expression in a subset of Barrett's esophageal lesions with dysplasia. (Almhana, K. et al., Appl. Immunohistochem. Mol. Morphor. 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 (9B): 3826-33 (2009) (Rossi et al.). In addition, the rate of HER2 expression correlates with the degree of dysplasia, suggesting related pathways in tumorigenesis.

Overexpression of RTK molecules, including members of the mesenchymal epithelial transitional factors HER family (HER1, HER2, and HER3) and cMET, has been identified in many of the more common malignancies, including breast, lung and gastrointestinal cancers. (Yokata, J. et al., Lancet 1: 765-767 (1986) and also confirmed in esophagogastric cancer. Identification of HER2 overexpression in a subset of breast cancer, association of HER2 overexpression with more aggressive ecology , And the effective targeting of HER2 with monoclonal antibodies was a central event in the evolution of targeted therapies for the treatment of solid tumors (Joensu, H. et al., N. Eng. J. Med. 354). (8): 809-20 (2006)). This experience provides the basis for further efforts to target RTK molecules in the treatment of other malignancies.

Overexpression of HER2 has been confirmed in a small number of gastric cancers and has been targeted by trastuzumab in a metastatic environment with little outcome effect (Bang, YJ et al., Lancet 376 (9742) 687). -97 (2010) (Bang et al.)). Overexpression of HER2 is more frequently expressed at the proximal gastric and gastroesophageal junctions as compared to more distal gastric adenocarcinomas (Rajagopal, I. et al., J. Clin. Diagn. Res. 9 (3): EC06-10 (2015)), with little impact on outcomes and targeted by trastuzumab (Bang et al. And Fitcher, CD 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 has been associated with a poor prognosis of esophageal adenocarcinoma, and inhibition of cMET-dependent signaling regulates the activity of HER1 and HER3 (Liu, X. et al., Clin. Cancer Res. 17 (22): 7127-38 (2011)). These data provide evidence for further efforts to characterize RTK expression in esophagogastric cancer and prodromal lesions. The studies described below aim to characterize RTK expression in dysplastic lesions of the gastroesophageal junction to identify potential targets for treatment and primary prevention.

METHODS: After approval by the University of Pennsylvania Institute Review Committee, clinical records and clinical records of 73 patients with Barrett's esophagus with dysplasia (low dysplasia (LGD), n = 32, severe dysplasia (HGD), n = 59) Histological specimens were retrospectively reviewed. Formalin-fixed paraffin-embedded tissue blocks from preserved endoscopic biopsy and mucosal resection specimens from 2003-2012 were sectioned at 5 μm on a plus slide (Fisher Scientific, Waltham, MA), followed by deparaffinization. Was rehydrated. All biopsy materials are HER1 (clone H11; 1:50; DAKO), HER2 (Hercept, DAKO, Carpinteria, CA) and HER3 (clone RTJ.2; 1:30; Santa Cruz Biotechnology, Dallas, TX) -III device) was immunostained and evaluated under a microscope (Leica Bond-III) by a single pathologist. The 3+ HER2 stained membrane was considered positive as well as the 2+ HER2-stained membrane in which more than 10% of the tumor cells were stained as seen in FIG. CMET immunohistochemical staining was performed in 42 cases in which sufficient tissue was available. More than 50% of tumor cells were considered positive for moderate or scleral staining. RTK overexpression was correlated with clinical data from either a dysplasia-adenocarcinoma biopsy specimen pair or adenocarcinoma diagnosis in a subsequent biopsy specimen to assess its association with invasive cancer.

Statistical analysis Two-sided tests were performed on all analyzes. Descriptive statistics are shown as the frequency of categorical variables and median (interquartile range (IQR)) of continuous variables. Categorical and continuous variables were analyzed using Pearson's chi-square test or Fisher's exact test and Wilcoxon rank sum test, respectively. A P-value of ≤0.05 was considered to be statistically significant. All tests were bilateral. The analysis was performed using SPSS v22.0 (IBM, Armonk, NY).

Results A total of 73 Barrett's esophageal patients with low-grade dysplasia (n = 32) or high-grade dysplasia (n = 59) were identified and analyzed for HER1, HER2, HER3 and cMET expression by immunohistochemistry. The median age of the cohort was 65 years (IQR 60-73 years). 81.9% were male and 87.5% were white. Alcohol usage in the cohort was 14.3% and active tobacco usage was 6.3%, whereas 55.6% were smokers. 26.4% had a family history of malignant tumors. 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 dysplasia (HGD) was overexpressed by HER1 (22.4% vs 3.1%, p = 0.016), HER2 (5.3% vs 0) compared to low dysplasia (LGD). It was associated with overexpression of 0.0%, p = 0.187) and overexpression of HER33 (45.6% vs. 9.4%, p <0.001).

Invasive esophageal adenocarcinoma lesions were associated with dysplastic lesions in 6 cases, all of which occurred in association with HGD (HGD: 10.2% vs. LGD: 0.0%, p <0. 001). An additional 9 patients were diagnosed with invasive esophageal adenocarcinoma on subsequent biopsies (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 lesions of invasive cancer (71.4% vs. 38.6%). p = 0.032), not significantly associated with HER1 or HER2 (HER1 increase: 26.7% vs. 20.5%, p = 0.616, HER2 increase: 14.3% vs. 2. 3%, p = 0.077).

Overexpression of cMET was observed in 18 of 42 evaluation specimens (42.9%) and more in HGD specimens compared to LGD specimens (58.3% vs. 36.7%, p = 0.200), and in most cases co-expressed with HER3 (62.5% of HER3-positive specimens vs. 38.2% of HER3-negative specimens (p = 0.212)). No similar tendency was observed in HER1-positive (p = 0.729) or HER2-positive (p = NA) samples. Invasive cancer was confirmed in 1 of 42 patients (5.6%). CMET was overexpressed in this patient (p = 0.243).

Discussion This analysis of RTK expression in dysplastic lesions of the gastroesophageal junction includes (1) that HER family proteins are upregulated in the dysplastic Barrett's esophagus, and (2) the frequency of HER family and cMET overexpression. (3) Confirm that upregulation of the HER protein is associated with an increased incidence of associated invasive cancers, especially in dysplastic lesions.

Therefore, HER3 may serve as a biomarker for potential invasive diseases in Barrett's esophagus and HGD patients. In addition, therapeutic agents targeting HER3 or CMET can result in secondary prevention of gastroesophageal cancer in a subset of patients.

Previous assessments of HER expression in Barrett's esophagus have been limited to assessments of HER2 overexpressing in a small number of 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 in which HER3 is more commonly overexpressed than HER2 (Fichter et al.). Increased overexpression of the HER3 protein with the progression from LGD to HGD, and in particular the frequent overexpression of HER3, is a new finding, not surprisingly. Homodimerization and heterodimerization of the HER receptor drive signal activation. Clustered overexpression of multiple members of the HER family has been observed in other tumor types. In addition, activated c-MET positively regulates the activity of HER1 and HER3 ¹¹ . In fact, the interaction between these receptors provides the rationale for multivalent therapies targeting 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))).

The data also suggest an opportunity for targeted secondary prevention of gastroesophageal cancer that has not yet been investigated. Previously targeted HER2 expression in ductal carcinoma in situ (DCIS) of the breast targeted promising results (US Patent Application Publication No. 2015/0323547; filed December 30, 2019. US Patent Application No. 14 / 985,303, Datta, J. et al., Onco Immunology 4: 8 e1022301 (2015) DOI: 10. 1080 / 2162402X. 2015.1022301; (1): See 71 (2015)). Such an approach remains a farther goal for gastrointestinal malignancies. Nonetheless, all current treatment options for Barrett's esophagus, including endoscopic resection and resection modalities and definitive surgery, have serious limitations. Alternative strategies are needed to eliminate the pathological condition and reduce the risk of invasive cancer. This analysis of RTK expression in dysplastic lesions of the gastroesophageal junction includes (1) that HER family proteins are upregulated in the dysplastic Barrett's esophagus, and (2) the frequency of HER family and cMET overexpression. It is confirmed that there is a positive correlation with the degree of dysplasia; (3) upregulation of the HER protein is associated with an increased incidence of associated invasive cancers, especially in dysplastic lesions.

Therefore, HER3 may serve as a biomarker for potential invasive diseases in Barrett's esophagus and HGD patients. In addition, therapeutic agents targeting HER3 or CMET can result in secondary prevention of gastroesophageal cancer in a subset of patients.

In summary, this data shows the relationship between frequent overexpression of HER3 and malignant transformation in highly dysplastic lesions at the gastroesophageal junction, especially with highly dysplastic lesions with latent invasive cancer. These findings may justify a more aggressive management approach for HER3-expressing dysplastic lesions, and as will be readily appreciated by those skilled in the art, future application of HER3-targeted therapies in early disease setting. Provide the basis for.

We have previously shown that native anti-HER-2 CD4 Th1 is progressively lost during the development of ^{HER-2 positive breast tumors.} This loss of response was associated with a lack of complete pathological response (“pCR”) to neoadjuvant therapy, correlated with an increased risk of breast cancer recurrence, and could be recovered by vaccination. Example 4 below examines whether there is a similar loss of anti-HER3 Th1 response during chest tumor development.

Loss of anti-HER3 CD4 Th1 occurs in chest tumorigenesis and is negatively associated with outcomes
Summary of Overall Example 4 We have previously shown that native anti-HER2 CD4 Th1 is progressively lost during the development of ^{HER2-positive breast tumors.} This loss of response was associated with a lack of complete pathological response (“pCR”) to neoadjuvant therapy, correlated with an increased risk of breast cancer recurrence, and could be recovered by vaccination. This example examines whether there is a similar loss of anti-HER3 Th1 response during breast tumor development.

Peripheral blood from 131 subjects, including healthy donors (“HD”), benign breast disease (“BD”), ductal carcinoma in situ (“DCIS”) and patients with invasive breast cancer (“IBC”) Collected. The immune response to the four different HER3 immunogenic peptides identified in Examples 1 and 2 above was tested via an enzyme-bound immunoadsorption (ELISpot) assay and all metrics of the immune response were analyzed.

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

Thus, CD4 Th1 anti-HER3 was found to be lost during thoracic tumor development, especially in TN IBC, a group with limited treatment options and overexpressing HER3 with a significantly poor prognosis. It was found that there is. These findings influence attempts to restore this response to prevent recurrence.

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

The immune system plays an important role in the regulation of HER2-expressing tumors. It has been previously shown that the native anti-HER2 CD4 T cell response gradually diminishes from healthy subjects towards HER2- ^positive DCIS, HER2- ^{positive IBC, but not in HER2-negative IBC.} In addition, a lower anti-HER2 immune response correlates with subsequent breast cancer recurrence, while a higher anti-HER2 immune response correlates with a complete pathological response to neoadjuvant chemotherapy and the role of the immune system in ^{HER2-positive tumorigenesis.} (Datta, J. et al., Onco Immuneology 4 (10): e1027474.DOI: 10.1080 / 2162402X.2015. 1022301 (2015) and US Patent Application Publication No. 2015/0323547A1 (collectively below). 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 US Publication No. 2015/0323547A1).

HER2 is a member of the EGFR family, which is an RTK group that also includes HER1 and HER3. Although it is well known that HER2 dimers itself, the role of HER3 in signal transduction is less clear and may act to dimerize both itself and HER2. Dimerization of HER3 with HER2 has been proposed as an escape mechanism in breast cancer patients treated with trastuzumab. Czopek, J. et al. 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 has recently been added to the market and is the first tumor treatment to receive early FDA approval for neoadjuvant therapy, inhibiting HER2 / HER3 dimerization and overall when used in combination with trastuzumab for breast cancer patients. (Javeri, 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).

There is overexpression found in some ER-positive, HER2-positive and triple-negative (“TN”) subtypes, but HER3 expression is less clearly delineated between breast cancer subtypes. Moder, C.I. Et al., Cancer 115 (11): 2400-9 (2009). Although HER3 overexpression ^{can be more common in HER2-positive} IBC, it is interesting that its prognostic value is more pronounced in TN IBC. HER3 expression in ER- ^positive / HER2- ^positive IBC did not affect disease-free survival (“DFS”) or overall survival (“OS”), whereas HER3 expression in TN IBC was 5 years DFS and 10 years OS. It was correlated with the deterioration of. Bae et al. And Czopek et al. Notably, a significant subset of TN IBC patients with HER3 overexpression (a group that, by definition, has no classical treatment options) can benefit from new recognizable targets. It is unclear whether an anti-HER3 CD4 Th1 response is present in healthy donors and whether this response changes during breast tumor development. This study seeks answers to these questions.

Method
Subject Enrollment A total of 131 subjects met the test criteria and received informed consent and were subsequently enrolled at the University of Pennsylvania. This study was approved by the University of Pennsylvania's Instrumental Review Board and the Abramson Cancer Center prior to subject enrollment. Of the 131 subjects, 9 lacked peripheral blood monocytes for the assay, resulting in 122 subjects for reviewing 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 The CD4 T cell responses to four different HER3 immunogenic peptides to the HER2 polypeptide were compared between triple negative IBCs (TN IBC, n = 27).

Collection of peripheral blood monocytes Peripheral blood was collected by venipuncture. Blood was diluted 1: 1 with Hanks buffer or PBS and lymphocyte isolation medium was overlaid under diluted blood in a conical tube. Blood was then separated by density centrifugation at 1200 rpm for 30 minutes. Mononuclear cell layers were collected and washed twice with Hanks buffer or PBS. Cells were counted, resuspended at 10 million cells per milliliter and frozen at −80 ° C. for 24-48 hours before transfer to −200 ° C. Cells were stored until experimental assay.

Measurement of Anti-HER3 CD4 Th1 Responses Anti-HER3 CD4 Th1 cell responses were measured by the 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 washed again with PBS and blocked with Iscover medium containing 10% human serum for 1 hour. Peripheral blood monocytes were thawed at 37 ° C., washed with PBS or Hanks buffer, resuspended at 1 million cells per milliliter, and four immunogenic HER3 peptides (p12 (peptide 56-70): CEVVMGNLEIVLTGH (sequence)). No. 4); p81 (Peptide 401-415): SWPPHMHNFFSVFSNL (SEQ ID NO: 5); p84 (Peptide 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6); and p91 (Peptide 451-465): AGRIYISANRQLCYH (SEQ ID NO: 7) Seeded at 200,000 cells / well with one of them, anti-CD3 / CD28 (polyclonal stimulation, positive control), tetanus Cruz Biotechnology, Dallas, TX, or blank (non-stimulation control). The plate was incubated at 38 ° C. for 48 hours. After 48 hours, the plate was washed with PBS, biotinylated anti-IFN-γ antibody was added, and the plate was incubated at 38 ° C. for 2 hours. The plate was incubated with PBS. Wash again with Streptavidin-HRP, incubate the plate at 38 ° C. for 1 hour, and finally wash the plate with PBS and then add TMB substrate solution. Color development is thoroughly washed with tap water. This allowed it to stop after 5 minutes. The plates were dried at 4 ° C. overnight.

Immune response analysis spots were counted by immunospot software. (1) Cumulative response, i.e. total response to all four HER3 immunogenic peptides per million spots, (2) repertoire, i.e. number of peptides per subject with 20 or more spots, (3) ) Three parameters (evaluation index) of responsiveness, that is, the proportion (%) of the target responding to at least one peptide (defining 20 or more spots as a threshold value) were quantified. The tetanus response with spots per 200,000 cells and the anti-CD3 / CD28 response with spots per 200,000 cells were also quantified as controls. All immune response metrics were analyzed with GraphPad Prism software.

result
Characteristics of the study A total of 131 subjects met the study criteria and received informed consent at a hospital at the University of Pennsylvania and were enrolled in succession. Nine subjects had insufficient cells for analysis, bringing the total number of subjects to 122. Of these, the average age was 50 years, between 25 and 83 years. Whites accounted for 72.1%, African Americans 18.0%, and other races 9.8%. Subjects were HD (n = 30), BD (n = 11), DCIS (n = 13), HER2- ^positive IBC (n = 21), ER- ^positive IBC (n = 20), TN IBC (n = 27). It belongs to one of the six groups. Of the 68 IBC subjects, 35 (51.5%) were stage I, 22 (32.4%) were stage II, 9 (13.2%) were stage IV, and 2 (13.2%) were stage IV. 2.9%). There were 52 (76.5%) patients who received chemotherapy and / or Herceptin and / or tamoxifen treatment, and 16 (23.5%) who had never been treated. Three DCIS patients and three HER2- ^positive IBC patients received HER2-pulse dendritic cell vaccination. Other properties are reported in Table 4 below.

Comparing HD, BD, DCIS, HER2- ^positive IBC, ER- ^positive IBC, and TN IBC with reduced anti-HER3 immune response of CD4 Th1 cells from healthy donors to a subset of invasive breast cancer, all three immune parameters Decreased and reached the lowest point in TN IBC: cumulative response (90: 80: 66: 79: 48: 40, p = 0.01, respectively, as shown in FIG. 24A), repertoire (shown in FIG. 24B, respectively). As such, 1.0 vs. 0.6 vs. 0.8 vs. 0.8 vs. 0.5 vs. 0.3, p = 0.003, respectively) and responsiveness (as shown in FIG. 24C, respectively, 76. 7% vs. 63.6% vs. 53.8% vs. 66.7% vs. 45.0% vs. 33.3%, p = 0.002). In particular, these differences were not only statistically significantly higher in HD but more than doubled in HD compared to TN IBC patients across all three immune parameters: cumulative response (90:40, p = 0). .002), repertoire (1.0 vs 0.3, p = 0.004), responsiveness (76.7% vs 33.3%, p = 0.001). Compared to TN IBC patients, BD had a significantly higher cumulative response (40:80, p = 0.007, respectively) and DCI patients showed a significantly higher repertoire (0.8: 0.3, respectively). , P = 0.04), HER2- ^positive IBC has a significantly higher repertoire (0.3 vs 0.8, p = 0.01) and responsiveness (33.3% vs 66.7%, respectively, p = 0. 04) was shown. Patients with ER- ^positive IBC had the second lowest anti-HER3 CD4 T cell response and showed a statistically significantly lower response compared to HD across all three immune parameters: cumulative response (48:90, respectively). p = 0.03), repertoire (0.5 vs 1.0, p = 0.008) and responsiveness (45.0% vs 76.7%, p = 0.03, respectively). Of note is the fact that the HER2- ^positive IBC anti-HER3 response 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 was analyzed for tetanus response and polyclonal stimulation with anti-CD3 / CD28 response , which is specific to HER3 and does not attribute to a broad deficiency of immune response. The overall immune responsiveness was compared and controlled. There was no difference in the anti-tetanus response of CD4 Th1 cells measured at spots per 200,000 cells via the ELISpot assay between HD, BD, DCIS, HER2- ^positive IBC, ER- ^{positive IBC or TN IBC patients (FIG. 25).} 37:30:19:34:24:29, p = 0.65, respectively, as shown in. Importantly, the anti-tetanus response between HD and TN IBC patients (the group with the most divergent anti-HER3 CD4 Th1 cell response) was similar (37:29, p = 0.37). Similarly, there was no difference in polyclonal stimulation with anti-CD3 / CD28, and the majority of subjects in each group had incredible spot development, which was innumerable. Among the calculable patients, HD, BD, DCIS, HER2- ^positive IBC, ER- ^positive IBC or TN IBC patients (688 vs. 549 vs. 804 vs. 699 vs. 629 vs. 675, respectively, p = 0. There was no statistically significant difference between 68).

Anti-HER3 CD4 Th1 Cell Response in Patients with Invasive Breast Cancer Correlates with Prognosis and Characteristics of Tumor Invasion To determine if the anti-HER3 CD4 T-cell response correlates with the characteristics of tumor invasion, the lymph nodes of the first surgery Status (lymph node positive (“LN ^positive ”) vs. lymph node negative (“LN ^negative ”), recurrence vs. non-recurrence in patients at least 1 year after diagnosis, and response to neoadjuvant chemotherapy (pathological completeness) The immune response of IBC patients was compared by response (“pCR”)) vs. disease persistence (“<pCR”) ^{. LN-positive} IBC patients (n = 28) ^{compared with LN-negative} patients (n = 31). Immune response was generally low across the three parameters, but none were statistically significant: cumulative response (40:56, p = 0.12, respectively), repertoire (as shown in FIG. 26A). respectively 0.4 vs. 0.6, p = 0.08) and responsive (respectively 35.7% vs. 54.8%, p = 0.19). Remarkably, the ^LN-positive subject, Subjects with lymph node metastasis after neoadjuvant chemotherapy were included. LN- ^negative subjects after neoadjuvant chemotherapy were excluded from the analysis as it was unclear whether they had positive lymph nodes before treatment. Among patients who have been diagnosed for at least 1 year, recurrent breast cancer (local or distant metastasis, n = 7) is significant in all three immune parameters compared to disease-free patients (n = 36). Showed low anti-HER3 response: cumulative response (17 vs. 66, p = 0.04, respectively), repertoire (0.0 vs. 0.6, p <0.05, respectively) and response, as shown in FIG. 26B. Gender (0% vs. 55.6%, p = 0.01, respectively). Finally, as shown in FIG. 26C, pCR (n = 11) of the patients (n = 16) who received neoadjuvant chemotherapy. The pCR (n = 5) had a significantly higher cumulative response (144 vs. 32, p = 0.004, respectively) and repertoire (0.8 vs. 0.4, p = 0.05, respectively). There was no statistically significant difference between pCR and <pCR (80.0% vs. 27.3%, p = 0.10, respectively). LN- ^positive and LN- ^negative patients There was no difference in rupture response between relapsed vs. non-recurrence patients and pCR vs. <pCR: rupture response (22:29, p = 0.35, respectively), recurrence vs. non-recurrence patients (27:35, respectively). p = 0.65) and pCR pair < pCR (17:59, p = 0.15, respectively). Therefore, the reduced CD4 Th1 cell response in IBC patients with more aggressive tumor features is HER3 specific.

Anti-HER3 CD4 Th1 cell response age (under 50 years (n = 25) or over 50 years (n = 16)), race (white (n = 29), African American (n =)) due to characteristics of healthy donors 12)), in HD and BD, depending on the pregnancy status (0 (n = 17) or 1 or more pregnancies (n = 24)) and the menopausal status (premenopausal (n = 30) or postmenopausal (n = 11)). Anti-HER3 CD4 Th1 reactions were compared. 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, postmenopausal women showed a significantly higher cumulative response and repertoire compared to premenopausal women, as shown in Figure 27D: cumulative response (136 spots vs. 70 spots per million cells, respectively). p = 0.005) (upper panel), repertoire (1.4 to 0.8 peptides, p = 0.03, respectively) (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 the tetanus response between pre- and post-menopausal women (lower panel), indicating that the difference in immune response depending on the menopausal state is HER3 specific.

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

Discussion Knowledge of the role of the immune system in the development, progression and prognosis of cancer is expanding rapidly. It is well established that immunodeficiency conditions increase the risk of developing cancer not only from tumors of viral origin, but also from tumors of nonviral origin. Boschoff, C.I. Et al., Nature Rev. Cancer 2: 373-82 (2002). Sheil, A.M. G. , World J. Surg. 10: 389-96 (1986); Penn, I. et al. , Transplantation 61: 274-78 (1996); and Penn, I. et al. , Transplantation 60: 1485-91 (1995). It is also known that certain immune phenotypes are associated with breast cancer. Circulatory inflammatory cytokines TNF-α and IL-6 are higher in breast cancer patients, low CD4- ^positive / CD8- ^positive T cell ratios are associated with a more aggressive breast cancer phenotype, but tumor-infiltrating lymphocytes are associated with more aggressive breast cancer phenotypes in some breast cancers. Associated with a better prognosis. Alocail, M. et al. S. Et al., Med. Oncol. 31 (8): 38 (2014) doi: 10.1007 / s12032-014-0038-0; Jai, Y. et al. 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 loss of immune recognition in immune response hosts to specific molecular carcinogenic drivers is relatively new. A reduction in the native anti-HER2 CD4 Th1 cell response from healthy donors to HER2- ^positive DCIS, HER2- ^positive IBC has only recently been shown to eliminate the loss of immune response to this specific carcinogenic driver in chest tumorigenesis. This is one of the first studies to show. Those skilled in the art will readily appreciate that identifying and understanding such specific losses is likely to lead to specific immune targeting therapies.

This study found that (1) ^{there was a decrease in anti-HER3 CD4 Th1 cell response from HD to ER positive} and TN IBC, and (2) anti-HER3 response was correlated with prognosis, specifically higher. Responses are associated with pCR to neoadjuvant therapy, but lower responses are associated with recurrence, (3) postmenopausal HD indicates a significantly higher anti-HER3 immune response. All of these findings will have diagnostic and clinical uses for anti-HET3 CD4 Th1 cell responses.

The anti-HER3 CD4 Th1 cell response was highest in HD and lowest in TN IBC, where the prognosis was more severely affected by HER3 overexpression than other types of IBC. Bae et al. And Czopek, J. et al. Et al. Although HER3 expression is unknown in the cohort of IBC patients in this study, the TN IBC and ER- ^positive IBC groups appear to have higher levels of HER3 expression ^{compared to the HER2-positive IBC cohort, which responded similarly to HD.} Is done. In fact, our previous studies showed that the anti-HER2 CD4 Th1 cell response was directly correlated with HER2 expression, with a significant reduction in ^{HER2-positive IBC but not in HER2-negative} IBC. Not only can HER3 have a greater effect on the prognosis of TN IBC compared to receptor-expressing breast cancer, but even in TN IBC it can have a greater effect on the prognosis of HER2 (0) compared to HER2 (1+) tumors. There is sex. Schmidt, G.M. Et al., Arch. Gynecol. Obstet. 290: 1221-29 (2014). This can partially explain the similarities in the immune response between ^{HER2-positive IBC and HD.} If the tumor is already infiltrated due to overexpression of HER2, tumor progression cannot circumvent immune surveillance. However, if the immune system has already recognized and targeted HER2, the tumor can be adapted by HER3 overexpression, where antigenic escape has an evolutionary advantage for tumor cell survival.

Interestingly, ER- ^positive IBC showed an anti-HER3 CD4 T cell response similar to TN IBC, significantly lower than ^{HD or HER2-positive IBC. Although HER3 expression has a less significant prognosis in ER-positive} IBC compared to TNF-IBC, there is evidence that HER3 mRNA expression is positively correlated with ER expression. Fujiwara, S.A. Et al., Breast Cancer 21: 472-81 (2014), which can explain the lower immune response seen in this subgroup of IBC.

Breast cancer patients with recurrent disease have lower anti-HER3 CD4 Th1 responses compared to those who remain disease-free, indicating that immune surveillance may be an important mechanism for successful long-term treatment. ing. Recurrent patients can more likely to have a tumor with high expression of HER3, this itself, recurrence, metastasis, and overall survival is poor risk correlates with high. Li, Q. Etc., 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. Volume 105 (No. 4): pp. 266-73 (2013). In addition, immune editing has been proposed as an escape mechanism by which new cells are selectively eliminated by the immune system until they have evolved to express molecular carcinogenic drivers such as HER3 that evade immune recognition. .. Dunn, G.M. P. Et al., Nature Immunology 3 (11): 991-8 (2008). Therefore, expression of HER3 is possible due to the lack of immune surveillance, which increases the risk of recurrence. Interestingly, recent evidence suggests that recurrent tumors may not be pathologically identical to the primary tumor. The rate of disagreement between primary and secondary tumors is particularly high for progesterone receptors, and any type of disagreement indicates a propensity for a poor prognosis of recurrent tumors. Idirisinghe, P.M. K. A. Et al., Am. J. Clin. Pathol. 133: 416-29 (2010); Bloom, R. et al. J. Et al., Anticancer Research 29: 1557-62 (2009); and Liedtke, C.I. Et al., Anals of Oncology 20: 1953-58 (2009). To date, no studies have compared HER3 expression between primary and secondary tumors. It is unclear whether HER3 expression is inconsistent between primary and recurrent tumors and whether it represents a mechanism for avoiding recurrence in which HER3 expression occurs. If so, targeting patients with low anti-HER3 CD4 T cell responses may help improve immune surveillance and prevent long-term recurrence.

Patients with pCR to neoadjuvant chemotherapy also had a significantly higher anti-HER3 CD4 T cell response than patients with <pCR, suggesting a role in the prognosis of the immune system. HER3 signaling has been shown to mediate acquired resistance to targeted therapies. Sergina, N. et al. 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 involved in early resistance rather than acquired resistance, making the anti-HER3 immune response a potential prognostic marker for patients who most benefit from neoadjuvant therapy. Further studies should clarify whether the anti-HER3 immune response can intervene not only in prognosis but also in facilitating the 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 response, 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 thoracic parenchyma are menopausal. It is unlikely to occur during the period. Press, M.M. F. Et al., Oncogene 5: 953-62 (1990). Imaging that mimics breast regression during pregnancy and exposes the immune system to cellular proteins that are also normally expressed in breast tissue has observed changes in mammary gland density due to hormonal changes (as in menopause). Clendenen, T. et al. V. Et al., Magnetic Resonance Imaging 31: 1-9 (2013). Alternatively, the higher anti-HER3 immune response in postmenopausal women may be explained by the difference in risk. Expression of various carcinogenic drivers is clearly age-dependent: HER2- ^positive IBC is less likely to decline with age, and ER- ^positive IBC is more likely to occur. Clark, G.M. 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). Moreover, these two receptors are not independent of each other. This is because age-related ER / PR expression is HER2-dependent and vice versa. Nevern, P.M. Et al., Breast Cancer Res. Treat. 110: 153-59 (2008). TN IBC patients, who have the lowest anti-HER3 immune response and the most sensitive prognosis to HER3 overexpression, occur more frequently in premenopausal women. Bae et al. And Howlander, N. et al. Et al. N. C. I. 106 (5): 1-8 (2014). Thus, while a subset of premenopausal HD is the group at high risk of developing TN IBC, postmenopausal HD has already exceeded this higher risk period and in fact overexpresses both HER2 and HER3. A group with a low risk of developing breast cancer. It is also worth noting that while TN IBC is more common in young women, its occurrence in the elderly predicts a good prognosis for unknown reasons. Aapro, M. et al. , Et al., anals 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 due to a biological mechanism that actually occurs during menopause rather than the risk aversion group. be able to.

CONCLUSIONS: This example demonstrated a decrease in anti-HER3 CD4 T cell response from HD, BD and DCIS towards ER ^{positive and TN IBC.} Furthermore, a lower anti-HER3 response correlates with <pCR for recurrence and neoadjuvant therapy, 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 is ^{reduced 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 the greater role of the immune system in patrols of molecular carcinogenic drivers. Interestingly, postmenopausal HD generally has a higher risk of developing breast cancer that overexpresses HER3 and is significantly higher than premenopausal HD, a group that potentially exhibits mechanisms that mediate this risk. Had a response. It is important to determine whether HER3 pulse 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.

CONCLUSIONS: The anti-HER-3 immune response of CD4 Th1 cells is lost from healthy donors in invasive breast cancer, especially in TN IBC, a group with limited treatment options and a significant prognosis for HER-3 overexpression. The anti-HER3 immune response also mitigates the response to treatment and prognosis and points to potential immunotherapeutic targets. Addition of the HER3 immunogenic peptide to the DC1 vaccine may increase the population of IBC patients who will benefit from vaccination. Most importantly, these results reflect previous findings, pointing out that the immune system plays a major role in the patrol of molecular carcinogenic drivers.

The findings of this example, namely the significant loss of anti-HER3 CD4 + Th1 in breast tumorigenesis from HDS to IBC, are useful for the diagnosis and treatment of HER3-expressing cancers, especially breast cancer, especially triple negative IBC. It is understood by those skilled in the art that it is useful. It would be possible to develop a blood test to detect the circulating anti-cancer CD4 + Th1 response in the subject and utilize these findings. Preferably, such a blood test is based on the type of cancer the patient is suffering from, the four HER3 immunogenic peptides used herein or any other MHC class II immunogenicity. Use a HER3 immunogenic peptide such as a peptide that can elicit an immune response in the patient. As a non-limiting example, patients with recurrent breast cancer and lack of pCR for neoadjuvant therapy should be monitored by such blood tests to determine their anti-HER3 CD4 Th1 response and be treated accordingly. Can be done.

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

All patents, patent application disclosures and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is the present invention that other embodiments and modifications of the invention can be devised without departing from the spirit and scope of the true nature of the invention. It is obvious to the trader. The appended claims shall be construed to include all such embodiments and equivalent modifications.

Claims (28)

p11-13 (Peptide 51-75): KLYERCEVVMGNLEIVLTGHNADLSFLQW (SEQ ID NO: 1), p81-83 (Peptide 401-425): SWPPHMHNFFSVFSNLTTIGGRSLYN (SEQ ID NO: 2), p84-86 (Peptide 416-440): NNRGLS , P12 (Peptides 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4), p81 (Peptides 401-415): SWPPHMHNFFSVFSNL (SEQ ID NO: 5), p84 (Peptides 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6), and p91 (Peptide No. 6). 451-465): An isolated peptide selected from the group consisting of AGRIYISANRQLCYH (SEQ ID NO: 7). An immunomodulatory agent comprising one or more peptides according to claim 1. A vaccine comprising one or more of the peptides according to claim 1 and a pharmaceutically acceptable salt. The vaccine according to claim 3, further comprising an adjuvant. A cell that has been contacted with one or more of the peptides according to claim 1. The cell according to claim 5, which is an antigen-presenting cell. The cell according to claim 5, which is a T cell. A method of eliciting an immune response in a subject, comprising the step of administering the composition of claim 1 to the subject. A method of treating cancer in a subject, comprising the step of administering to the subject one or more of the peptides according to claim 1. The method of claim 9, wherein the subject is a human and has cancer. 10. 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 of the peptides of claim 1. The method of claim 12, wherein the cell is an antigen presenting cell. The method of claim 12, wherein the cell is a T cell. A method of producing activated dendritic cells (DCs) that have incorporated peptides for use in immunotherapy.
A step of pulse-adding one or more of the peptides according to claim 1 to the DC.
A method comprising activating the DC with at least one TLR agonist. 15. The method of claim 15, comprising contacting the DC with an agent that increases the intracellular calcium concentration in the DC. 15. The method of claim 15, wherein the agent comprises calcium ionophore. 15. The method of claim 15, further comprising the step of cryopreserving the DC, when the DC is thawed, the DC produces an effective amount of at least one cytokine to generate a T cell response. A cell produced by the method of claim 15. A vaccine comprising cells produced by the method of claim 15. The vaccine according to claim 20, wherein the vaccine is in the form of a multi-dose vaccine for injection. A method of eliciting an immune response in a mammal, comprising the step of administering a cell population generated from the method of claim 20 to a mammal in need thereof. A method of treating a disease or disorder in a mammal, comprising the step of administering a 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 the gastroesophageal junction in a subject having Barrett's esophagus, the biomarker comprising detecting overexpression of HER3 in the subject. A method of treating a patient who has lost anti-HER3 CD4 + Th1 and is an antigen pulsed DC1 vaccine derived from the monocytic dendritic cell (DC) precursor of the patient pulsed with a HER3 MHC class II immunogenic peptide. The peptide comprises administering at least one dose of the above to the patient: p12 (peptide 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4), p81 (peptide 401-415): SWPPHMMHNFFSVFSNL (SEQ ID NO: 5), p84 ( Peptide 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6), and p91 (Peptide 451-465): AGRIYISANRQLCYH (SEQ ID NO: 7), and a method selected from the group consisting of any combination thereof. 25. 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.
Peripheral blood mononuclear cells derived from the patient were pulsed with a HER3 MHC class II immunogenic peptide, and the peptide was p12 (peptide 56-70): CEVVMGNLEIVLTGH (SEQ ID NO: 4), p81 (peptide 401-415) :. SWPPHMHNFFSVFSNL (SEQ ID NO: 5), p84 (Peptide 416-430): TTIGGRSLYNRGFSL (SEQ ID NO: 6), and p91 (Peptide 451-465): AGRIYISANRQLCYH (SEQ ID NO: 7), selected from the group consisting of any combination thereof. , And a method of detecting the resulting immune response. 28. The method of claim 28, wherein detection of the anti-HER3 CD4 Th1 response is measured by the IFN-γELISpot assay.

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